Miami Lakes Award Program Honors the Achievement
MIAMI LAKES November 14, 2019 — Global Stem Cells Group has been selected for the 2019 Best of Miami Lakes Award in the Biotechnology Company category by the Miami Lakes Award Program.
Each year, the Miami Lakes Award Program identifies companies that we believe have achieved exceptional marketing success in their local community and business category. These are local companies that enhance the positive image of small business through service to their customers and our community. These exceptional companies help make the Miami Lakes area a great place to live, work and play.
Various sources of information were gathered and analyzed to choose the winners in each category. The 2019 Miami Lakes Award Program focuses on quality, not quantity. Winners are determined based on the information gathered both internally by the Miami Lakes Award Program and data provided by third parties.
About Miami Lakes Award Program
The Miami Lakes Award Program is an annual awards program honoring the achievements and accomplishments of local businesses throughout the Miami Lakes area. Recognition is given to those companies that have shown the ability to use their best practices and implemented programs to generate competitive advantages and long-term value.
The Miami Lakes Award Program was established to recognize the best of local businesses in our community. Our organization works exclusively with local business owners, trade groups, professional associations and other business advertising and marketing groups. Our mission is to recognize the small business community’s contributions to the U.S. economy.
SOURCE: Miami Lakes Award Program
Miami Lakes Award Program
by James R. Edgar
Exosomes are extracellular vesicles first described as such 30 years ago and since implicated in cell–cell communication and the transmission of disease states, and explored as a means of drug discovery. Yet fundamental questions about their biology remain unanswered. Here I explore what exosomes are, highlight the difficulties in studying them and explain the current definition and some of the outstanding issues in exosome biology.
What is the current definition of an exosome?
That is a very good question. Since the original description of exosomes over 30 years ago, the term has been loosely used for various forms of extracellular vesicle, muddying the field and contributing to the scepticism with which the research has sometimes been met. Exosomes are best defined as extracellular vesicles that are released from cells upon fusion of an intermediate endocytic compartment, the multivesicular body (MVB), with the plasma membrane. This liberates intraluminal vesicles (ILVs) into the extracellular milieu and the vesicles thereby released are what we know as exosomes (Fig. 1).
Exosomes correspond to intraluminal vesicles of multivesicular bodies. A transmission electron micrograph of an Epstein–Barr virus-transformed B cell displaying newly expelled exosomes at the plasma membrane. Multivesicular bodies (MVB) can be seen which can deliver content to lysosomes for degradation or can fuse with the cell surface to release intraluminal vesicles as exosomes, indicated by the arrows at the top of the picture
There are other types of microvesicle, including apoptotic bodies and ectosomes, which are derived from cells undergoing apoptosis and plasma membrane shedding, respectively. Although apoptotic bodies, ectosomes and exosomes are all roughly the same size (typically 40–100 nm) and all also contain ‘gulps’ of cytosol, they are different species of vesicles and understanding differences between them is of paramount importance but has too often been overlooked.
How were exosomes first recognized as distinct entities?
The presence of membranous vesicles outside cells was first recognized 50 years ago, although these were originally assumed to be waste products released via shedding of the plasma membrane. The recognition of what we now call exosomes didn’t come until 1983, from studies on the loss of transferrin during the maturation of reticulocytes into erythrocytes . These studies showed, by following transferrin-gold conjugates through the endocytic system, that ILVs generated in MVBs can be released to the extracellular space through fusion with the plasma membrane , although it was not until 1987 that the term ‘exosome’ was coined for them .
Even then, however, these extracellular vesicles were largely ignored, forgotten or, again, dismissed as a means of cellular waste disposal. It is only in the past decade that interest in exosomes has exploded, with a nearly tenfold increase in publications in as many years (115 in 2006, 1010 in 2015).
Why this explosion of interest?
For at least three reasons. First, they are thought to provide a means of intercellular communication and of transmission of macromolecules between cells. Second, in the past decade, exosomes have been attributed roles in the spread of proteins, lipids, mRNA, miRNA and DNA and as contributing factors in the development of several diseases. And third, they have been proposed to be useful vectors for drugs because they are composed of cell membranes, rather than synthetic polymers, and as such are better tolerated by the host. In fact, some of the earliest exosome research indicated that they can carry the MHC–peptide complexes that are recognized by T lymphocytes  and that secretion of such exosomes could promote antitumour immune responses in mice in vivo . Exosome therapies are now being explored in anti-cancer clinical trials and recent reports claim taxol-filled exosomes can be used to treat cancers in mice at 50-fold lower doses than conventional treatments, with the additional benefit that exosomes do not invoke an immune response .
Yet despite 20 years of research, the very basics of exosome biology are in their infancy and we know little of the part they play in normal cellular physiology.
So do we know how they are generated?
Yes and no. We do know that they are made as ILVs; but first of all, not all ILVs finish up as exosomes, and second, the mechanism of their generation in endosomes is not fully understood. Most conventional membrane budding processes deform membrane from an organelle into the cytoplasm but in ILV formation the membrane buds away from the cytoplasm and into the endosome. This unconventional budding process is not limited to ILV generation but also takes place during enveloped virus budding from the cytosol and during cytokinesis , and it requires specialised machinery.
ILVs (and thus exosomes) can be generated at the endosomal limiting membrane by at least two mechanisms, one of which depends on the ESCRT machinery (ESCRT stands for endosomal sorting complexes required for transport) whereas the other is ESCRT-independent (Fig. 2).
ILVs are generated by invagination of the endosomal membrane and have three possible fates. Inset: intraluminal vesicles (ILV) are formed by invagination of the endosomal membrane by either ESCRT-dependent or ESCRT-independent mechanisms. Matured endosomes accumulate ILVs within their lumen and have three distinct fates. They may deliver content that contributes to the biogenesis of specialized lysosome-related organelles (for example, melanosomes, Weibel-Palade bodies, azurophilic granules), they may fuse with lysosomes or they may fuse with the plasma membrane where released ILVs are now termed ‘exosomes’
The ESCRT machinery consists of a set of cytosolic protein complexes that are recruited to endosomes by membrane proteins that have been tagged, usually with ubiquitin on their cytosolic domains. The ubiquitin tag is recognized by the first of the ESCRT complexes, ESCRT-0, which is thus recruited to the endosomal membrane and passes ubiquitinated cargos to ESCRT-I, one of whose components, Tsg101, also recognizes ubiquitin. The recruitment of the ESCRT machinery acts to both cluster the ubiquitinated cargo proteins on the endosome and induce curvature of the endosomal membrane to form ILVs.
But ILVs are still able to form in the absence of ESCRTs , so other means of generating ILVs must exist, although the mechanisms for their generation are less clear. Generation of these ESCRT-independent ILVs requires the tetraspanin CD63—a protein abundant on ILVs but with unclear function —and may be facilitated by cone-shaped bending properties of lipids such as ceramide .
If not all ILVs become exosomes, what determines the fate of an ILV?
The destiny of ILVs is directed by the fate of the MVB they reside in. Confusingly, in addition to different types of ILVs, there are also different types of MVBs  and what regulates the fate of these endosomes is another interesting question. MVBs have several potential fates (Fig. 2) and can either fuse with lysosomes (where contents are degraded and recycled), fuse with the plasma membrane (where ILVs are released as exosomes), as I have already mentioned, or contribute to the generation of specialised organelles, such as melanosomes (in melanocytes), Weibel-Palade bodies (endothelial cells), azurophilic granules (in neutrophils) and secretory granules (in mast cells). The levels of cholesterol on MVBs appear to play a part in regulating their fate, cholesterol-rich MVBs being directed to the plasma membrane for exosome release, while cholesterol-poor MVBs are targeted to the lysosome .
But what regulates the balance between exosome release and alternative fates of ILVs remains engimatic.
What about differences between cells: do all cells release exosomes?
Well, not all cells have an endomembrane system, so no. But most mammalian cells contain endomembranes and generate ILVs within MVBs, though remarkably little is known about exosome release in most cell types.
Some cells—for example, the B cells, dendritic cells and mast cells of the immune system—appear to release exosomes constitutively; in fact, most of the data we have on exosomes comes from immune cells. As well as releasing exosomes constitutively, these cells may also be stimulated to secrete exosomes by cellular interactions. For example, murine dendritic cells, which are specialized to activate T lymphocytes, secrete higher levels of exosomes upon interaction with antigen-specific CD4+ T lymphocytes . In fact, lymphocyte interactions generally can be accompanied by exosome release; human T cells (including primary T cells from blood, T cell clones and Jurkat cell lines) release exosomes upon activation of their antigen receptors  and B cells release more exosomes upon engagement with antigen-specific CD4+ T cells .
Other cell types can be pushed to secrete exosomes by means of calcium ionophores or other stimuli[16, 17], but the extent of physiological exosome secretion in non-immune cells is largely unknown.
What happens when exosomes reach an acceptor cell?
We don’t know exactly. Exosomes that transfer membrane proteins or luminal content to the acceptor cell may be engulfed whole or the exosome membrane may fuse directly with the host plasma membrane (Fig. 3). Alternatively, exosomes may not need to be taken up by cells to elicit a physiological response: follicular dendritic cells, for example, carry on their cell surface exosomes that bear MHC–peptide complexes and other proteins that they do not express and are thereby enabled to activate immune cells with which they interact .
Exosome uptake by recipient cells. Fusion of MVBs with the cell surface releases ILVs as exosomes. In order for exosomes to elicit a response from recipient cells they might either fuse with plasma membrane (a) or be taken up whole via endocytosis (b), following which the exosome must be delivered to the cytosol, for example, via a back-fusion event (c). Alternatively, exosomes may attach to the surface of recipient cells to elicit a signalling response (d)
For intercellular transmission, various mechanisms of phagocytosis and endocytosis of extracellular vesicles have been described and which mechanism operates may depend upon vesicle size, which may in turn depend upon the cargo carried by the vesicle. In order for material to be released to an acceptor cell, exosomes must fuse with the host cell and this takes place via either direct fusion with the plasma membrane or a ‘back-fusion’ step from within a host endocytic organelle after the exosome has been engulfed. The process of back-fusion is not entirely clear, although it appears to require the unconventional lipid LBPA and protein Alix  (and is exploited by anthrax toxin lethal factor to escape from endosomes to the cytosol ).
Whether exosomes fuse with target cells or act via interactions with cell-surface proteins, or both, is another fundamental cell biology question that will need to be addressed if we are to understand the functions of exosomes.
So what are the consequences of all this information transfer? What biological functions have been established for exosomes?
There are many proposed functions for exosomes, the best-established being in immune responses. Exosomes isolated from B lymphocytes and bearing MHC class II molecules were shown in early experiments  to activate T lymphocytes in vitro, suggesting that they were communicating with the T lymphocytes in just the way that the parent B cells did. I have already mentioned later work by the same group, who showed that exosomes derived from dendritic cells, which are specialized to activate T cells in the initiation of immune responses, could promote antitumour immune responses in mice , exciting interest in the possibility of practical applications.
Or, as with follicular dendritic cells, exosome-associated MHC II can be found on the surface of cell types that neither express MHC II nor secrete exosomes, indicating that exosomes are delivered from one cell type to another .
However, exosomes may have roles other than in immune responses as several non-immune cells secrete exosomes. The only physiological role so far reported for non-immune cells is in keratinocyte-derived exosomes, which have been shown to modulate melanin synthesis by increasing the expression and activity of proteins within the melanosomes that modulate skin pigmentation .
How exactly would exosomes from one cell influence the expression and activity of proteins in an acceptor cell?
Exosomes transfer not only protein and lipids but mRNA and microRNA into acceptor cells and these RNAs have been shown in experiments in vitro to have functional effects in recipient cells. For example, exosomes from mice can be transferred to human cells and mRNA can be translated into mouse protein . Similarly, microRNAs—double-stranded RNA fragments that can regulate specific sets of mRNA (and so protein levels)—can act functionally in acceptor cells. The mode of action of exosomes has been a focus of special interest in cancer biology. Exosomes from breast cancer cell lines, for example, have been shown to be enriched for miRNAs relative to nontumorigenic breast cell lines and exposure of normal cells to exosomes derived from breast cancer cell lines increased both cell survival and proliferation, accompanied by loss of expression of some tumour-suppressor proteins . Exosome levels are elevated in the serum of some cancer patients versus controls. However, whether these vesicles are exosomes or other forms of extracellular vesicle, or a mix, is unclear—I have already mentioned this persistent problem in exosome research.
So exosomes can also contribute to disease?
Yes indeed. As exosomes provide a means of intercellular communication, they may also act as vehicles for ‘bad’ communication or spread. As well as miRNAs in the case of cancer, exosomes have been shown to contain numerous disease-associated cargos—for example, neurodegenerative-associated peptides, such as Aβ  (in Alzheimer’s disease), tau  (in numerous neurodegenerative diseases), prions  (in transmissible spongiform encephalopathies), alpha-synuclein  (in synucleinopathies, including Parkinson’s disease) and superoxide dismutase 1  (in amyotrophic lateral sclerosis). Exosomes have thus been suggested to be propagators of neurodegenerative protein spread, although some cargos are easier to envisage than others.
Of the neurodegenerative-associated proteins, only some are integral membrane proteins, that is, proteins inserted into lipid bilayers, rather than cytosolic. Sorting of proteins into ILVs (and thus exosomes) is easier to envisage for membrane proteins, where tags such as ubiquitin regulate where they end up. So far, the presence of both Aβ  and PrPc  has in fact been shown in ILVs, though this has not been demonstrated for other membrane proteins, such as alpha-synuclein and tau.
The mechanism whereby cytosolic proteins may be sorted to ILVs/exosomes, however, is not clear. In order for cytosolic proteins to become concentrated in ILVs, they would require positive incorporation and sorting, possibly by membrane-associated components on endosomes. All we can say is that there is evidence that this does in fact happen; cytosolic factors such as miRNAs are enriched in exosomes relative to cytosol, indicating that sorting must occur whereby certain miRNAs are concentrated and others are not .
The means by which disease-associated factors spread between cells remains poorly understood and exosomes would provide a means for such transmission. The presence of exosomal proteins, such as Alix, in association with Alzheimer’s senile plaques strengthens the circumstantial case for exosomes as a mediator in such spread. The hope is that having a means to regulate exosome release and spread may be useful in combatting some of these diseases but much more basic biology needs to be established before then.
Now I’m confused—what determines what exosomes contain?
Exosomes will contain whatever is sorted into them during their formation (as ILVs). For membrane proteins, this usually occurs through ubiquitination, which acts as a substrate for recruitment of the ESCRT machinery and subsequent generation of ESCRT-dependent ILVs.
The mechanisms that concentrate cytosolic factors are currently unknown. Although it seems clear that miRNAs, for example, are enriched relative to the amount in their parent cells, and are not randomly incorporated into exosomes, it is not clear how some are enriched more than others. There are currently a few hypotheses for miRNA sorting, including sorting via sumoylated heterogeneous nuclear ribonucleoproteins  or by a miRNA-induced silencing complex (miRISC) .
Because of the difficulties in separating exosomes from other extracellular vesicles, it is likely that some cargos reported to be enriched in ‘exosomes’ may in fact be contained in contaminant vesicles that are not exosomes. While many researchers are very stringent about applying the labels ‘exosomes’ and ‘extracellular vesicles’ correctly, others unfortunately are not. In addition, as I have said before, cytosolic proteins are likely to be found in exosome preparations because the exosome lumen is made of cytosol.
So how exactly can you be sure that a given extracellular vesicle is an exosome and not something else?
This is an interesting question that has a complex answer. Ideally, an intracellular compartment is identified by a specific biological marker, as, for example, in the case of the Golgi, nucleus or mitochondria, all of which carry proteins not found, or found at much lower levels, elsewhere.
One problem is that ILVs, and thus exosomes, represent an intermediate compartment of an intermediate. MVBs are not static organelles but rather undergo continuous maturation, in the course of which they gain and lose proteins. There will never be an exclusive marker for exosomes because any cargo on the ILV/exosome membrane must first be on the limiting membrane of the endosome and anything found inside must first come from the cytosol. A cargo may be concentrated on ILVs/exosomes but it will also be elsewhere. CD63 could be thought of as a pseudo-marker for exosomes. ILVs and exosomes are enriched in several such tetraspanins and my colleagues and I have show that CD63 is required for ESCRT-independent ILV formation . Alix also appears to be concentrated in ILVs/exosomes , as does Tsg101, a component of ESCRT-I, which has been used as a marker of exosomes in numerous studies [33,34], although the presence of Tsg101 in ILVs or exosomes does not fit with conventional models of ILV formation. Although Tsg101 is involved in ESCRT-dependent ILV formation, as mentioned earlier, it, along with other ESCRT components, should disassociate from the endosomal membrane prior to an ILV pinching off the endosomal membrane to allow it to participate in further events . Exactly when ESCRT-I components ‘fall off’ the membrane is unknown but it is conventionally thought to be prior to ILV formation, so Tsg101 should remain cytosolic and available for subsequent rounds of ILV formation. It is possible that some Tsg101 may be ‘swallowed’ into the forming ILV lumen, but levels should be negligible.
So are you saying there is no reliable marker for endosomes?
There may not be—not a single reliable one. Ultimately, perhaps the best method of defining exosomes biochemically may be through a combination of markers, including tetraspanins, Alix and others, with a concomitant exclusion of resident plasma membrane proteins. Although ILVs/exosomes will by their nature contain some plasma membrane proteins and the plasma membrane will contain some ILV/exosomal proteins, it should be possible to define relative levels and/or enrichment of proteins of exosomes that distinguish them from other microvesicles. Cargos such as MHC II from B cells and other cell type-specific antigens may also help to distinguish exosomes from other forms of extracellular vesicle. Common exosomal cargos include tetraspanins (CD63, CD81, CD9), antigen presentation molecules (MHC I and MHC II) and others (Alix, flotillin-1). An online database exists  where proteins, lipids and RNA are catalogued from published and unpublished exosomal studies.
If they are so hard to characterize reliably, how are exosomes isolated and studied?
Exosomes are rarely imaged by conventional methods as they are too small to be resolved by fluorescence microscopy and their release may be a rare event. A few studies have imaged exosome release occurring in cell cultures by various electron microscopic techniques but, more commonly, exosomes are pooled from cellular supernatant or animal fluids. Traditionally, they have been isolated by differential centrifugation from culture medium whereby larger contaminants are first excluded by pelleting out through increasing speeds of centrifugation before exosomes, small extracellular vesicles and even protein aggregates are pelleted at very high speeds (~100,000 × g) . These preparations therefore represent an enrichment rather than a purification. Enriched preparations are commonly analysed by biochemistry, mass spectrometry or electron microscopy. Electron microscopy of isolated fractions as ‘whole mounts’ make it possible to immuno-label vesicles, with the limitation that isolated preparations do not provide the same internal controls as labelling sections of cells. Remarkably little attention has been paid to the characterization of exosomes, although efforts are being made to repair this omission with guidelines and criteria for defining groups of extracellular vesicles .
What would you say are the most important issues in exosome research?
Without doubt the single most important issue is actually understanding the biological significance of these structures. With so little known about their basic physiological functions, it may seem hard to understand how exosomes have been implicated in the pathogenesis of so many disparate disease states. Fundamental questions remain about exosome generation, fate and normal function but, ultimately, in order to understand exosomes, one must first understand ILVs, a fact that is too often overlooked. Meanwhile, it is important that publications on exosomes give a careful and explicit account of the criteria used for distinguishing them from other extracellular vesicles to avoid confusing the field and encouraging scepticism.
Harding C, Heuser J, Stahl P. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol. 1983;97(2):329–39.
Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol. 1985;101(3):942–8.
Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem. 1987;262(19):9412–20.
Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996;183(3):1161–72.
Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, et al. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med. 1998;4(5):594–600.
Kim MS, Haney MJ, Zhao Y, Mahajan V, Deygen I, Klyachko NL, et al. Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine. 2015;12(3):655–64.
McDonald B, Martin-Serrano J. No strings attached: the ESCRT machinery in viral budding and cytokinesis. J Cell Sci. 2009;122(Pt 13):2167–77.
Stuffers S, Sem Wegner C, Stenmark H, Brech A. Multivesicular endosome biogenesis in the absence of ESCRTs. Traffic. 2009;10(7):925–37.
Edgar JR, Eden ER, Futter CE. Hrs- and CD63-dependent competing mechanisms make different sized endosomal intraluminal vesicles. Traffic. 2014;15(2):197–211.
Trajkovic K, Hsu C, Chiantia S, Rajendran L, Wenzel D, Wieland F, et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science. 2008;319(5867):1244–7.
White IJ, Bailey LM, Aghakhani MR, Moss SE, Futter CE. EGF stimulates annexin 1-dependent inward vesiculation in a multivesicular endosome subpopulation. EMBO J. 2006;25(1):1–12.
Mobius W, Ohno-Iwashita Y, van Donselaar EG, Oorschot VM, Shimada Y, Fujimoto T, et al. Immunoelectron microscopic localization of cholesterol using biotinylated and non-cytolytic perfringolysin O. J Histochem Cytochem. 2002;50(1):43–55.
Buschow SI, Nolte-’t Hoen EN, van Niel G, Pols MS, ten Broeke T, Lauwen M, et al. MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways. Traffic. 2009;10(10):1528–42.
Blanchard N, Lankar D, Faure F, Regnault A, Dumont C, Raposo G, et al. TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex. J Immunol. 2002;168(7):3235–41.
Muntasell A, Berger AC, Roche PA. T cell-induced secretion of MHC class II-peptide complexes on B cell exosomes. EMBO J. 2007;26(19):4263–72.
Savina A, Furlan M, Vidal M, Colombo MI. Exosome release is regulated by a calcium-dependent mechanism in K562 cells. J Biol Chem. 2003;278(22):20083–90.
Guo BB, Bellingham SA, Hill AF. Stimulating the release of exosomes increases the intercellular transfer of prions. J Biol Chem. 2016;291(10):5128–37.
Denzer K, van Eijk M, Kleijmeer MJ, Jakobson E, de Groot C, Geuze HJ. Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J Immunol. 2000;165(3):1259–65.
Bissig C, Gruenberg J. ALIX and the multivesicular endosome: ALIX in Wonderland. Trends Cell Biol. 2014;24(1):19–25.
Abrami L, Lindsay M, Parton RG, Leppla SH, van der Goot FG. Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway. J Cell Biol. 2004;166(5):645–51.
Lo Cicero A, Delevoye C, Gilles-Marsens F, Loew D, Dingli F, Guere C, et al. Exosomes released by keratinocytes modulate melanocyte pigmentation. Nat Commun. 2015;6:7506.
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654–9.
Melo SA, Sugimoto H, O’Connell JT, Kato N, Villanueva A, Vidal A, et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell. 2014;26(5):707–21.
Rajendran L, Honsho M, Zahn TR, Keller P, Geiger KD, Verkade P, et al. Alzheimer’s disease beta-amyloid peptides are released in association with exosomes. Proc Natl Acad Sci U S A. 2006;103(30):11172–7.
Saman S, Kim W, Raya M, Visnick Y, Miro S, Saman S, et al. Exosome-associated tau is secreted in tauopathy models and is selectively phosphorylated in cerebrospinal fluid in early Alzheimer disease. J Biol Chem. 2012;287(6):3842–9.
Fevrier B, Vilette D, Archer F, Loew D, Faigle W, Vidal M, et al. Cells release prions in association with exosomes. Proc Natl Acad Sci U S A. 2004;101(26):9683–8.
Emmanouilidou E, Melachroinou K, Roumeliotis T, Garbis SD, Ntzouni M, Margaritis LH, et al. Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival. J Neurosci. 2010;30(20):6838–51.
Gomes C, Keller S, Altevogt P, Costa J. Evidence for secretion of Cu, Zn superoxide dismutase via exosomes from a cell model of amyotrophic lateral sclerosis. Neurosci Lett. 2007;428(1):43–6.
Guduric-Fuchs J, O’Connor A, Camp B, O’Neill CL, Medina RJ, Simpson DA. Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types. BMC Genomics. 2012;13:357.
Edgar JR, Willen K, Gouras GK, Futter CE. ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-beta accumulation. J Cell Sci. 2015;128(14):2520–8.
Villarroya-Beltri C, Gutierrez-Vazquez C, Sanchez-Cabo F, Perez-Hernandez D, Vazquez J, Martin-Cofreces N, et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun. 2013;4:2980.
Fabian MR, Sonenberg N. The mechanics of miRNA-mediated gene silencing: a look under the hood of miRISC. Nat Struct Mol Biol. 2012;19(6):586–93.
Bobrie A, Colombo M, Krumeich S, Raposo G, Thery C. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J Extracell Vesicles. 2012;1:18397.
Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J, et al. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol. 2001;166(12):7309–18.
Hurley JH. The ESCRT complexes. Crit Rev Biochem Mol Biol. 2010;45(6):463–87.
Thery C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protocols Cell Biol. 2006;Chapter 3:Unit 3 22.
Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014;3:26913.
The author wishes to thank Scottie Robinson, Paul Luzio and Paul Manna for critical reading of this article.
The author declares that he has no competing interests.
The awards recognize the physicians’ leadership in education and instruction in regenerative medicine
MIAMI LAKES, Florida—Three faculty members with the International Society for Stem Cell Application (ISSCA) were honored with awards at the organization’s recent regenerative medicine conference held at the University of Miami on October 24-27. The awards were sponsored by the Sociedad Internacional en Investigación, Salud, Desarrollo Empresarial y Tecnologías (SISSDET) and lauded the honorees for their commitment to leadership and education in the field of regenerative medicine.
The three recipients of the awards are Dr. Damian Ariel Siano, Dra. Maritza Novas, and Dra. Silvina Pastrana. All three have continued to partner with the ISSCA and have notable contributions to the field by offering courses in regenerative medicine, helping thousands of doctors around the world add stem cells therapies to their medical practices.
Dr. Damian Ariel Siano is an orthopedic physician from Argentina who has dedicated his professional life to treat sports injuries. He is one of the most renowned sports medicine specialists in South America. Dr. Siano currently works with one of the most famous soccer teams in Argentina, using cell therapies to help professional athletes avoid unnecessary surgery and recuperate quicker from injuries.
Dra. Maritza Novas currently serves as the Director of Research and Development for the Global Stem Cells Group. For the past 10 years, she has dedicated herself to educating doctors in the latest stem cells advancements and conducting stem cell research. Dra. Novas has visited all continents, sharing her knowledge as a stem cells practitioner and researcher.
Dra. Silvina Pastrana is one of the first doctors that helped form the ISSCA. She has become a visionary in the field and is noted for creating her own stem cells protocols and using complementary therapies to get better results in patient who utilize cell therapies. Dra. Pastrana combines both ozone and vitamin C therapies before employing stem cell protocols, obtaining excellent results in treating patients with arthritis.
“The ISSCA is committed to helping physicians who want to add regenerative medicine to their practices gain the education and tools to do that,” said Benito Novas, Vice President of Public Relations for ISSCA. “The three doctors recognized at our recent event at the University of Miami are prime examples of the high-quality instructors that physicians can anticipate working with when then attend one of our conferences. Congratulations to our faculty on receiving this prestigious award, and thank you to SISSDET for recognizing their accomplishments.”
ISSCA is a global leader in stem cells research, applications, and education, partnering with major global institutions and locations worldwide to host its independent medical congresses. To learn more about the ISSCA and its all of its past and upcoming events, visit http://www.issca.us
The organization seeks to improve education and access to stem cells therapies for physicians and patients worldwideThe organization seeks to improve education and access to stem cells therapies for physicians and patients worldwide
MIAMI LAKES, Florida—Following two recent highly successful conferences, the International Society for Stem Cell Application (ISSCA) is set to offer six additional events on its training calendar to round out 2019. Recent events attracting physicians across the globe include a conference held at the University of Miami and the ISSCA’s 6th Annual Regenerative Medicine Symposium in Buenos Aires. With its upcoming slate of events, the group is hoping to receive the same positive response.
One of the ISSCA’s missions is to share the latest in stem cells protocols and clinical applications with physicians across the globe, so they can implement stem cells therapies into their practices. By adding these protocols to their practices, physicians can help alleviate suffering and improve recuperation times for patients diagnosed with a host of degenerative diseases and injuries.
To accomplish its mission, the ISSCA will host the following events in 2019:
- Uccle, Belgium – November 15-16
- Mexico City – November 22-23
- Cochabamba, Bolivia – November 22-23
- Miami, FL – November 29-30
- Buenos Aires, Argentina – December 6-7
- Bogota, Colombia – December 13-14
“We invite all physicians interested in adding stem cells therapies to their practices to join us at one of our upcoming conferences,” said Benito Novas, Vice President of Public Relations for ISSCA. “Attendees will learn from highly trained physicians and researchers in the field of regenerative medicine and take away the necessary education and tools to implement the latest stem cells therapies into their own practices.”
The ISSCA is a global leader in stem cells research, applications, and education, partnering with major global institutions and locations worldwide to host its independent medical congresses. To learn more about the ISSCA and its all of its past and upcoming events, visit http://www.issca.us
ISSCA Conference at University of Miami Attracts Regenerative Medicine Experts and Physicians from across the Globe
The regenerative medicine symposium was held on October 24-27 with nearly 200 attendees and over 20 expert speakers
MIAMI LAKES, Florida—The International Society for Stem Cell Application (ISSCA), in collaboration with SISDET, held a highly successful three-day medical conference on the University of Miami campus on October 24-26. The conference featured a host of international experts in regenerative medicine and introduced new standards in regenerative medicine protocols to those in attendance. The Miami conference is part of the ISSCA’s growing commitment to increasing the awareness and practice of regenerative medicine across the globe in an effort to help alleviate suffering for those diagnosed with degenerative diseases.
Around 200 physicians, scientists, and researchers interested in regenerative medicine traveled to the University of Miami campus for the event. The conference focused on providing attendees with information on today’s most successful stem cells treatment protocols and the latest advances in regenerative medicine. Attendees heard from more than 20 expert speakers within the stem cells field, with lecturers from Europe, the US, and Latin America on the conference agenda.
“This three-day event included recognized keynote speakers, as well as aspiring young physicians discussing the latest advances in stem cell biology in an informal and collaborative setting,” said Benito Novas, Vice President of Public Relations for ISSCA. “Our goal with all of our events is to strengthen the cooperative and dynamic spirit in this research area. We would also like to thank the University of Miami for hosting this event, as it was a great honor partnering with such a prestigious university.”
ISSCA is a global leader in stem cells research, applications, and education, partnering with major global institutions and locations worldwide to host its independent medical congresses. To learn more about the ISSCA and its all of its past and upcoming events, visit http://www.issca.us
The courses will target physicians based in the Americas, providing them with critical stem cells education and tools needed to implement protocols in their own practices
The International Society for Stem Cell Application (ISSCA), a multi-disciplinary community of scientists and physicians collaborating to treat diseases and lessen human suffering through science, technology, and regenerative medicine, is hosting two upcoming courses in Miami. The courses will inform physicians on a range of topics about the latest stem cells protocols and how they can be incorporated into physicians’ own practices.
In continuing its commitment to help alleviate human suffering through expanding physician access to today’s cutting-edge stem cells technologies, the ISSCA is bringing its globally renowned courses to Miami, offering physicians two opportunities to participate. The course offered on November 1-2 is sold out, but seats still remain for the November 29-30 session for physicians looking to incorporate stem cells treatment protocols into their practices.
Doctors attending the Miami courses will learn about the latest trends and breakthroughs in in-office stem cells protocols. The ISSCA has also added a new topic into the Miami sessions: previously, the course covered autologous cell therapy, but in this newest course iteration, speakers will discuss how to add heterologous cell therapy into medical practices. In this training, physicians will learn about treatment protocols utilizing stem cells derived from neonatal tissue, such as cord blood, exosomes, and amniotic liquid. Attendees will learn both how these products are obtained as well as their most common clinical applications.
For those interested in learning more about the ISSCA’s upcoming courses in Miami or to register, visit https://www.stemcelltraining.net/hands-on-course/
“The ISSCA is pleased to bring our highly lauded courses on stem cells protocols to physicians in the Miami area and beyond,” said Benito Novas, ISSCA VP of Public Relations. “Our goal is to help physicians better treat their patients suffering from degenerative diseases by giving them the tools and education they need to implement these highly effective protocols in their own practices. Like all of our educational sessions, our Miami courses have been met with a great response, proving the growing interest in and efficacy of stem cells treatments.”
To learn more about all of the ISSCA’s latest news and innovations, visit http://www.stemcellsgroup.com/
The ISSCA sponsored symposium is in its sixth year, sharing recent advancements in stem cells therapies with physicians looking to implement them into their own practices
The International Society for Stem Cell Application (ISSCA), a multi-disciplinary community of scientists and physicians collaborating to treat diseases and lessen human suffering through science, technology, and regenerative medicine, recently hosted a symposium in Buenos Aires, aimed at physicians looking to incorporate regenerative medicine into their practices.
The 6th Simposio Mundial sobre Medicina Regenerativa y Terapias Celulares was held in Buenos Aires, Argentina on October 12-13, with 300 physicians in attendance. The event has become a premier global event in the world of regenerative medicine, attracting a growing number of returning physicians each year.
The 6th annual event featured 13 expert speakers from five countries, including Argentina, Chile, Colombia, Bolivia, Cuba, and the US. The panel of speakers included practitioners within the regenerative medicine field as well as scientists currently working on developing new stem cells protocols and allogeneic compounds. Throughout the weekend, physicians attended lectures providing information on how stem cell therapies can help patients suffering from degenerative diseases.
The event was sponsored by the City of Buenos Aires, which was pleased to host the event in the city and has invited the group back next year to host its 7th annual event.
All of the symposium’s lectures will be posted to ISSCA’s YouTube channel, with free viewing access to the public. Those interested in viewing the symposium’s lectures can visit
“For six years now, ISSCA has proudly sponsored the Simposio Mundial sobre Medicina Regenerativa y Terapias Celulares, bringing physicians from across the globe together to learn about the newest advancements in stem cells therapies,” said Benito Novas, ISSCA VP of Public Relations. “This year’s event was a great success, and we would like to thank all of the attending physicians, our panel of expert speakers, and the City of Buenos Aires for graciously hosting us.”
To learn more about all of the ISSCA’s latest news and innovations, visit http://www.stemcellsgroup.com/.
The certification will target physicians in the Middle East and Europe, providing them with critical education to implement stem cells treatments in their own practices
The International Society for Stem Cell Application (ISSCA), a multi-disciplinary community of scientists and physicians collaborating to treat diseases and lessen human suffering through science, technology, and regenerative medicine, is continuing its mission to become a world leader in regenerative medicine education by adding a new Stem Cells Certification course to its portfolio.
Over the past few months, ISSCA has expanded its reach both through offering new certification courses and by opening new stem cells treatment centers across the globe. The group’s latest Stem Cells Certification course will be offered in Brussels, Belgium, giving physicians in both Europe and the Middle East who have an interest in incorporating regenerative medicine into their practices easier access to critical educational opportunities led by some of the world’s leading experts in the field.
The Brussels course is a collaboration between Belgium-based physician Dr. Samy Joheir, ISSCA Europe, and ISSCA. Participants in the upcoming course are expected to come from the Middle East, specifically from Pakistan and India. The participating physicians are eager to add cell therapies to their practices to better serve patients who are suffering from degenerative conditions.
Physicians attending the certification will learn easy-to-implement protocols that will allow them to harvest and reintegrate stem cells in their own medical offices. The practices shared in the certification will focus on utilization of two different resources: fat tissue and bone marrow tissue.
Physicians interested in learning more about any of ISSCA’s Stem Cells Certification courses are invited to visit https://www.stemcelltraining.net/belgium-hands-on-course/
“The ISSCA is continuing to make good on its mission to alleviate human suffering due to degenerative diseases by providing more physicians across the globe access to critical stem cells training courses,” said Benito Novas, ISSCA VP of Public Relations. “The upcoming course in Belgium will give physicians across Europe and the Middle East critical opportunities to obtain the education needed to utilize today’s cutting-edge stem cells therapies and give them the tools they need to incorporate these life-changing protocols into their own practices.”
To learn more about all of the ISSCA’s latest news and innovations, visit http://www.stemcellsgroup.com/.
Phase I/II Study of Safety and Preliminary Efficacy of Intravenous Allogeneic Mesenchymal Stem Cells in Chronic Stroke
Phase I/II Study of Safety and Preliminary Efficacy of Intravenous Allogeneic Mesenchymal Stem Cells in Chronic Stroke
Michael L. Levy, MD, PhD; John R. Crawford, MD; Nabil Dib, MD; Lev Verkh, PhD; Nikolai Tankovich, MD, PhD; Steven C. Cramer, MD
Background and Purpose—Stroke is a leading cause of long-term disability. Limited treatment options exist for patients with chronic stroke and substantial functional de?cits. The current study examined safety and preliminary ef?cacy estimates of intravenous allogeneic mesenchymal stem cells in this population.
Methods—Entry criteria included ischemic stroke >6 months prior and substantial impairment (National Institutes of Health Stroke Scale score =6) and disability. Enrollees received a single intravenous dose of allogeneic ischemia-tolerant mesenchymal stem cells. Phase 1 used a dose-escalation design (3 tiers, n=5 each). Phase 2 was an expanded safety cohort. The primary end point was safety over 1-year. Secondary end points examined behavioral change.
Results—In phase 1 (n=15), each dose (0.5, 1.0, and 1.5 million cells/kg body weight) was found safe, so phase 2 subjects (n=21) received 1.5 million cells/kg. At baseline, subjects (n=36) averaged 4.2±4.6 years poststroke, age 61.1±10.8 years, National Institutes of Health Stroke Scale score 8 (6.5–10), and Barthel Index 65±29. Two were lost to follow-up, one was withdrawn and 2 died (unrelated to study treatment). Of 15 serious adverse events, none was possibly or probably related to study treatment. Two mild adverse events were possibly related to study treatment, a urinary tract infection and intravenous site irritation. Treatment was safe based on serial exams, electrocardiograms, laboratory tests, and computed tomography scans of chest/abdomen/pelvis. All behavioral end points showed signi?cant gains over the 12-months of follow-up. For example, Barthel Index scores increased by 6.8±11.4 points (mean±SD) at 6-months (P=0.002) and by 10.8±15.5 points at 12-months (P<0.001) post-infusion; the proportion of patients achieving excellent functional outcome (Barthel score =95) increased from 11.4% at baseline to 27.3% at 6-months and to 35.5% at 12-months.
Conclusions—Intravenous transfusion of allogeneic ischemia-tolerant mesenchymal stem cell in patients with chronic stroke and substantial functional de?cits was safe and suggested behavioral gains. These data support proceeding to a randomized, placebo-controlled study of this therapy in this population.
Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identi?er: NCT01297413. (Stroke. 2019;50:00-00. DOI: 10.1161/STROKEAHA.119.026318.)
Key Words: abdomen ? brain ischemia ? neuroprotection ? pelvis ? reperfusion
Stroke is perennially among the leading causes of human disability1 and the leading neurological cause of lost dis-ability-adjusted life years.2 The mean survival after stroke is 6 to 7 years, and indeed more than 85% of patients live past the ?rst year poststroke,3 many with years of enduring disability. Many restorative therapies are under study to improve outcomes after stroke.4 Restorative therapies aim to improve patient outcomes by promoting the neural processes under-lying behavioral recovery,5 and are distinguished from acute therapies, such as reperfusion or neuroprotection, that aim to reduce initial injury. As such, restorative therapies often have a time window measured in days-months, or in some cases6–9 in years.
Mesenchymal stem cells (MSC), also known as mes-enchymal stromal cells, are among the leading restorative therapy candidates. Substantial preclinical data support the safety and ef?cacy of MSC as a restorative therapy to improve outcomes after stroke. For example, a meta-analysis reported that 44 of 46 preclinical stroke studies found MSC to be supe-rior to placebo,10 with effect sizes >1.0.
Initial human studies of MSC (or MSC-like cells) after stroke focused on autologous cell therapies,11–13 whereby bone marrow is taken from each patient to produce his/her own MSC batch, and found MSC infusion to be safe. MSC are rela-tively immunoprivileged given their very low levels of human leukocyte antigen molecule expression,14 a fact that opens the door to administration of allogeneic MSC. Allogeneic MSC Table 1. Entry Criteria have been found to be safe without use of concomitant im-munosuppression,15 and can be manufactured in a manner that enables broad clinical application. Studies of allogeneic MSC (or MSC-like cells) poststroke have focused on early time points (administration 24–48 hours poststroke)16 or used an invasive procedure to implant cells intracerebrally.17 Each approach has its relative advantages and disadvantages, and an intravenous method of introducing MSC if comparably ef-?cacious might facilitate widespread implementation and also avoid adverse events attributable to invasive procedures.
The current study was a phase I/II dose-escalation trial that examined effects of a single intravenous infusion of al-logeneic ischemia-tolerant MSC. The target population was patients with chronic ischemic stroke and substantial func-tional de?cits, a group for whom treatment options remain limited. The primary outcome was safety, based on serial measures of behavior, computed tomography (CT) scans, and laboratory testing. Preliminary estimates of treatment ef?cacy were also examined.
Table 1. Entry Criteria
|1. Age =18 y|
|2. Ischemic stroke =6 mo prior, radiologically confirmed at initial diagnosis and at study enrolment|
|3. Severe disability resulting from the index stroke, operationally defined as subject confined to a wheelchair or required to have home nursing care or needing assistance with activities of daily living|
|4. No substantial improvement in neurological or functional status for the 2 mo before study enrolment|
|5. NIHSS score 6–20|
|6. Life expectancy >12 mo|
|7. Patient receiving standard of care secondary stroke prevention before enrolment|
|8. Patient or a surrogate able to provide informed consent|
|9. Reasonable expectation that the patient will receive standard posttreatment care and attend all scheduled study visits|
|10. Adequate systemic organ function, specifically:
Serum aspartate aminotransferase =2.5× upper limit of normal
Serum alanine aminotransferase =2.5× upper limit of normal
Total serum bilirubin =1.5× upper limit of normal
Prothrombin time and partial thromboplastin time =1.25× upper limit of
normal in subjects who are not receiving anti-thrombotic therapy
Serum albumin =3.0 g/dL
Absolute neutrophil count =1500/µL
Platelet count =150 000/µL
Hemoglobin =9.0 g/dL
Serum creatinine =1.5× upper limit of normal
Serum amylase or lipase =1.0× upper limit of normal
|1. History of uncontrolled seizure disorder|
|2. History of cancer within the past 5 y, with the exception of localized basal or squamous cell carcinoma|
|3. History of cerebral neoplasm|
|4. Positive for hepatitis B, C, or HIV|
|5. Myocardial infarction within 6 months of study entry|
|6. Presence of any other clinically significant medical or psychiatric condition, or laboratory abnormality, for which study participation would pose a safety risk in the judgment of the Investigator or Sponsor|
|7. Findings on baseline computed tomography suggestive of subarachnoid or intracerebral hemorrhage within past 12 mo.|
|8. Participation in another investigational drug or device study in the 3 mo before treatment|
|9. History within the past year of drug or alcohol abuse|
|10. Pregnant or lactating, or expectation to become pregnant during the study|
|11. Allergy to bovine or porcine products|
NIHSS indicates National Institutes of Health Stroke Scale
This was a phase I/II multi-center, open-label study that aimed to evaluate the safety and preliminary ef?cacy of a single intra-venous infusion of marrow-derived allogeneic ischemia-tolerant MSC. Entry criteria appear in Table 1 and in sum describe enroll-ment of adults with radiologically veri?ed chronic stable ischemic stroke and substantial impairment and functional de?cits. Patients were followed for one year after MSC infusion. The study made no restrictions on, and did not provide any forms of, medication or therapy (occupational, physical, or speech) during the follow-up year after infusion. All patients signed consent in accordance with local Institutional Review Board approval. This study was approved by the Food and DrugAdministration and was registered at clinical-trials.gov. The data that support the ?ndings of this study are avail-able from the corresponding author on reasonable request.
The study occurred in 2 parts, with part 1 being a dose-escala-tion study and part 2 being an expanded safety study based on part 1 ?ndings. Part 1 consisted of 3 cohorts (n=5 per cohort) enrolled sequentially in a dose-escalation manner, with subjects receiving one of 3 doses based on body weight, with a maximum dosage of 150 million cells. Cohort 1 received 0.5 million cells/kg of body weight; Cohort 2, 1.0 million cells/kg; and Cohort 3, 1.5 million cells/kg. The dose-escalation plan in part 1 required a review by the Data Safety Monitoring Board once the 5 subjects in Cohort 1 were treated and evaluated through study day 10. If safety was established, Cohort 2 was to proceed at the next highest dose, followed by a similar safety review before escalation to the highest dose in Cohort 3. Part 2 aimed to enroll an additional minimum of 20 subjects at the highest safe dose level determined in part 1. An additional interim review was conducted by the Data Safety Monitoring Board after the ?rst 5 patients were treated in part 2. Detailed stopping rules appear in the online-only Data Supplement (see Stopping Rules and Determination of Maximum Tolerated Dose).
The target dose of 1.5 million cells/kg corresponds to allometric scaling from animal studies. Our meta-analysis of preclinical studies of MSC after experimental ischemic stroke10 identi?ed 9 rodent stud-ies that transfused MSC using the intravenous route in the post-acute period. In each study, MSC provided substantial behavioral gains (effect sizes >1.0), using doses ranging from 3.6 to 12.4×106 MSC/kg body weight (mean dose of 10.1×106 MSC/kg). The approach to al-lometric scaling from animals to humans recommended by the Food and Drug Administration 18 uses a body surface area normalization, which for the mean value in rodents yields a comparable human dose of 1.6×106 MSC/kg.
Cell Manufacturing and Shipping
Manufacturing of MSC was performed at the GMP-compliant fa-cility of the sponsor, Stemedica Cell Technologies, Inc (San Diego, CA). MSC were grown from the bone marrow of a single human donor and are from the same batch used in prior preclinical19,20 and clinical21 studies. Cells were grown under low oxygen (5%) condi-tions. Such ischemia-tolerant MSC have advantages compared with those grown under normoxic conditions, for example, showing higher proliferation rate, expression of stem cell-related genes, production of key cytokines, and migration activity.21,22 Cells were harvested at passage 4 and expressed CD105, CD73, and CD90 surface markers, consistent with the International Society for Cellular Therapy de?-nition.23 Cells were cryopreserved by suspending in Cryostar CS10 freezing medium (BioLife Solutions, Bothell, WA) then stored in the vapor phase of liquid nitrogen. This parent cell bank was then tested for quality control including cell count, viability, appearance, and quantitative polymerase chain reaction for viruses including HIV, Epstein-Barr virus, cytomegalovirus, hepatitis B virus, parvovirus B19, and hepatitis C virus. Cryovials were shipped at =-150° C in a vapor phase liquid nitrogen shipper with temperature monitor.
Infusion of Investigational Product
Each site’s pharmacy prepared MSC for infusion per a study-pro-vided protocol. Cryovials (the number of which was based on the dose to be infused) were thawed and MSC were washed in, and then suspended in, Lactated Ringer’s solution at a concentration of 1×106 cells/mL using one to three 60 mL syringes. The suspension then un-derwent ?nal testing before being released for intravenous infusion, consisting of cell count, endotoxin, Gram stain, and review of appear-ance. Cell count was performed using 0.1% Trypan Blue and a hema-cytometer, which also yielded % cell viability. The minimum percent cell viability was required to be =70% for the cells to be released. A sample was also sent for subsequent sterility testing. After release by the pharmacy, the ?nal formulation was stored at 2° to 8°C and infused within 8 hours of preparation.
Before MSC infusion, a 0.1 mL aliquot of the ?nal MSC formulation was injected intradermally; any subject showing a positive reaction (eg, wheal with erythema) would not be infused. Cells were admin-istered intravenously via metered-dose syringe pump at 2 mL/min. Patients remained in the inpatient telemetry unit for observation until clinically stable.
Patients had frequent monitoring until discharged from the telem-etry unit. After discharge, patients had safety evaluations on day 2, 3, 4, and 10, then again on month, 1, 3, 6, 9, and 12. Adverse events were coded according to the MedDRA adverse event dic-tionary. The relationship that adverse events had to the investiga-tional product was assessed by the site investigator. Patients were followed for one year using tests of behavior, serology, blood chem-istry and cell counts, electrocardiogram, urine, and CT of chest, abdomen, and pelvis. The full schedule of assessments appears in Table SI in the online-only Data Supplement.
The primary study end point was safety and tolerability, evaluated in all subjects who received any portion of an infusion, and determined by the incidence/severity of adverse events, clinically signi?cant changes on laboratory and imaging tests, vital signs, and physical plus neurological examinations. Four secondary end points were scored serially to derive preliminary estimates of ef?cacy: National Institutes of Health Stroke Scale, Barthel Index (BI), Mini-Mental Status Exam, and Geriatric Depression Scale. For each, the change from baseline was evaluated using Wilcoxon signed-rank test, with primary analysis of preliminary ef?cacy being change from baseline to 6 months post-infusion, and analysis including all subjects who re-ceived an infusion except for one subject who failed to return after the day 10 visit for all visits (except for month 9 follow-up). For any sub-ject missing 6-month data, 9-month or 12-month data were substituted for this analysis, otherwise missing data were not imputed. Data were analyzed using R statistical software. Given the exploratory nature of this study, sample size was selected as appropriate for detection of any safety concerns in an early phase clinical trial.
Of 50 subjects who seemed eligible on prescreening, 36 were enrolled and received treatment from March 14, 2011 to December 15, 2016 (Figure and Table 2). There were 13 subjects enrolled at the University of California, San Diego, 19 subjects at Arizona, and 4 subjects at the University of California, Irvine. Interim safety reviews disclosed no con-cerns, and so 5 subjects received 0.5×106 cells/kg in part 1/ Cohort 1, 5 subjects received 1.0×106 cells/kg in part 1/Cohort 2, 5 subjects received 1.5×106 cells/kg in part 1/Cohort 3, and all 21 subjects in part 2 received 1.5×106 cells/kg. For the 15 subjects in part 1, 12 completed the study, 2 died of unrelated causes (coronary artery disease 6 months post-infusion and sepsis 1 month after infusion), and 1 was lost to follow-up after day 10 (reappearing only for the month 9 follow-up visit). For the 21 subjects in part 2, 19 completed the study, 1 was lost to follow-up after month 6, and 1 was withdrawn by the site PI after month 6 due to treatment with another in-vestigational product. Of the 36 subjects enrolled, the planned dose was delivered within 2 mL (ie, within 2×106 cells) of the target in 26 subjects, whereas in 10 subjects a median of 7.6 (interquartile range, 4.4–10.25) mL (ie, 7.6×106 cells) was not infused as planned, which represented a median of 6.5% (5.3– 9.8) of the intended dose. A total of 179 protocol deviations were reported, mainly related to scheduling study visits or study testing (Table SII in the online-only Data Supplement).
Figure. CONSORT diagram.
Table 2. Baseline Subject Characteristics
|Cohort 1||Cohort 2||Cohort 3|
|Male||5 (100%)||4 (80%)||4 (80%)||14 (66.67%)||27 (75%)|
|Female||0 (0%)||1 (20%)||1 (20%)||7 (33.33%)||9 (25%)|
|Age, y||50.8:±9.8 [40—62]||56.8±11.1 [39—69]||68.8±11.58 [53—84]||62.8±9.2 [51—83]||61.1±10.8 [39—84]|
|White||4 (80%)||3 (60%)||5 (100%)||17 (80.95%)||29 (80.56%)|
|Asian||0 (0%)||1 (20%)||0 (0%)||0 (0%)||1 (2.78%)|
|American Indian/Alaskan Native||0 (0%)||1 (20%)||0 (0%)||0 (0%)||1 (2.78%)|
|Native Hawaiian/Pacific Islander||0 (0%)||0 (0%)||0 (0%)||0 (0%)||0 (0%)|
|Black||1 (20%)||0 (0%)||0 (0%)||1 (4.76%)||2 (5.56%)|
|Other||0 (0%)||0 (0%)||0 (0%)||3 (14.29%)||3 (8.33%)|
|Hispanic or Latino||0 (0%)||0 (0%)||0 (0%)||2 (9.52%)||2 (5.56%)|
|Non-Hispanic or Non-Latino||5 (100%)||5 (100%)||5 (100%)||19 (90.48%)||34 (94.44%)|
|At home||5 (100%)||5 (100%)||3 (60%)||19 (90.48%)||32 (88.89%)|
|In a living facility||0 (0%)||0 (0%)||2 (40%)||2 (9.52%)||4 (11.11%)|
|Time from stroke to infusion, y||1.6±0.9 [0.6—2.9]||7.7±5.0 [1.1—14.5]||4.1±2.2 [1.7—7.0]||4.0±5.0 [0.7—24.8]||4.2±4.6 [0.6—24.8]|
Values are counts (%) else mean±SD. Values in brackets indicate range.
A total of 15 serious adverse events were reported.These were wide-ranging in nature, for example, infections, vascular dis-orders, and pain syndromes (for full details, see Table SIII in the online-only Data Supplement). All serious adverse events were deemed unrelated or unlikely related to the investiga-tional product. A total of 109 adverse events were reported, of which 2, both mild, were considered by the site investigator to be possibly related to the investigational product: one uri-nary tract infection and one report of intravenous site irrita-tion. Both adverse events recovered completely.
Study testing disclosed no safety concerns. No subject showed a preinfusion positive reaction to intradermal testing. Serial physical exams and blood testing did not disclose any signi?cant ?ndings. Only one of the serial electrocardiograms was thought to have clinically signi?cant ?ndings, in a subject with moderate intraventricular conduction delay, only at the 1-month follow-up visit. Similarly, across serial CT scans of the chest, abdomen, and pelvis, only one was considered clinically signi?cant, a soft tissue density in the anterior abdominal wall seen at 6-months that was stable when reimaged at 12-months.
Across all subjects, improvements were seen in National Institutes of Health Stroke Scale, BI, Mini-Mental Status Exam, and Geriatric Depression Scale scores at both the 6-month and the 12-month follow-up visits (Table 3). These were statistically signi?cant, generally stable over time, and clinically modest in magnitude. Most ?ndings would sur-vive correction for multiple comparisons. Changes in the BI suggest clinical utility, with a 6.8 point gain by 6-months that grew to a 10.8 point gain by 12-months post-infusion (P<0.001), and with the proportion of patients achieving ex-cellent functional outcome (Barthel score =95) increasing from 11.4% (4/35) at baseline to 9/33 (27.3%) at 6-months to 35.5% (11/31) at 12-months.
Stroke is a major cause of human disability. This can be reduced by acute therapies that are introduced in the early hours poststroke to reduce initial injury, and by restorative therapies that are introduced days, months, or years poststroke to promote neural repair. Allogeneic MSC show substantial favorable effects in preclinical studies, including when intro-duced via the intravenous route.10 The current study found a single intravenous infusion of allogeneic MSC to be safe and potentially associated with functional improvement.
The current study is the largest trial of intravenous MSC in patients with chronic stroke and the ?rst to evaluate alloge-neic MSC therapy in this population. It is also the ?rst human stroke study to evaluate MSC grown under hypoxic conditions, which favorably affects cell proliferation, gene expression, cy-tokine production, and migration.21,22 Intravenous infusion of MSCwasfoundtobesafein36patientswhohadchronicstroke with substantial functional de?cits. Across 3 escalating doses, treatment-related adverse events were infrequent, mild, and transient. Serial assessments of exam, laboratory testing, elec-tro cardiogram, and CT scans of chest/abdomen/pelvis disclosed no safety concerns, with limited subject dropout. These results are consistent with the overall excellent safety record that MSC have in clinical trials of human subjects across numerous non-cerebrovascular diagnoses15,24–27 and in stroke trials.11–13,16,17,28,29
Patients with stroke in the chronic stage generally show functional decline; however, enrollees in the current study showed 12 months of continued functional improvement. In general, recovery from stroke-related de?cits shows a bimodal time course. Initially, most stroke survivors show some degree of spontaneous recovery, for example, during the initial months for the motor system.30 Within a year of stroke onset, however, a signi?cant decline in function is commonly seen.31–34 This is signi?cant given that few treat-ment options are available to improve function in patients in the chronic phase of stroke. In the current study, behav-ioral gains were seen, though were modest in magnitude. However, a 2-point improvement in the National Institutes of Health Stroke Scale score (Table 3) in the setting of chronic stroke, if veri?ed in a larger controlled study, might be regarded as important. Also, the mean gain in BI from base-line grew to 10.8 points by 12 month-poststroke (P<0.001), higher than the BI minimal clinically important difference of 9.25 points.35 Furthermore, the proportion of patients with an excellent functional outcome (BI score =95) increased from 11.4% at baseline to 27.3% at 6-months and to 35.5% at 12-months (Table 3). This 12-month period of continued functional improvement is consistent with preclinical studies examining the distribution of systemically administered MSC: intravenous MSC given early after stroke initially localize to lungs then spleen, then increase within the region of brain ischemia,36 and by 30 days poststroke are concen-trated in the peri-infarct region.37 At one year, most surviving MSC are in the peri-infarct region, with very few present in other organs.38 Patients also showed signi?cant improvement in the Mini-Mental Status Exam and Geriatric Depression Scale (Table 3), changes that were largely sustained at 12 months post-infusion, suggesting that MSC have broad effects on brain function. These ?ndings require veri?cation in a larger, controlled study but raise hope that this interven-tion could improve functional status in the chronic stroke setting. Future studies might also incorporate modality-spe-ci?c outcome measures30 to provide more granular assessments of behavioral gains in individual neural systems.
Table 3. Behavioral Change Over Time
|Mini-Mental Status Exam score||n||P Value|
|Change to 6 mo||1.8±2.8||32||<0.001|
|Change to 12 mo||1.3±2.7||31||0.017|
|Baseline||8 [6.5 to 10]||35|
|Change to 6 mo||-1 [-2.25 to 0]||32||<0.001|
|Change to 12 mo||-2 [-3.5 to -0.5]||21||<0.001|
|Geriatric depression scale score|
|Change to 6 mo||-1.6±3.8||32||0.015|
|Change to 12 mo||-1.4±3.8||31|
|Barthel Index (score)|
|Change to 6 mo||6.8±11.4||33||0.002|
|Change to 12 mo||10.8±15.5||31||<0.001|
|Barthel Index (% =95)|
|Proportion at baseline||4 (11.4%)||35|
|Proportion at 6 mo||9 (27.3%)||33||0.015|
|Proportion at 12 mo||11 (35.5%)||31||0.01|
Values are mean±SD or median (interquartile range) across all enrollees. Specific data for part 1 and part 2 appear in Table SIV in the online-only Data Supplement. NIHSS indicates National Institutes of Health Stroke Scale.
Meta-analysis of MSC effects in animals with experimental ischemic stroke10 showed large effect sizes that remained sub-stantial after adjusting for potential publication bias and was robust across species, delivery route, time of administration in relation to stroke, and dose. The longest time period when MSC have been introduced poststroke in preclinical studies is 1 month39 or 4 to 6 weeks40 post-infarct. The current ?ndings in patients who were many months poststroke (Table 2) sug-gest the need for bidirectional translation, that is, translation of bedside experience to inform preclinical studies.41,42
There are several strengths to this study. Enrollees had substantial functional de?cits in the chronic stage of stroke, a population that numbers in the millions, for whom treatment options remain limited. The infused cells were allogeneic, an approach made possible by the relatively immunoprivileged nature of MSC,14 which eliminates the need for immuno-suppression15 and which, as compared with autologous cell therapies, enables treatment protocols that can be broadly implemented in the stroke population. A dose-escalation study design was used to evaluate safety. Cell culture was limited to 4 passages, a potential advantage given that higher number of passages (and thus cell divisions) adversely affect MSC features such as proliferation, differentiation, homing, and viability.43–45 Safety was assessed across multiple modalities, including chest/abdomen/pelvis CT and extensive laboratory testing, for a 1-year period.
There are also important weaknesses. As this study was focused on safety, no control group was included, which complicates interpretation of observed behavioral gains (Table 3). Mechanism of action was not studied. Cell thera-pies improving outcomes in the chronic phase likely act via multiple mechanisms that include release of growth factors and anti-in?ammatory effects, and possibly exosomes,46,47 which can be evaluated in subsequent trials. Restorative ther-apies after stroke often provide maximal bene?t when paired with appropriate training,48 but this was not provided in the current safety study.
The current study demonstrated safety of intravenous MSC in patients with chronic stroke who had substantial functional de?cits. Results also suggest functional bene?t, although this requires veri?cation in a controlled study. Together, these ?ndings support further study of intravenous allogeneic MSC in patients with chronic stroke, including evaluation of mech-anism of action.
Sources of Funding
This study was supported by Stemedica Cell Technologies, Inc.
Dr Levy is on the Scienti?c Advisory Board for KOH Robotics. Dr Dib has served as a consultant for J&J Consulting and ISCTR Consulting. Dr Verkh is Chief Regulatory and Clinical Development Of?cer at Stemedica Cell Technologies, Inc. Dr Tankovich is the President and Chief Medical Of?cer for Stemedica Cell Technologies, Inc. Dr Cramer has served as a consultant for Abbvie, Constant Therapeutics, MicroTransponder, Neurolutions, Regenera, SanBio, Stemedica, Biogen, Fuji?lm Toyama Chemical, and TRCare. The other authors report no con?icts.
- Feigin VL, Lawes CM, Bennett DA, Anderson CS. Stroke epidemiology: a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century. Lancet Neurol. 2003;2:43–53.
- Johnston SC, Hauser SL. Neurological disease on the global agenda. Ann Neurol. 2008;64:A11–A12.
- Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone G, et al. Heart disease and stroke statistics–2010 update: a report from the American Heart Association. Circulation. 2010;121:e46–e215.
- Lin DJ, Finklestein SP, Cramer SC. New directions in treat-ments targeting stroke recovery. Stroke. 2018;49:3107–3114. doi: 10.1161/STROKEAHA.118.021359
- Cramer SC. Repairing the human brain after stroke. II. Restorative thera-pies. Ann Neurol. 2008;63:549–560. doi: 10.1002/ana.21412
- Wolf SL, Winstein CJ, Miller JP, Taub E, Uswatte G, Morris D, et al; EXCITE Investigators. Effect of constraint-induced movement therapy on upper extremity function 3 to 9 months after stroke: the EXCITE randomized clinical trial. JAMA. 2006;296:2095–2104. doi: 10.1001/jama.296.17.2095
- McCabe J, Monkiewicz M, Holcomb J, Pundik S, Daly JJ. Comparison of robotics, functional electrical stimulation, and motor learning methods for treatment of persistent upper extremity dysfunction after stroke: a randomized controlled trial. Arch Phys Med Rehabil. 2015;96:981–990. doi: 10.1016/j.apmr.2014.10.022
- Dodakian L, McKenzie AL, Le V, See J, Pearson-Fuhrhop K, Burke Quinlan E, et al. A home-based telerehabilitation program for patients with stroke. Neurorehabil Neural Repair. 2017;31:923–933. doi: 10.1177/1545968317733818
- Ward NS, Brander F, Kelly K. Intensive upper limb neurorehabilita-tion in chronic stroke: outcomes from the Queen Square programme. J Neurol Neurosurg Psychiatry. 2019;90:498–506. doi: 10.1136/jnnp-2018-319954
- Vu Q, Xie K, Eckert M, Zhao W, Cramer SC. Meta-analysis of preclinical studies of mesenchymal stromal cells for ischemic stroke. Neurology. 2014;82:1277–1286. doi: 10.1212/WNL.0000000000000278
- Bang OY, Lee JS, Lee PH, Lee G. Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol. 2005;57:874–882. doi: 10.1002/ana.20501
- Honmou O, Houkin K, Matsunaga T, Niitsu Y, Ishiai S, Onodera R, et al. Intravenous administration of auto serum-expanded autologous mes-enchymal stem cells in stroke. Brain. 2011;134(pt 6):1790–1807. doi: 10.1093/brain/awr063
- Bhasin A, Srivastava MV, Kumaran SS, Mohanty S, Bhatia R, Bose S, et al. Autologous mesenchymal stem cells in chronic stroke. Cerebrovasc Dis Extra. 2011;1:93–104. doi: 10.1159/000333381
- Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringdén O. HLA expression and immunologic properties of differentiated and undifferen-tiated mesenchymal stem cells. Exp Hematol. 2003;31:890–896.
- Lalu MM, McIntyre L, Pugliese C, Fergusson D, Winston BW, Marshall JC, et al; Canadian Critical Care Trials Group. Safety of cell therapy with mesenchymal stromal cells (SafeCell): a systematic re-view and meta-analysis of clinical trials. PLoS One. 2012;7:e47559. doi: 10.1371/journal.pone.0047559
- Hess DC, Wechsler LR, Clark WM, Savitz SI, Ford GA, Chiu D, et al. Safety and ef?cacy of multipotent adult progenitor cells in acute ischaemic stroke (MASTERS): a randomised, double-blind, pla-cebo-controlled, phase 2 trial. Lancet Neurol. 2017;16:360–368. doi: 10.1016/S1474-4422(17)30046-7
- Steinberg GK, Kondziolka D, Wechsler LR, Lunsford LD, Coburn ML, Billigen JB, et al. Clinical outcomes of transplanted modi?ed bone marrow-derived mesenchymal stem cells in stroke: a phase 1/2a study. Stroke. 2016;47:1817–1824. doi: 10.1161/STROKEAHA.116.012995
- Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. 2005. www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatory Information/Guidances/ucm078932.pdf.Accessed June 22, 2019.
- Luger D, Lipinski MJ, Westman PC, Glover DK, Dimastromatteo J, Frias JC, et al. Intravenously delivered mesenchymal stem cells: sys-temic anti-in?ammatory effects improve left ventricular dysfunction in acute myocardial infarction and ischemic cardiomyopathy. Circ Res. 2017;120:1598–1613. doi: 10.1161/CIRCRESAHA.117.310599
- Harach T, Jammes F, Muller C, Duthilleul N, Cheatham V, Zufferey V, et al. Administrations of human adult ischemia-tolerant mesenchymal stem cells and factors reduce amyloid beta pathology in a mouse model of Alzheimer ’s disease. Neurobiol Aging. 2017;51:83–96. doi: 10.1016/j.neurobiolaging.2016.11.009
- Butler J, Epstein SE, Greene SJ, Quyyumi AA, Sikora S, Kim RJ, et al. Intravenous allogeneic mesenchymal stem cells for nonischemic car-diomyopathy: safety and ef?cacy results of a phase II-a randomized trial. Circ Res. 2017;120:332–340. doi: 10.1161/CIRCRESAHA. 116.309717
- Vertelov G, Kharazi L, Muralidhar MG, Sanati G, Tankovich T, Kharazi A. High targeted migration of human mesenchymal stem cells grown in hypoxia is associated with enhanced activation of RhoA. Stem Cell Res Ther. 2013;4:5. doi: 10.1186/scrt153
- Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for de?ning multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position state-ment. Cytotherapy. 2006;8:315–317. doi: 10.1080/14653240600855905
- Devine SM. Mesenchymal stem cells: will they have a role in the clinic? J Cell Biochem Suppl. 2002;38:73–79.
- Hil?ker A, Kasper C, Hass R, Haverich A. Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine: is there a future for transplantation? Langenbecks Arch Surg. 2011;396:489–497. doi: 10.1007/s00423-011-0762-2
- Williams AR, Hare JM. Mesenchymal stem cells: biology, pathophys-iology, translational ?ndings, and therapeutic implications for cardiac disease. Circ Res. 2011;109:923–940. doi: 10.1161/CIRCRESAHA. 111.243147
- Figueroa FE, Carrión F, Villanueva S, Khoury M. Mesenchymal stem cell treatment for autoimmune diseases: a critical review. Biol Res. 2012;45:269–277. doi: 10.4067/S0716-97602012000300008
- Tsang KS, Ng CPS, Zhu XL, Wong GKC, Lu G, Ahuja AT, et al. Phase I/II randomized controlled trial of autologous bone marrow-derived mesenchymal stem cell therapy for chronic stroke. World J Stem Cells. 2017;9:133–143. doi: 10.4252/wjsc.v9.i8.133
- Savitz SI,Yavagal D, Rappard G, Likosky W, Rutledge N, Graffagnino C, et al.A phase 2 randomized, sham-controlled trial of internal carotid artery infusion of autologous bone marrow-derived ALD-401 cells in patients with recent stable ischemic stroke (RECOVER-Stroke). Circulation. 2019;139:192–205. doi: 10.1161/CIRCULATIONAHA.117.030659
- Cramer SC, Koroshetz WJ, Finklestein SP. The case for modality-speci?c outcome measures in clinical trials of stroke recovery-pro-moting agents. Stroke. 2007;38:1393–1395. doi: 10.1161/01.STR. 0000260087.67462.80
- van de Port IG, Kwakkel G, van Wijk I, Lindeman E. Susceptibility to deterioration of mobility long-term after stroke: a prospective cohort study. Stroke. 2006;37:167–171.
- Dhamoon MS, Moon YP, Paik MC, Boden-Albala B, Rundek T, Sacco RL, et al. Long-term functional recovery after ?rst ischemic stroke: the Northern Manhattan Study. Stroke. 2009;40:2805–2811. doi: 10.1161/STROKEAHA.109.549576
- Wondergem R, Pisters MF, Wouters EJ, Olthof N, de Bie RA, Visser-Meily JM, et al. The course of activities in daily living: who is at risk for decline after ?rst ever stroke? Cerebrovasc Dis. 2017;43:1–8. doi: 10.1159/000451034
- Sennfalt S, Norrving B, Petersson J, Ullberg T. Long-term sur-vival and function after stroke. Stroke. 2019;50:53–61. doi: 10.1161/STROKEAHA.118.022913
- Hsieh YW, Wang CH, Wu SC, Chen PC, Sheu CF, Hsieh CL. Establishing the minimal clinically important difference of the Barthel Index in stroke patients. Neurorehabil Neural Repair. 2007;21:233– 238. doi: 10.1177/1545968306294729
- Detante O, Moisan A, Dimastromatteo J, Richard MJ, Riou L, Grillon E, et al. Intravenous administration of 99mTc-HMPAO-labeled human mes-enchymal stem cells after stroke: in vivo imaging and biodistribution. Cell Transplant. 2009;18:1369–1379. doi: 10.3727/096368909X474230
- Sykova E, Jendelova P. In vivo tracking of stem cells in brain and spinal cord injury. Prog Brain Res. 2007;161:367–383. doi: 10.1016/S0079-6123(06)61026-1
- Shen LH, Li Y, Chen J, Cui Y, Zhang C, Kapke A, et al. One-year follow-up after bone marrow stromal cell treatment in mid-dle-aged female rats with stroke. Stroke. 2007;38:2150–2156. doi: 10.1161/STROKEAHA.106.481218
- Shen LH, Li Y, Chen J, Zacharek A, Gao Q, Kapke A, et al. Therapeutic bene?t of bone marrow stromal cells administered 1 month after stroke. J Cereb Blood Flow Metab. 2007;27:6–13. doi: 10.1038/sj.jcbfm.9600311
- Yasuhara T, Matsukawa N, Hara K, Maki M, Ali MM, Yu SJ, et al. Notch-induced rat and human bone marrow stromal cell grafts reduce ischemic cell loss and ameliorate behavioral de?cits in chronic stroke an-imals. Stem Cells Dev. 2009;18:1501–1514. doi: 10.1089/scd.2009.0011
- Bosetti F, Koenig JI, Ayata C, Back SA, Becker K, Broderick JP, et al. Translational stroke research: vision and opportunities. Stroke. 2017;48:2632–2637. doi: 10.1161/STROKEAHA.117.017112
- Armstead WM, Vavilala MS. Translational approach towards deter-mining the role of cerebral autoregulation in outcome after traumatic brain injury. Exp Neurol. 2019;317:291–297. doi: 10.1016/j.expneurol. 2019.03.015
- Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells. 2004;22:675– 682. doi: 10.1634/stemcells.22-5-675
- Izadpanah R, Kaushal D, Kriedt C, Tsien F, Patel B, Dufour J, et al. Long-term in vitro expansion alters the biology of adult mesenchymal stem cells. Cancer Res. 2008;68:4229–4238. doi: 10.1158/0008-5472. CAN-07-5272
- Galipeau J. The mesenchymal stromal cells dilemma–does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy. 2013;15:2–8. doi: 10.1016/j.jcyt.2012.10.002
- Wechsler LR, Bates D, Stroemer P, Andrews-Zwilling YS, Aizman I. Cell therapy for chronic stroke. Stroke. 2018;49:1066–1074. doi: 10.1161/STROKEAHA.117.018290
- Chen J, Chopp M. Exosome therapy for stroke. Stroke. 2018;49:1083– 1090. doi: 10.1161/STROKEAHA.117.018292
- Cramer SC, Sur M, Dobkin BH, O’Brien C, Sanger TD, Trojanowski JQ, et al. Harnessing neuroplasticity for clinical applications. Brain. 2011;134(pt 6):1591–1609. doi: 10.1093/brain/awr039
Biodistribution, migration and homing of systemically applied mesenchymal stem/stromal cells
Mesenchymal stem/stromal cells (MSCs) are increasingly used as an intravenously applied cellular therapeutic. They were found to be potent in situations such as tissue repair or severe inflammation. Still, data are lacking with regard to the biodistribution of MSCs, their cellular or molecular target structures, and the mechanisms by which MSCs reach these targets. This review discusses current hypotheses for how MSCs can reach tissue sites. Both preclinical and clinical studies using MSCs applied intravenously or intra-arterially are discussed in the context of our current understanding of how MSCs might work in physiological and pathological situations.
In the 1970s, Friedenstein and colleagues  first reported that locally applied culture-expanded populations of bone marrow stroma-derived fibroblastic cells remained at their injection sites under the kidney capsule, where an ectopic hematopoiesis was initiated. Later, Arnold Caplan’s group described mesenchymal stem/stromal cells (MSCs) as multipotent mesenchymal cell populations which can differentiate into several tissue types, and demonstrated roles for MSCs in the regeneration of bone, cartilage or ligaments in animal and clinical studies [2–4]. In these studies, however, transplanted cells were followed, if at all, at the site of transplantation, and biodistribution was not an issue.
By the year 2000, clinicians had become increasingly interested in intravenously applied MSCs. Pivotal studies by the group of Horwitz in children with osteogenesis imperfecta, an inherited enzyme deficiency of collagen synthesis by mesenchymal cells in bone, opened the field for intravenous use of MSCs. This concept started from the observation that bone marrow transplantation can provide stromal cells able to synthesize intact collagen type I, replacing deficient patient cell function and ameliorating disease symptoms . Therefore, the authors concluded that transplantation of isolated healthy allogeneic MSCs might cure the disease. This implies homing of transplanted MSCs to sites in bone marrow and/or bone. Efficacy was noted in all six infants treated . Children who received transplants showed improved growth rates and started to synthesize intact bone. Engraftment of donor-type MSC-derived osteoblasts was shown using bone specimens and microsatellite DNA marker analysis. In a second study , these authors showed that autologous, enzyme-deficient MSCs transduced with a copy of the intact gene resulted in normal collagen production in bone cavities. Moreover, children who received transplants approached growth curves similar to the children transplanted with allogeneic complete bone marrow . This pioneering work provided the basis for the successful application of MSCs using the intravenous route in other clinical entities.
Establishment of methods to track intravenously administered MSCs
After 2000, the therapeutic use of MSCs by intravenous administration was explored by a number of studies in animals and also humans. These studies used various ways to label culture-expanded MSCs, and to track them in different tissues over time. The tissue source of the MSCs was in most cases not decisive, and cells from various tissue sources were explored. The labeling methodologies used included radioactive labeling of MSCs, labeling with fluorescent vital dyes, contrast agents, transduction with reporter genes, or the use of donor cell-specific DNA markers such as microsatellites [7–11] (reviewed in ). The labeling methodologies were, in part, designed to detect only short-term homing of MSCs. In addition, they do not enable the determination of whether detected cells are still alive. These studies were mainly conducted in rodents and nonhuman primates and mostly in non-injury situations. The main common results of these studies were that: MSCs distribute to a variety of tissues after intravenous (i.v.) injection; MSCs are detectable at low or very low frequencies in tissues after transplantation; and signals from the injected cells were found early after administration of the MSCs at the highest frequencies in the lungs, followed by liver and spleen.
The observed biodistribution patterns were confirmed by studies in humans. In patients with mammary carcinoma, Koç et al.  demonstrated that i.v. MSCs were well-tolerated in patients at a dose of one million MSCs/kg body weight; however, the cells were trackable in blood only. The data were confirmed in patients with liver cirrhosis using 111In-oxine labeled MSCs, which were found to first accumulate in the lungs followed by continuous increases in liver and spleen up to day 10 after administration . The proportion of accumulation in lung decreased from about 35 % early after transplantation to 2 % or less by day 10, whereas spleen had the highest signals by day 10 after transplant. These results confirm a similar overt biodistribution of MSCs in lung, liver and spleen in humans to that observed in animal models.
Expression of cell adhesion molecules by MSCs as a basis for their interaction with endothelial cells and tissue-directed extravasation
In theory, the main prerequisite for the interaction of transplanted MSCs with endothelial cells are adhesion molecules present on the cell surface of MSCs, and expression of appropriate adhesion counter-receptors on endothelial cells. MSCs (most investigations were performed in human MSCs (hMSCs)) have shown deficits in receptor binding to selectins and/or their ligands. They lack expression of L-selectin, and their E-selectin ligand (CD44) is not functional . MSCs can bind to P-selectin through a fucosylated ligand, which nevertheless is not P-selectin glycoprotein ligand (PSGL)-1 . Thankamony and Sackstein  have, however, defined an enzymatic fucosylation procedure which causes the CD44 epitope on MSCs to strongly bind to endothelial E-selectin, resulting in effective rolling of MSCs on endothelial cells and, moreover, extravasation into bone marrow sites. Of the integrins, alpha4beta1 (VLA-4) and alpha5beta1 (VLA-5) have been found to be expressed by MSCs, whereas the beta2 integrins alphaLbeta2 (LFA-1) and alphaMbeta2 (Mac1) could not be detected [15, 16, 18–20] (reviewed in [12, 21]). Interestingly, several chemokine receptors have been found to be expressed on MSCs, including CXCR4, which has been described as a major mediator of the homing and mobilization of hematopoietic cell types [12, 19, 20]. In summary, these findings indicate that MSCs have a deficit with regard to the expressing and/or employing adhesion receptors for coordinated extravasation and tissue-specific homing, as do leukocyte populations.
Emergence of common themes in exploring the biodistribution of MSCs
Subsequent to the first reports on the homing and migration of transplanted MSCs to tissues, additional questions about MSC biodistribution have been addressed, including quantification of MSCs, their preferential homing to several target sites, and the involvement of cues, such as regeneration or inflammation, and the size of MSCs in determining their biodistribution (Table 1).
Common themes in MSC biodistribution research
|Theme||Targeted tissues (possible mechanism)||References|
|Increased homing after intra-arterial delivery compared with intravenous delivery?||Kidney||[33, 34]|
|Other (many) tissues|||
|Side effects of intra-arterial versus intravenous delivery?||Incorporation into vessel wall||[23, 35]|
|Obstruction of microvessels|||
|Targeting of vessel wall and vessel-associated tissues?||Lungs, lymph nodes, intestine|||
|Targeting of tissues for regeneration||Myocardium||[18, 48–55]|
|Beta1 integrins||[48, 49]|
|Gut and liver||[64–67]|
|P/E selectin (CD44)|||
|Homing to bone marrow||Bone marrow||[76–81]|
|Biodistribution to the immune system?||Macrophages||[37, 41, 42]|
|Unknown target cells|
|Prostaglandin E2||[37, 41]|
|Elimination mechanisms?||Antibody formation|||
|Influence of radiation on homing?||Increased in brain, heart, bone marrow, and muscles||[43, 82]|
|Homing in malignancies?||Tumor||[83–85, 87–92]|
|Mediated by CCL25|||
|Mediated by sodium iodide symporter under the control of RANTES/CCL-5 promoter|||
|Homed MSCs form tumor-associated fibroblasts|||
|Formation of microvesicles||Microvesicles may contribute to/be part of MSC biodistribution||[14, 63, 93–97]|
|Mediated by horizontal transfer of microRNAs|||
In many of the earlier studies, the target sites as well as the molecular mechanisms governing the interactions of MSCs with the local environment after transplantation (e.g., endothelial cells, target tissue), such as adhesion molecules or signaling mechanisms, were either not addressed or were analyzed only to a minor degree. Moreover, MSCs were often evaluated by microscopy, a method relatively prone to artifacts. Many studies also did not quantify the numbers of MSCs in target or other tissues. Likewise, only few studies reported on the size of the identified MSCs. Despite this lack of information, other themes have emerged, especially research on cues that may regulate the biodistribution of systemically applied MSCs; these include first pass tissues, specifically the lungs, inflammation, irradiation, sites of hypoxia or repair, and cancer (Table 1). As a result, concepts have been raised which imply an ability of MSCs to migrate to specific sites—e.g., MSCs as an “injury drugstore” for several acute clinical situations [21, 22].
First-line accumulation of intravenously administered MSCs in the lungs
The first hurdle for intravenously transplanted MSCs is the lung capillary bed. After culture expansion, MSCs are relatively large cells with an estimated average size of around 30 µm in suspension (ranging from 16–53 µm) . Their size may also vary depending on the osmolarity of the culture media, passage number, and/or cell density during seeding as well as general culture conditions (two-dimensional versus three-dimensional culture). In comparison with MSCs, hematopoietic stem cells have a much smaller diameter, ranging from 4–12 µm depending on the subfraction analyzed [24, 25]. Therefore, obstructive events during lung passage are expected after intravenous administration of MSCs. Lee et al.  presented a kinetic study of MSCs accumulating in murine lungs in which up to 80 % of injected cells were found in the lungs within a few minutes after injection. Moreover, formation of emboli in lung vessels was noted. The MSC signal (an Alu sequence DNA marker) fell exponentially, with a half-life of about 24 h and practically complete disappearance after 4 days . Barbash and colleagues  confirmed the detection of the overall MSC load in the lungs using 99mTc-labeled MSCs in a rat model with induced myocardial infarction. Murine MSCs also showed deleterious effects in mice, including post-injection lethality, which was not the case after administration of hMSCs . Interaction of human or murine MSCs with lung endothelial cells was dependent on the suspension medium in which the transplanted cells were administered . Adhesion of the MSCs to endothelial cells was found to involve the integrin ligand vascular cell adhesion molecule (VCAM)-1. When comparing MSCs with mononuclear cells from bone marrow, neural stem cells and multipotent adult progenitor cells, Fischer et al.  found that MSCs showed the highest interaction with lung endothelia, which could be inhibited by pretreatment with anti-CD49d antibody. In a study by Kerkelä et al. , adhesion of MSCs to lung tissue (probably endothelial cells) was dependent on the enzyme treatment used during harvesting of confluent MSCs in culture before transplantation; after treatment with pronase, MSCs more readily cleared the lungs and could be found in other tissues compared with trypsinization treatment. Taken together, these data indicate an active role of the adhesion molecules VLA-4/VCAM-1 on MSCs/endothelial cells during interaction of MSCs with lung tissue. It remains to be clarified, however, whether this is a passive or active process. Also, relatively little is known about possible adhesion molecules other than VLA-4/VCAM-1 which may be operative in the interaction of MSCs with endothelial cell surfaces in the lung. This includes the fucosylation of CD44 to HCELL, a highly active E-selectin ligand on MSCs, which is relevant in bone marrow endothelia but seemingly did not affect lung interactions .
In summary, presently there is strong evidence that accumulation of MSCs in the lungs is a key determining factor for their biodistribution. The major adhesion molecule involved seems to be VLA-4/VCAM1. Still, it is not clear to what degree the findings in animal studies are quantitatively transferable to humans (Table 1).
Biodistribution of MSCs after intra-arterial versus intravenous administration
Studies comparing intra-arterial and intravenous application of MSCs have demonstrated a major association between intravenous application and retention of MSCs in the lungs, and their increased accumulation in therapeutic target tissues after intra-arterial injection. Walczak et al.  in a rat transient ischemia stroke model applied two independent detection methods (magnetic resonance imaging and Doppler flowmetry). They demonstrated that higher cerebral engraftment rates are associated with impeded cerebral blood flow, and that intra-arterial delivery may be advantageous in ischemic stroke to deliver MSCs to the site of injury. Mäkelä et al.  compared intra-arterial and intravenous administration of MSCs labeled with 99mTc, and also found that the intra-arterial transplantation route has a positive impact on the biodistribution of bone marrow-derived MSCs (BM-MSCs) to peripheral tissues. They found that intra-arterial transplantation decreased the deposition of BM-MSCs in the lungs and increased uptake in other organs, especially in the liver. In a study looking at human adipose tissue-derived MSCs in SCID mice, Toupet et al.  showed that 15 % of intra-arterially injected MSCs accumulate in inflamed joints during the first month, and 1.5 % over a longer term of >6 months, also favoring intra-arterial over intravenous application for, in their case, anti-inflammatory MSCs. Therapeutic effects of MSCs in kidney have been generally achieved after intra-arterial delivery [33, 34]. Although more studies will be needed, these data suggest that the intra-arterial route of administration is effective in avoiding pulmonary entrapment of BM-MSCs, and may thus improve the biodistribution and bioavailability of transplanted MSCs in clinically relevant tissues for, e.g., tissue repair.
Interactions of MSCs with the blood vessel wall: integration into the vessel wall or transmigration?
As described above, the majority of intravenously injected MSCs are generally detected in the lungs, and in no other tissue at comparable numbers even at later time points. Some groups asked whether MSCs may directly target vessels or perivascular tissue and investigated the fate of MSCs in and around blood vessels. These studies followed the cells using intravital microscopy and histologic examination in different tissues after intra-arterial [23, 30, 35] administration. In the cremaster muscle intravital microscopy model, Furlani et al.  observed that the microcirculation was disturbed, with some MSCs obstructing small vessels. In addition, pulmonary emboli were found. Toma et al.  also observed occlusion of microvessels and entrapment of the injected MSCs. Moreover, they observed stable integration of some transplanted cells into the vessel wall. Cui et al.  reported a risk of vascular occlusion in their rat stroke infarction model after intra-arterial injection, pointing to the fact that local intravasal entrapment of MSCs may frequently occur, and MSCs may obstruct the microcirculation. Currently, however, we lack conclusive data that MSCs that are entrapped in capillaries and/or are incorporated into the vessel wall or adjacent to endothelial cells would relocate (i.e., “home”) to their main tissue of origin, pericytes.
Transplanted MSCs interact with cells of the immune system
Transplanted MSCs have been shown to rapidly interact with immune cell types, which are—at least in part—present also in the bloodstream. In a lung sepsis model, Nemeth et al.  observed that MSCs co-localize with lung-resident macrophage cells and induce them to produce anti-inflammatory interleukin (IL)-10 via release of prostaglandin E by MSCs as part of their therapeutic effect. Chiesa et al.  showed that interstitial dendritic cells (DCs) decrease their physiological migration from skin to lymph nodes rapidly after intravenous administration of MSCs. They describe that MSCs inhibit Toll-like receptor (TLR)-4-induced activation of DCs, which results in the inhibition of cytokine secretion by DCs, downregulation of adhesion molecules involved in the migration of DCs to the lymph nodes, suppression of DC antigen presentation to CD4+ T cells, and cross-presentation to CD8+ T cells. Akiyama et al.  demonstrated that both human and murine MSCs can induce immune suppression by attracting and killing autoreactive T cells through FasL, thereby stimulating transforming growth factor beta production by macrophages and generation of regulatory T cells. The interaction has been shown to involve the secretion of MCP-1 by MSCs. The dying T cells in turn activate macrophages to produce transforming growth factor beta, thus stimulating regulatory T cells and promoting immune tolerance. Possibly, the secretion of anti-inflammatory protein TSG-6 by activated MSCs, which has been described in a zymosan-induced mouse peritonitis model, involves an interaction via TLR2/reduction of NF-?B signaling in resident macrophages .
Another type of potential interaction between MSCs and immune cells is suggested by data from Kim et al. , who used an in vitro system showing that murine MSCs inhibit functionality of DCs through TLR-4-mediated signals in co-culture with monocytes. During this study, hMSCs revealed a unique immunophenotype of alternatively activated human monocytes which are CD206-high, IL-10-high, IL-6-high, IL12-low, and tumor necrosis factor (TNF)-alpha-low . The immune suppressive effects of MSCs have been shown to depend on induction of indoleamine 2,3-dioxygenase , or production of prostaglandin E2 as a main effector to dampen inflammation [37, 44]. These data indirectly support the hypothesis that MSCs interact directly with monocytic and/or antigen-presenting cells in vivo.
The successful therapeutic use of MSCs in patients with severe immune dysregulations, such as graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, has attracted high interest by hematologists (reviewed in ). The studies were based on a number of in vitro findings that MSCs can either interact with or affect the function of various types of immune effector cells such as antigen-presenting cells, B or T lymphocytes, or natural killer (NK) cells (reviewed in ). In all these studies, identification of MSCs at target sites has been cumbersome, and often no transplanted MSCs were identified. von Bahr et al.  reported that MSC donor DNA was detectable at low levels in 8 out of 18 patients in vessel-associated tissues in the patients, including lungs, lymph nodes, and intestine. Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection.
Together, these studies strongly indicate the existence of interactions between transplanted MSCs and cells of the immune system. This way, MSCs also biodistribute to the immune system through contact with different types of leukocytes in the circulation or various tissues such as skin, spleen, and lymph nodes.
Potential mechanisms of elimination of MSCs from the circulation
A relevant aspect of the interaction between transplanted MSCs and immune system cells, in both animal models and humans, is the induction of xenogeneic and allogeneic immune responses, resulting in antibody formation or T-cell responses against the transplanted MSCs. Induction of antibody formation explains the failure to identify transplanted MSCs in patients upon repeated administration of allogeneic MSCs that had been cultured in fetal bovine serum-containing media . Anti-fetal calf serum antibody formation has been demonstrated in patients that did not respond to repeated MSC applications . Elimination of xenogeneic MSCs in some of the animal models studied may occur in ways analogous to those in the allogeneic situation.
Despite the fact that several target tissues of MSCs have been established, there are few data as to the place to which systemically applied MSCs will finally migrate, or where they end up before or when they are eliminated. The fact that the transplanted MSCs are often not detectable at all, or only a small fraction of them is traced, underscores the potential relevance of the lung as a “first pass” tissue, and may indicate an involvement of lung trapping in elimination of MSCs. On the other hand, the fact that MSCs are barely or not at all detectable in patients after transplantation demonstrates that systemic pathways to eliminate transplanted MSCs may be operating in humans, leading to barely detectable long-term engraftment.
Tissue repair situations which provide cues to attract transplanted MSCs
The interactions of MSCs with different types of immune cells point to their ability to respond to signals from the immune system. Since aspects of tissue repair have been associated with (adaptive) immune responses, it is likely that inflammatory and tissue repair signals influence MSC responses in vivo, including their biodistribution.
The VLA-4/VCAM receptor axis has been shown to be involved in MSC migration in myocardial infarction. Pre-treatment of MSCs with TNF-1alpha stimulated migration of MSCs through heart endothelia mediated through VCAM-1, indicating that beta1 integrins are actively involved in this process . Confirming this hypothesis, Ip et al.  demonstrated in a murine model that alpha4 integrin is required for migration of MSCs to myocardium, whereas the chemokine receptor CXCR4 was dispensable for the entry of transplanted cells into ischemic tissue.
Intravenously administered MSCs have been observed to, at least transiently, accumulate in areas of myocardial ischemia [18, 50, 51]. To this end, Belema-Bedada et al.  employed a transgenic mouse model expressing the monokine CC-chemokine ligand (CCL)2 under a cardiac specific promotor, increasing CCL2 expression in heart muscle. These authors observed that i.v. MSCs accumulate rapidly and selectively in the heart. They showed that the migration of the MSCs to heart is preceded by monocyte emigration to the myocardium, and involves G-protein-coupled receptors, pointing also towards the involvement of chemokine signals. Kraitchman et al.  confirmed the accumulation of i.v. MSCs into myocardial infarction areas using a radioimaging tracer and single-photon emission computed tomography in a dog model. Wang et al.  traced MSCs at later stages after infarction, and saw markers of newly regenerated cardiomyocytes. It is also not clear whether MSCs steadily incorporate into cardiac tissue. Other studies have failed to detect any homed MSCs in cardiac tissue over the long term (e.g., ). Jasmin et al.  injected MSCs i.v. after nanoparticle labeling in a model of heart inflammation caused by the Chagas disease parasite Trypanosoma cruzi. They observed that although most MSCs migrated to the lungs, liver and spleen, a few cells homed to the inflamed heart. In conclusion, some mechanisms seem to recruit, mostly transiently, some MSCs to inflamed or ischemic heart, including VLA-4/VCAM-1 and the CCL2 and possibly other chemokine receptor signals.
Despite the wide range of beneficial effects seen with the therapeutic use of MSCs in animal models, only a few clinical trials have tested the efficacy of MSCs for renal diseases. Reinders and colleagues  used intravenous injection of 1?×?106 autologous BM-MSCs/kg in six kidney allograft recipients to dampen rejection of the graft and/or decrease interstitial fibrosis and tubular atrophy. Likewise, Tan et al. investigated autologous BM-MSCs (1–2?×?106/kg) at kidney reperfusion and 2 weeks after application; the incidence of acute rejection decreased and renal function at 1 year improved compared with anti-IL-2 receptor antibody induction therapy . In a clinical phase I safety trial, five patients aged >65 years with underlying renal disease and multiple comorbidities were infused with allogenic MSCs during coronary artery bypass or cardiac surgery. Although the follow-up period was short and one of the patients died, none of the patients required dialysis, supporting the beneficial influence of MSCs on repair of kidney damage [58, 59].
In animal studies, MSCs were also associated with repair of the permeability barrier of the glomerulus in an Alport disease model  and improved kidney function in an experimental sepsis mouse model through reprogramming of macrophages via release of prostaglandin E2 . Morigi and colleagues [61, 62] have shown that treatment with murine BM-MSCs (2?×?105 per mouse) in an acute renal failure mouse model induced by cisplatin (a nephrotoxic anti-cancer drug) protected the animals from renal function impairment and tubular injury. Intriguingly, the effects of MSCs in stimulating proliferation and inhibiting apoptosis of tubular epithelial cells in a glycerol-induced acute kidney injury SCID mouse model could also be achieved by using microvesicles derived from hMSCs . In addition to these human studies, several studies demonstrate that MSCs localize within injured kidneys when injected in mice with acute kidney injury (e.g., [34, 63]; reviewed in ). The presence of MSCs at later stages of kidney injury or regeneration has not been studied, but the therapeutic benefits have been measured, and intra-arterial injection of MSCs seems to be more favorable [33, 34, 61].
Gholamrezanezhad et al.  studied i.v. infused 111In-oxine-labeled MSCs in patients with liver cirrhosis. The radioactivity was first observed to accumulate in the lungs. During the following hours to days, the radioactivity gradually increased in the liver and spleen, with spleen uptake exceeding that in the liver in all patients. In the liver and spleen, radioactivity increased by day 10 post-infusion, whereas residual activity in the lungs decreased approximately tenfold. In contrast, Briquet et al.  saw no recruitment of hMSCs to liver damaged by CCl4 intoxication in immune-deficient mice. A study by Zhang et al.  indicates that corticosteroids and the SDF-1/CXCR4 axis are involved in MSC migration in a carbon tetrachloride-induced liver fibrosis model. Another liver regeneration model in mice indicated that MSC homing to liver was associated with regeneration, but the mechanisms for this were not investigated . In summary, although many of the published studies have not addressed aspects of MSC biodistribution, there is some evidence for biodistribution to injured or diseased livers, but the underlying mechanisms are mostly unclear.
Gut and skin
Only a few studies have analyzed MSC accumulation in epithelial tissues so far. Inflammatory bowel disease models have addressed homing of i.v. MSCs. Parekaddan et al.  demonstrated the presence of MSC-derived signals not only in lungs and spleen but also in the gut of affected animals. Sasaki et al.  assessed whether homed MSCs can differentiate into skin cells, including keratinocytes, and whether they could contribute to wound repair. They i.v. injected green fluorescence protein (GFP) transgenic MSCs and identified GFP-positive cells associated with specific markers for keratinocytes, endothelial cells, and pericytes. They attribute the extravasation to inflamed areas to the presence of the chemokine CCL21 in vessels in the inflamed tissue. Still, numbers of detected MSCs in the wounded skin areas were low. MSCs have been found in wound tissues several days after transplantation in animal models [68–71] but their engraftment efficiency ranged from <0.01 % when MSCs were intravenously injected to 3.5 % in a study where MSCs were locally applied. This points to a minor role of i.v. injected MSCs in skin repair. One study reported that, after intravenous injection of GFP transgenic MSCs, keratinocytes, endothelial cells, pericytes and macrophages within the healed wound were all found to be GFP-positive. The authors concluded that they might be derived from donor MSCs .
Some studies have investigated whether transplanted MSCs migrate into inflamed brain tissue. In murine stroke models, MSCs migrated into ischemic areas after intravenous delivery [72, 73]. The latter study mentions that the MSCs are recruited to these sites via endothelial expressed P- and E-selectin, and that CD44 is present on the MSCs. In their rat brain ischemia model, Wei et al.  found that i.v. MSCs localize to ischemic zones and deliver neurotrophic factors. This occurs at an increased rate when MSCs have been exposed to hypoxia before transplantation. The extravasation efficiency of the MSCs correlated with increased expression of CXCR4, flk-1 and the erythropoietin receptors, and downregulation of pro-inflammmatory regulators in the homing MSCs. The activity of microglia formation was suppressed in animals after MSC therapy, and NeuN-positive and Glut1-positive cells were increased. Constantin et al.  used intravital microscopy in a murine experimental autoimmune encephalitis model. They found, using bioluminescence, accumulation of a subset of transplanted MSCs in inflamed brain venules in inflammatory foci of experimental autoimmune encephalomyelitis 16 and 30 days after transplantation, and showed a role for alpha4 integrin in the migration process of MSCs into brain tissue. Although absolute numbers of transplanted MSCs were not determined and may be low, the results indicate that active inflammation may switch the homing behavior of transplanted MSCs from unspecific entrapment to specific recruitment.
Together, these data indicate that MSCs can migrate into ischemic and proinflammatory regions in certain disease models. Mostly short- (within the first 3 days) and mid-term (3 days to 3 months) homing has been reported, whereas long-term persistence (>3 months) of MSCs is rarely detected. Due to the technologies used to detect transplanted cells, there is only limited evidence to indicate whether the MSCs home as intact cells into their target environments. The data are in favor of transient homing and locally acting MSCs in the investigated pathologies.
Homing of transplanted MSCs to bone marrow
Several decades of clinical and experimental work in the field of bone marrow transplantation have shown that donor type MSCs will generally not engraft in allogeneic hosts, including the precursor cell type for MSCs, fibroblast colony-forming units [76–78]. Rombouts and Ploemacher  demonstrated that prolonged time in culture induces a defect in MSCs that affects their engraftment into bone marrow in a classic bone marrow transplantation situation. In contrast, as reported above, Horwitz and colleagues [5, 6] demonstrated that MSCs engraft into bone marrow of children with osteogenesis imperfecta. Possibly, engraftment of MSCs therefore requires a “niche” which is not free in normal bone marrow transplant recipients, but is created in a deficiency state such as the collagen synthase defect found in osteogenesis imperfecta. Follenzi et al.  recently demonstrated that mice suffering from hemophilia A, when transplanted with normal healthy total bone marrow cells, show engraftment not only of hematopoietic cells but also of subendothelial MSC-like cells. Interestingly, these MSCs had not been cultured before transplantation. Functional MSCs may, therefore, engraft, at least in the case of certain deficiencies in the transplanted hosts. Interestingly, the group of Horwitz more recently showed that non-plastic-adherent bone marrow cells engraft in a murine model and give rise to osteoprogenitors, which are more potent osteoprogenitors than “classic” plastic-adherent MSCs in mice . This underscores the possibility that the culture period induces the engraftment defect, and that, in addition, cells other than “classic” MSCs can mediate stromal engraftment. On the other hand, “classic” plastic-adherent MSCs have been shown to remain as a source of hematopoietic environment when transplanted into tissues other than bone marrow . In contrast to these findings, the model by Sackstein et al. , where an active E-selectin ligand was engineered on the surface of plastic-adherent MSCs, resulted in efficient homing to bone marrow, indicating the possibility of BM-MSCs (or MSCs from other tissue sources) distributing to bone marrow.
Influence of irradiation on migration and biodistribution of MSCs
In a murine study, Francois et al.  showed that both total body irradiation and local irradiation (e.g., selective irradiation of abdomen or legs) affected the distribution of i.v. infused hMSCs in NOD/SCID mice compared with untreated animals. Intravenously infused hMSCs were found only in minimal amounts exclusively in the lung, bone marrow, and muscles in non-irradiated control animals. Mice after total body irradiation had increased absolute numbers of hMSCs in brain, heart, bone marrow, and muscles. Moreover, selective radiation of limbs or the abdomen yielded increased engraftment of hMSCs in the exposed skin or muscles than with total body irradiation alone. hMSC engraftment outside the locally irradiated regions was also increased, pointing to both local and systemic effects of irradiation on MSC engraftment. The study did not investigate long-term engraftment. Sémont et al.  looked at the engraftment and efficacy of transplanted MSCs in an immunodeficient mouse model of radiation-induced gastrointestinal tract failure. They demonstrated accelerated recovery in the group receiving hMSCs, with decreased apoptosis of epithelial cells and increased proliferation within the small intestinal mucosa. Yet, transplanted MSCs were not detected at significant amounts.
A special case: migration and engraftment of MSCs into tumors
Tumor-associated fibroblasts have been described as a form of MSCs, which are recruited from the MSC pool and are an integral part of the microenvironment of many different solid tumors [83, 84]. Tumor tissue therefore also represents a target for the homing of i.v. injected MSCs. In experimental studies, both beneficial and adverse effects have been reported. Beckermann et al.  verified the migration of i.v. MSCs into areas close to the vessel wall in human pancreatic tumors in immunodeficient mice. Alieva et al.  followed locally implanted adipose tissue-derived MSCs with a genetic modification induced by lentiviral transduction and traced them by bioluminescence in a glioblastoma model. After incorporation of the transplanted MSCs, administration of gancyclovir activates the thymidine kinase transgene, resulting in death and elimination of the transplanted MSCs and tumor regression. A PECAM-Promotor-driven second transgene as reporter construct served to indicate that the transplanted MSCs can acquire endothelial-like characteristics. Similarly, Knoop et al.  used i.v. MSCs expressing sodium iodide symporter under the control of the RANTES/CCL-5 promoter; when loaded with 131I compound these conferred significant anti-tumor effects.
Xu et al. , in a myeloma model, showed that MSCs are chemoattracted by the chemokine CCL25, thus supporting myeloma growth. In a Ewing sarcoma nude mouse model, i.v. injected MSCs expressing IL-12 were effective in treating the sarcomas . Interestingly, the transplanted MSCs themselves were not identified, while the secreted IL-12 was. Kidd et al.  showed that tumor-associated fibroblasts originating from transplanted MSCs in syngeneic ovarian and breast cancers are recruited from the bone marrow, whereas the bulk of the vascular and fibrovascular stromal cells (pericytes, a-smooth muscle actin-positive myofibroblasts, and endothelial cells) were recruited from adipose tissue. These data indicate a process whereby, once bone marrow homing of transplanted MSCs is established, these MSCs may be (genetically) directed along pre-established pathways of endogenous MSCs that circulate from bone marrow to the tumor. Further work by Grisendi et al.  demonstrated that the process of MSC incorporation into tumors implies the formation of epithelial–mesenchymal or endothelial–mesenchymal transitions, and requires the formation of fibroblasts derived from mesenchymal progenitors.
MSCs were also found to enhance angiogenesis, as shown in models of B16 melanoma cells and Lewis lung carcinoma . Co-injection of tumor cells and MSCs led to increased tumor size compared with injection of tumor cells alone. Tumor vessel areas were greater in tumors after co-injection of tumor cells with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs localized close to vascular walls, and also expressed the endothelial marker CD31/PECAM-1.
In conclusion, MSCs show a clear tumor tropism. Many data indicate that they are incorporated into the tumor microenvironment and can stimulate tumor growth. Their biodistribution and tumor tropism, however, may also be exploited to target tumors, e.g., using a suicide transgene approach.
Recent developments: exosomes, microparticles and MSCs
As with many other cell types, MSCs are capable of forming exosomes [63, 93, 94]. Exosomes are small membrane vesicles (40–100 nm in diameter) of endosomal origin derived from MSCs. Exosomes have been found to accumulate in target cells of MSC therapy, such as tubular cells in acute kidney injury , or after recovery from traumatic brain injury . In other studies, microvesicles have been found to contain signaling molecules which are hypothesized to be important for MSC-mediated therapeutic effects by horizontal transfer, such as miR-133b in a rodent stroke model , or insulin-like growth factor receptor in renal tubular injury . Kordelas et al.  administered exosomes isolated from MSCs to a patient with severe graft-versus-host disease; this patient showed marked improvement after the exosome infusion. This field is currently expanding rapidly, and can only be covered briefly by this review. One of the relevant open questions for the biodistribution of MSCs is whether exosomes are indeed formed by intravasally administered MSCs.
Summary: possible ways for MSCs to interact within the local environment of the bloodstream to direct their biodistribution
A summary of the possible ways MSC might interact within the blood circulation is shown in Fig. 1. MSC surface marker profiling has revealed no expression of the co-stimulatory molecules CD40, CD86, and CD80 needed for correct T-cell responses leading to T-cell anergy. In vitro studies also showed that CD4+ T cells in contact with MSCs were arrested in the G1/G0 phase and stopped proliferating whereas regulatory T cell proliferation was favored and IgG production by plasma cells seemed to be affected . In addition, MSCs only express a low amount of major histocompatibility complex (MHC) I and almost no MHC II (except after interferon-? treatment), making them more evasive to NK cell cytoxicity in an allogenic/xenogenic setting. Interactions between NK cells and MSCs in general have been controversial, as discussed by different groups (e.g., [99–101]). MSCs seem to lower NK cell cytoxicity through downregulation of interferon-? expression and production of anti-inflammatory IL-4 and IL 10, but NK cells were associated with the ability to lyse MSCs from allogenic donors . Additionally, the so-called instant blood-mediated inflammatory response might be triggered by the innate immune response caused by tropism of dying MSCs within the blood circulation, resulting in complement activation and opsonization of injected MSCs following uptake of marked MSC cell fragments by primary/secondary phagocytes, as was shown by Moll et al. . Intravital microscopy of MSCs in a cremaster muscle mouse model (our unpublished data) revealed that MSCs are likely to be disrupted by the shear force of the blood flow, resulting in fragmentation of the cell and creation of small extracellular vesicles able to influence paracrine secretion of immunomodulatory molecules or cause phagocytosis of these fragments by macrophages and endothelial cells, subsequently followed by clearance of disrupted MSCs in the liver and spleen within a few days. MSCs that find a niche and survived the journey through the bloodstream might interact actively or passively with the endothelial wall and may extravasate after interacting with the extracellular matrix (e.g., with MMP 2 and gelatinase) and reside in a pericyte-like location in the long term.
The final fate of the bulk of i.v. injected MSCs remains elusive, since preclinical animal studies and some human data have been able to detect only small proportions, if any, of injected MSCs. A number of open questions remain. These include: Which contacts are made between MSCs and other cells upon infusion in the bloodstream and what are the consequences of these? What is the fate of MSCs that do not migrate into inflamed tissue and are there physiological clearance pathways for transplanted MSCs? Given that many therapeutic effects have been observed without detectable MSCs in the target tissues, are intact MSCs therefore relevant for the observed effects?
We believe that further careful analysis of animal disease models, including investigation of the role of mediators such as exosomes, signaling proteins, and microRNAs, will help further advance our understanding of why we have so far not obtained clear answers about how MSCs biodistribute, migrate and home, and how these cells exert their beneficial effects, and what might be the potential of these new insights for the development of further improvements of MSC-derived therapies.
This article is part of a thematic series ‘Mesenchymal Stem/Stromal Cells—An update’. Other articles in this series can be found at http://www.biomedcentral.com/series/mesenchymal
The authors thank the team of the Institute of Transfusion Medicine and Immune Hematology, German Red Cross Blood Donor Service, University of Frankfurt for their constant support.
|BM-MSC||Bone marrow-derived mesenchymal stem/stromal cell|
|GFP||Green fluorescence protein|
|hMSC||Human mesenchymal stem/stromal cell|
|MHC||Major histocompatibility complex|
|MSC||Mesenchymal stem/stromal cell|
|TNF||Tumor necrosis factor|
|VCAM||Vascular cell adhesion molecule|
The authors declare that they have no competing interests.
All authors read and approved the final manuscript.
- Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV. Stromal cells responsible for transferring the micro-environment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation. 1974;17:331–40. doi: 10.1097/00007890-197404000-00001. [PubMed] [CrossRef] [Google Scholar]
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7. doi: 10.1126/science.284.5411.143. [PubMed] [CrossRef] [Google Scholar]
- Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem. 2006;98:1076–84. doi: 10.1002/jcb.20886. [PubMed] [CrossRef] [Google Scholar]
- Caplan AI. Why are MSCs therapeutic? New data: new insight. J Pathol. 2009;217:318–24. doi: 10.1002/path.2469. [PubMed] [CrossRef] [Google Scholar]
- Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A. 2002;99:8932–7. doi: 10.1073/pnas.132252399. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL, Neel M, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med. 1999;5:309–13. doi: 10.1038/6529. [PubMed] [CrossRef] [Google Scholar]
- Devine SM, Bartholomew A, Mahmud N, Nelson M, Patil S, Hardy W, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol. 2001;29:244–55. doi: 10.1016/S0301-472X(00)00635-4. [PubMed] [CrossRef] [Google Scholar]
- Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates. Blood. 2003;101:2999–3001. doi: 10.1182/blood-2002-06-1830. [PubMed] [CrossRef] [Google Scholar]
- Gao J, Dennis JE, Muzic RF, Lundberg M, Caplan AI. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs. 2001;169:12–20. doi: 10.1159/000047856. [PubMed] [CrossRef] [Google Scholar]
- Barbash IM, Chouraqui P, Baron J, Feinberg MS, Etzion S, Tessone A, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation. 2003;108:863–8. doi: 10.1161/01.CIR.0000084828.50310.6A. [PubMed] [CrossRef] [Google Scholar]
- Kraitchman D, Tatsumi M, Gilson WD, Ishimori T, Kedziorek D, Walczak P, et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation. 2005;112:1451–61. doi: 10.1161/CIRCULATIONAHA.105.537480. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Karp JM, Leng Teo GS. Mesenchymal stem cell homing: the devil is in the details. Cell Stem Cell. 2009;4:206–16. doi: 10.1016/j.stem.2009.02.001. [PubMed] [CrossRef] [Google Scholar]
- Koç ON, Gerson SL, Cooper BW, Dyhouse SM, Haynesworth SE, Caplan AI, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol. 2000;18:307–16. [PubMed] [Google Scholar]
- Gholamrezanezhad A, Mirpour S, Bagheri M, Mohamadnejad M, Alimoghaddam K, Abdolahzadeh L, et al. In vivo tracking of 111In-oxine labeled mesenchymal stem cells following infusion in patients with advanced cirrhosis. Nucl Med Biol. 2011;38:961–7. doi: 10.1016/j.nucmedbio.2011.03.008. [PubMed] [CrossRef] [Google Scholar]
- Sackstein R, Merzaban JS, Cain DW, Dagia NM, Spencer JA, Lin CP, et al. Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone. Nat Med. 2008;14:181–7. doi: 10.1038/nm1703. [PubMed] [CrossRef] [Google Scholar]
- Ruster B, Gottig S, Ludwig RJ, Bistrian R, Muller S, Seifried E, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood. 2006;108:3938–44. doi: 10.1182/blood-2006-05-025098. [PubMed] [CrossRef] [Google Scholar]
- Thankamony SP, Sackstein R. Enforced hematopoietic cell E- and L-selectin ligand (HCELL) expression primes transendothelial migration of human mesenchymal stem cells. Proc Natl Acad Sci U S A. 2011;108:2258–63. doi: 10.1073/pnas.1018064108. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Schenk S, Mal N, Finan A, Zhang M, Kiedrowski M, Popovic Z, et al. Monocyte chemotactic protein-3 is a myocardial MSC homing factor. Stem Cells. 2007;25:245–51. doi: 10.1634/stemcells.2006-0293. [PubMed] [CrossRef] [Google Scholar]
- Shi M, Li J, Liao L, Chen B, Li B, Chen L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment: role in homing efficiency in NOD/SCID mice. Haematologica. 2007;92:897–904. doi: 10.3324/haematol.10669. [PubMed] [CrossRef] [Google Scholar]
- Potapova IA, Brink PR, Cohen IS, Doronin SV. Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells. J Biol Chem. 2008;283:13100–7. doi: 10.1074/jbc.M800184200. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Caplan A, Correa D. The MSC: an injury drugstore. Cell Stem Cell. 2011;9:11–5. doi: 10.1016/j.stem.2011.06.008. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Stappenbeck TS, Miyoshi H. The role of stromal stem cells in tissue regeneration and wound repair. Science. 2009;324:1666–9. doi: 10.1126/science.1172687. [PubMed] [CrossRef] [Google Scholar]
- Furlani D, Ugurlucan M, Ong L, Bieback K, Pittermann E, Westien I, et al. Is the intravascular administration of mesenchymal stem cells safe? Mesenchymal stem cells and intravital microscopy. Microvasc Res. 2009;77:370–6. doi: 10.1016/j.mvr.2009.02.001. [PubMed] [CrossRef] [Google Scholar]
- Sharma S, Krishan A. Hematopoietic Stem Cells. In: Krishan A, Krishnamurthy H, Totey S, editors. Applications of flow cytometry in stem cell research and tissue regeneration. New Jersey: Wiley-Blackwell; 2010. pp. 103–14. [Google Scholar]
- Radley JM, Ellis S, Palatsides M, Williams B, Bertoncello I. Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status. Exp Hematol. 1999;27:365–9. doi: 10.1016/S0301-472X(98)00017-4. [PubMed] [CrossRef] [Google Scholar]
- Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell. 2009;5:54–63. doi: 10.1016/j.stem.2009.05.003. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Deak E, Rüster B, Keller L, Eckert K, Fichtner I, Seifried E, et al. Suspension medium influences interaction of mesenchymal stromal cells with endothelium and pulmonary toxicity after transplantation in mice. Cytotherapy. 2010;12:260–4. doi: 10.3109/14653240903401840. [PubMed] [CrossRef] [Google Scholar]
- Fischer UM, Harting MT, Jimenez F, Monzon-Posadas WO, Xue H, Savitz SI, et al. Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect. Stem Cells Dev. 2009;18(5):683–92. doi: 10.1089/scd.2008.0253. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Kerkelä E, Hakkarainen T, Mäkelä T, Raki M, Kambur O, Kilpinen L, et al. Transient proteolytic modification of mesenchymal stromal cells increases lung clearance rate and targeting to injured tissue. Stem Cells Transl Med. 2013;2(7):510–20. doi: 10.5966/sctm.2012-0187. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Walczak P, Zhang J, Gilad AA, Kedziorek DA, Ruiz-Cabello J, Young RG, et al. Dual-modality monitoring of targeted intraarterial delivery of mesenchymal stem cells after transient ischemia. Stroke. 2008;39:1569–74. doi: 10.1161/STROKEAHA.107.502047. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Mäkelä T, Takalo R, Arvola O, Haapanen H, Yannopoulos F, Blanco R, et al. Safety and biodistribution study of bone marrow-derived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model. Cytotherapy. 2015;17:392–402. doi: 10.1016/j.jcyt.2014.12.004. [PubMed] [CrossRef] [Google Scholar]
- Toupet K, Maumus M, Luz-Crawford P, Lombardo E, Lopez-Belmonte J, van Lent P, et al. Survival and biodistribution of xenogenic adipose mesenchymal stem cells is not affected by the degree of inflammation in arthritis. PLoS One. 2015;10:e0114962. doi: 10.1371/journal.pone.0114962. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Tögel F, Yang Y, Zhang P, Hu Z, Westenfelder C. Bioluminescence imaging to monitor the in vivo distribution of administered mesenchymal stem cells in acute kidney injury. Am J Physiol Renal Physiol. 2008;295:F315–21. doi: 10.1152/ajprenal.00098.2008. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Morigi M, De Coppi P. Cell therapy for kidney injury: different options and mechanisms—mesenchymal and amniotic fluid stem cells. Nephron Exp Nephrol. 2014;126:59. doi: 10.1159/000360667. [PubMed] [CrossRef] [Google Scholar]
- Toma C, Wagner WR, Bowry S, Schwartz A, Villanueva F. Fate of culture-expanded mesenchymal stem cells in the microvasculature: in vivo observations of cell kinetics. Circ Res. 2009;104(3):398–402. doi: 10.1161/CIRCRESAHA.108.187724. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Cui LL, Kerkelä E, Bakreen A, Nitzsche F, Andrzejewska A, Nowakowski A, et al. The cerebral embolism evoked by intraaterial delivery of allogeneic bone marrow mesenchymal stem cells in rats is related to cell dose and infusion velocity. Stem Cell Res Ther. 2015;6:11. doi: 10.1186/scrt544. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Nemeth K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, Doi K, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009;15:42–9. doi: 10.1038/nm.1905. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Chiesa S, Morbelli S, Morando S, Massollo M, Marini C, Bertoni A, et al. Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells. Proc Natl Acad Sci U S A. 2011;108:17384–9. doi: 10.1073/pnas.1103650108. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Akiyama K, Chen C, Wang D, Xu X, Qu C, Yamaza T, et al. Mesenchymal stem cell-induced immunoregulation involves Fas ligand/Fas-mediated T cell apoptosis. Cell Stem Cell. 2012;10(5):544–55. doi: 10.1016/j.stem.2012.03.007. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Choi H, Lee RH, Bazhanov N, Oh JY, Prockop DJ. Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-?B signaling in resident macrophages. Blood. 2011;118:330–8. doi: 10.1182/blood-2010-12-327353. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Kim J, Hematti P. Mesenchymal stem celleducated macrophages: a novel type of alternatively activated macrophages. Exp Hematol. 2009;37:1445–53. doi: 10.1016/j.exphem.2009.09.004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Maggini J, Mirkin G, Bognanni I, Holmberg J, Piazzón IM, Nepomnaschy I, et al. Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile. PLoS One. 2010;5(2):e9252. doi: 10.1371/journal.pone.0009252. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Francois M, Romieu-Mourez R, Li M, Galipeau J. Human MSC suppression correlates with cytokine induction of indoleamine 2,3-dioxygenase and bystander M2 macrophage differentiation. Mol Ther. 2011;20:187–95. doi: 10.1038/mt.2011.189. [PubMed] [CrossRef] [Google Scholar]
- Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol. 2008;180:2581–7. doi: 10.4049/jimmunol.180.4.2581. [PubMed] [CrossRef] [Google Scholar]
- Le Blanc K, Mougiakanos D. Multipotent mesenchymal stromal cells and the innate immune system. Nat Rev Immunol. 2012;12:383–96. doi: 10.1038/nri3209. [PubMed] [CrossRef] [Google Scholar]
- Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007;110:3499–506. doi: 10.1182/blood-2007-02-069716. [PubMed] [CrossRef] [Google Scholar]
- von Bahr L, Batsis I, Moll G, Hägg M, Szakos A, Sundberg B, et al. Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation. Stem Cells. 2012;30:1575–8. doi: 10.1002/stem.1118. [PubMed] [CrossRef] [Google Scholar]
- Segers VFM, Van Riet I, Andries LJ, Lemmens K, Demolder MJ, De Becker AJML, et al. Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms. Am J Physiol Heart Circ Physiol. 2006;290:H1370–7. doi: 10.1152/ajpheart.00523.2005. [PubMed] [CrossRef] [Google Scholar]
- Ip JE, Wu Y, Huang J, Zhang L, Pratt RE, Dzau VJ. Mesenchymal stem cells use integrin beta1 not CXC chemokine receptor 4 for myocardial migration and engraftment. Mol Biol Cell. 2007;18:2873–82. doi: 10.1091/mbc.E07-02-0166. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Wu Y, Zhao RC. The role of chemokines in mesenchymal stem cell homing to myocardium. Stem Cell Rev. 2012;8:243–50. doi: 10.1007/s12015-011-9293-z. [PubMed] [CrossRef] [Google Scholar]
- Zhang M, Mal N, Kiedrowski M, Chacko M, Askari AT, Popovic ZB, et al. SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction. FASEB J. 2007;21:3197–207. doi: 10.1096/fj.06-6558com. [PubMed] [CrossRef] [Google Scholar]
- Belema-Bedada F, Uchida S, Martire A, Kostin S, Braun T. Efficient homing of multipotent adult MSCs depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell. 2008;2:566–75. doi: 10.1016/j.stem.2008.03.003. [PubMed] [CrossRef] [Google Scholar]
- Wang Y, Zhang D, Ashraf M, Zhao T, Huang W, Ashraf A, et al. Combining neuropeptide Y and mesenchymal stem cells reverses remodeling after myocardial infarction. Am J Physiol Heart Circ Physiol. 2010;298:H275–86. doi: 10.1152/ajpheart.00765.2009. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Laurila JP, Laatikainen L, Castellone MD, Trivedi P, Heikkila J, Hinkkanen A, et al. Human embryonic stem cell-derived mesenchymal stromal cell transplantation in a rat hind limb injury model. Cytotherapy. 2009;11:726–37. doi: 10.3109/14653240903067299. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Jasmin, Jelicks LA, Tanowitz HB, Peters VM, Mendez-Otero R, de Carvalho AC C, et al. Molecular imaging, biodistribution and efficacy of mesenchymal bone marrow cell therapy in a mouse model of Chagas disease. Microbes Infect. 2014;16:923–35. doi: 10.1016/j.micinf.2014.08.016. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Reinders ME, de Fijter JW, Roelofs H, Bajema IM, de Vries DK, Schaapherder AF, et al. Autologous bone marrow-derived mesenchymal stromal cells for the treatment of allograft rejection after renal transplantation: results of a phase I study. Stem Cells Transl Med. 2013;2:107–11. doi: 10.5966/sctm.2012-0114. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Tan J, Wu W, Xu X, Liao L, Zheng F, Messinger S, et al. Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. JAMA. 2012;307:1169–77. doi: 10.1001/jama.2012.316. [PubMed] [CrossRef] [Google Scholar]
- Humphreys BD, Bonventre JV. Mesenchymal stem cells in acute kidney injury. Annu Rev Med. 2008;59:311–25. doi: 10.1146/annurev.med.59.061506.154239. [PubMed] [CrossRef] [Google Scholar]
- Gooch A, Doty J, Flores J, Swenson L, Toegel FE, Reiss GR, et al. Initial report on a phase I clinical trial: prevention and treatment of post-operative acute kidney injury with allogeneic mesenchymal stem cells in patients who require on-pump cardiac surgery. Cell Ther Transplant. 2008;1:e.000028.01. [Google Scholar]
- Sugimoto H, Mundel TM, Sund M. Bone-marrow-derived stem cells repair basement membrane collagen defects and reverse genetic kidney disease. Proc Natl Acad Sci U S A. 2006;103:7321–6. doi: 10.1073/pnas.0601436103. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate M, et al. Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am Soc Nephrol. 2004;15(7):1794–804. doi: 10.1097/01.ASN.0000128974.07460.34. [PubMed] [CrossRef] [Google Scholar]
- Imberti B, Morigi M, Tomasoni S, Rota C, Corna D, Longaretti L, et al. Insulin-like growth factor-1 sustains stem cell mediated renal repair. J Am Soc Nephrol. 2007;18:2921–8. doi: 10.1681/ASN.2006121318. [PubMed] [CrossRef] [Google Scholar]
- Bruno S, Grange C, Deregibus MC, Calogero RA, Saviozzi S, Collino F, et al. Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. J Am Soc Nephrol. 2009;20:1053–67. doi: 10.1681/ASN.2008070798. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Briquet A, Grégoire C, Comblain F, Servais L, Zeddou M, Lechanteur C, et al. Human bone marrow, umbilical cord or liver mesenchymal stromal cells fail to improve liver function in a model of CCl4-induced liver damage in NOD/SCID/IL-2R?(null) mice. Cytotherapy. 2014;16:1511–8. doi: 10.1016/j.jcyt.2014.07.003. [PubMed] [CrossRef] [Google Scholar]
- Zhang S, Lv C, Yang X, Han Z, Zhang S, Zhang J, et al. Corticosterone mediates the inhibitory effect of restraint stress on the migration of mesenchymal stem cell to carbon tetrachloride-induced fibrotic liver by downregulating CXCR4/7 expression. Stem Cells Dev. 2015;24(5):587–96. doi: 10.1089/scd.2014.0243. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Li Q, Zhou X, Shi Y, Li J, Zheng L, Cui L, et al. In vivo tracking and comparison of the therapeutic effects of MSCs and HSCs for liver injury. PLoS One. 2013;8(4) doi: 10.1371/journal.pone.0062363. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Parekkadan B, Upadhyay R, Dunham J, Iwamoto Y, Mizoguchi E, Mizoguchi A, et al. Bone marrow stromal cell transplants prevent experimental enterocolitis and require host CD11b?+?splenocytes. Gastroenterology. 2011;140:966–75. doi: 10.1053/j.gastro.2010.10.013. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Wu M, Ji S, Xiao S, Kong Z, Fang H, Zhang Y, et al. JAM-A promotes wound healing by enhancing both homing and secretion activities of mesenchymal stem cells. Clin Sci (Lond). 2015. [Epub ahead of print]. [PubMed]
- Kidd S, Spaeth E, Dembinski JL, Dietrich M, Watson K, Klopp A, et al. Direct evidence of mesenchymal stem cell tropism for tumor and wounding microenvironments using in vivo bioluminescent imaging. Stem Cells. 2009;27:2614. doi: 10.1002/stem.187. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, et al. Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair. Stem Cells. 2004;22:812. doi: 10.1634/stemcells.22-5-812. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Wu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells. 2007;25:2648. doi: 10.1634/stemcells.2007-0226. [PubMed] [CrossRef] [Google Scholar]
- Wu J, Sun Z, Sun H-S, Wu J, Weisel RD, Keating A, et al. Intravenously administered bone marrow cells migrate to damaged brain tissue and improve neural function in ischemic rats. Cell Transplant. 2008;16:993–1005. doi: 10.3727/000000007783472435. [PubMed] [CrossRef] [Google Scholar]
- Yilmaz G, Vital S, Yilmaz CE, Stokes KY, Alexander JS, Granger DN. Selectin-mediated recruitment of bone marrow stromal cells in the postischemic cerebral microvasculature. Stroke. 2011;42:806–11. doi: 10.1161/STROKEAHA.110.597088. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Wei L, Fraser JL, Lu ZY, Hu X, Yu SP. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats. Neurobiol Dis. 2012;46:635–45. doi: 10.1016/j.nbd.2012.03.002. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Constantin G, Marconi S, Rossi B, Angiari S, Calderan L, Anghileri E, et al. Adipose-derived mesenchymal stem cells ameliorate chronic experimental autoimmune encephalomyelitis. Stem Cells. 2009;27:2624–35. doi: 10.1002/stem.194. [PubMed] [CrossRef] [Google Scholar]
- Simmons PJ, Przepiorka D, Thomas ED, Torok-Storb B. Host origin of marrow stromal cells following allogeneic bone marrow transplantation. Nature. 1987;328:429–32. doi: 10.1038/328429a0. [PubMed] [CrossRef] [Google Scholar]
- Cilloni D, Carlo-Stella C, Falzetti F, Sammarelli G, Regazzi E, Colla S, et al. Limited engraftment capacity of bone marrow-derived mesenchymal cells following T-cell-depleted hematopoietic stem cell transplantation. Blood. 2000;96:3637–43. [PubMed] [Google Scholar]
- Rieger K, Marinets O, Fietz T, Körper S, Sommer D, Mücke C, et al. Mesenchymal stem cells remain of host origin even a long time after allogeneic peripheral blood stem cell or bone marrow transplantation. Exp Hematol. 2005;33:605–11. doi: 10.1016/j.exphem.2005.02.004. [PubMed] [CrossRef] [Google Scholar]
- Rombouts WJ, Ploemacher RE. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia. 2003;17:160–70. doi: 10.1038/sj.leu.2402763. [PubMed] [CrossRef] [Google Scholar]
- Follenzi A, Raut S, Merlin S, Sarkar R, Gupta S. Role of bone marrow transplantation for correcting hemophilia A in mice. Blood. 2012;119:5532–42. doi: 10.1182/blood-2011-07-367680. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Otsuru S, Gordon PL, Shimono K, Jethva R, Marino R, Phillips CL, et al. Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms. Blood. 2012;120:1933–41. doi: 10.1182/blood-2011-12-400085. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Sémont A, Mouiseddine M, François A, Demarquay C, Mathieu N, Chapel A, et al. Mesenchymal stem cells improve small intestinal integrity through regulation of endogenous epithelial cell homeostasis. Cell Death Differ. 2010;17:952–61. doi: 10.1038/cdd.2009.187. [PubMed] [CrossRef] [Google Scholar]
- Fakhrejahani E, Toi M. Tumor angiogenesis: pericytes and maturation are not to be ignored. J Oncol. 2012;2012:261750. doi: 10.1155/2012/261750. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- D’souza N, Burns JS, Grisendi G, Candini O, Veronesi E, Piccinno S, et al. MSC and tumors: homing, differentiation, and secretion influence therapeutic potential. Adv Biochem Eng Biotechnol. 2013;130:209–66. [PubMed] [Google Scholar]
- Beckermann BM, Kallifatidis G, Groth A, Frommhold D, Apel A, Mattern J, et al. VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma. Br J Cancer. 2008;99:622–31. doi: 10.1038/sj.bjc.6604508. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Alieva M, Bagó JR, Aguilar E, Soler-Botija C, Vila OF, Molet J, et al. Glioblastoma therapy with cytotoxic mesenchymal stromal cells optimized by bioluminescence imaging of tumor and therapeutic cell response. PLoS One. 2012;7:e35148. doi: 10.1371/journal.pone.0035148. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Knoop K, Schwenk N, Schmohl K, Müller A, Zach C, Cyran C, et al. Mesenchymal stem cell (MSC)-mediated, tumor stroma-targeted radioiodine therapy of metastatic colon cancer using the sodium iodide symporter as theranostic gene. J Nucl Med. 2015;pii:jnumed.114.146662. [PubMed]
- Xu S, Menu E, De Becker A, Van Camp B, Vanderkerken K, Van Riet I. Bone marrow-derived mesenchymal stromal cells are attracted by multiple myeloma cell-produced chemokine CCL25 and favor myeloma cell growth in vitro and in vivo. Stem Cells. 2012;30:266–79. doi: 10.1002/stem.787. [PubMed] [CrossRef] [Google Scholar]
- Duan X, Guan H, Cao Y, Kleinerman ES. Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into Ewing sarcoma tumors. Cancer. 2009;115:13–22. doi: 10.1002/cncr.24013. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Kidd S, Spaeth E, Watson K, Burks J, Lu H, Klopp A, et al. Origins of the tumor microenvironment: quantitative assessment of adipose-derived and bone marrow-derived stroma. PLoS One. 2012;7:e30563. doi: 10.1371/journal.pone.0030563. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Grisendi G, Bussolari R, Veronesi E, Piccinno S, Burns JS, De Santis G, et al. Understanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: the Mesenkillers. Am J Cancer Res. 2011;1:787–805. [PMC free article] [PubMed] [Google Scholar]
- Suzuki K, Sun R, Origuchi M, Kanehira M, Takahata T, Itoh J, et al. Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization. Mol Med. 2011;17:579–87. doi: 10.2119/molmed.2010.00157. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 2014;15:4142–57. doi: 10.3390/ijms15034142. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Akyurekli C, Le Y, Richardson RB, Fergusson D, Tay J, Allan DS. A systematic review of preclinical studies on the therapeutic potential of mesenchymal stromal cell-derived microvesicles. Stem Cell Rev. 2015;11:150–60. doi: 10.1007/s12015-014-9545-9. [PubMed] [CrossRef] [Google Scholar]
- Zhang Y, Chopp M, Meng Y, Katakowski M, Xin H, Mahmood A, et al. Effect of exosomes derived from multipluripotent mesenchymal stromal cells on functional recovery and neurovascular plasticity in rats after traumatic brain injury. J Neurosurg. 2015;16:1–12. [PMC free article] [PubMed] [Google Scholar]
- Xin H, Li Y, Liu Z, Wang X, Shang X, Cui Y, et al. MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles. Stem Cells. 2013;31:2737–46. doi: 10.1002/stem.1409. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Tomasoni S, Longaretti L, Rota C, Morigi M, Conti S, Gotti E, et al. Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells. Stem Cells Dev. 2013;22:772–80. doi: 10.1089/scd.2012.0266. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Kordelas L, Rebmann V, Ludwig AK, Radtke S, Ruesing J, Doeppner TR, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia. 2014;28:970–3. [PubMed] [Google Scholar]
- Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood. 2006;107:1484–90. doi: 10.1182/blood-2005-07-2775. [PubMed] [CrossRef] [Google Scholar]
- Giuliani M, Bennaceur-Griscelli A, Nanbakhsk A, Oudrhiri N, Chouaib S, Azzarone B, et al. TLR ligands stimulation protects MSC from NK killing. Stem Cells. 2014;32:290–300. doi: 10.1002/stem.1563. [PubMed] [CrossRef] [Google Scholar]
- Reinders ME, Hoogduijn MJ. NK cells and MSCs: possible implications for MSC therapy in renal transplantation. J Stem Cell Res Ther. 2014;4:10000166. doi: 10.4172/2157-7633.1000166. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Moll G, Rasmusson-Duprez I, Von Bahr L, Conolly-Andersen AM, Elgue G, Funke L, et al. Are therapeutic human mesenchymal stromal cells compatible with human blood. Stem Cells. 2012;30:1565–74. doi: 10.1002/stem.1111. [PubMed] [CrossRef] [Google Scholar]
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