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Focus on Mesenchymal Stem Cell-Derived Exosomes: Opportunities and Challenges in Cell-Free Therapy

Lin Cheng,1 Kun Zhang,2  Shuying Wu,1  Manhua Cui,1 and  Tianmin Xu1

1Gynecology and Oncology Department of the Second Hospital of Jilin University, Ziqiang Street 218, Changchun 130000, China
2Medical Research Center, Second Clinical College, Jilin University, Ziqiang Street 218, Changchun 130000, China

Received 11 August 2017; Revised 5 November 2017; Accepted 22 November 2017; Published 19 December 2017

Academic Editor: Changwon Park

Abstract

Mesenchymal stem cells have been at the forefront of regenerative medicine for many years. Exosomes, which are nanovesicles involved in intercellular communication and the transportation of genetic material transportation that can be released by mesenchymal stem cells, have been recently reported to play a role in cell-free therapy of many diseases, including myocardial infarction, drug addiction, and status epilepticus. They are also thought to help ameliorate inflammation-induced preterm brain injury, liver injury, and various types of cancer. This review highlights recent advances in the exploration of mesenchymal stem cell-derived exosomes in therapeutic applications. The natural contents, drug delivery potency, modification methods, and drug loading methods of exosomes are also discussed.

1. Introduction

Mesenchymal stem cells (MSCs) originate from the mesoderm of many tissues, including bone marrow, liver, spleen, peripheral blood, adipose, placenta, and umbilical cord blood, and have the capacity to self-renew and the ability to generate differentiated cells. Over the last decade, MSCs have emerged as a popular research topic because of their potential role in regenerative medicine, immunoregulation, neuroprotection, and antitumor effects originally attributed to direct cell replacement. However, experimental data indicates that most MSCs are largely cleared, while a small proportion will integrate into injured tissue after intravenous injection [1]. Furthermore, the “cell replacement theory” does not account for the sufficient durations in a variety of disease models [2, 3]. Recently, several mechanisms have been put forward regarding the therapeutic potential of MSCs, including (1) paracrine factors involving proteins/peptides and hormones and (2) the transfer of mitochondria or exosomes/microvesicles packaging multitudinous molecules [4].

Exosomes are a family of nanoparticles with a diameter in the range of 40–100 nm that are generated inside multivesicular endosomes or multivesicular bodies (MVBs) and are secreted when these compartments fuse with plasma membrane [5]. Exosomes are enriched in endosome-derived components as well as many bioactive molecules such as proteins, lipids, mRNAs, microRNAs (miRNAs), long noncoding RNAs (lncRNAs), transfer RNA (tRNA), genomic DNA, cDNA, and mitochondrial DNA (mtDNA) [6–12]. It has also been reported that exosomes may be released from multiple cell types, including reticulocytes [13], immunocytes, tumor cells, and MSCs [14]. This suggests that the secretion of exosomes is a general cellular function that plays an important role in the intercellular transfer of information.

In this review, we focus on the mechanisms of exosomes/microvesicles, covering the current knowledge on biological characteristics and their potential cell-free therapeutic applications for MSC-derived exosomes.

2. Characterization and Isolation of Exosomes

Exosomes were first discovered by Harding’s group as “a garbage can” in maturing sheep reticulocytes [13]. Originally, they were thought to have a typical “cup-shaped” or “saucer-like” morphology when analyzed by electron morphology [15, 16]. Zabeo’s group revealed a wide diversity in exosome morphology when purified from homogeneous cell types (the human mast cell line HMC-1). They classified exosome morphology into nine categories: (1) single vesicle; (2) double vesicle; (3) triple vesicle or more; (4) small double vesicle; (5) oval vesicle; (6) small tubule; (7) large tubule; (8) incomplete vesicle; and (9) pleomorphic vesicle [17]. This categorization suggested that different morphologies of exosomes may be accompanied by various and specific functions. Exosomes also contain surface proteins unique to the endosomal pathway, which are generally used to characterize exosomes and distinguish them from microvesicles (MVs), apoptotic bodies, and other vesicles (Table 1), such as tetraspanins (CD63, CD81, and CD9), heat shock proteins (Hsc70), lysosomal proteins (Lamp2b), the tumor-sensitive gene 101 (Tsg101), and fusion proteins (CD9, flotillin, and annexin) [12, 18]. Exosomes are released in almost all types of extracellular fluids, including blood, urine, amniotic fluid, ascites, hydrothorax, saliva, breast milk, seminal fluid, and cerebrospinal fluid. Exosomal content greatly depends on cellular origin. For example, exosomes derived from B lymphocytes that bring functional MHCI, MHCII, and T cell costimulatory molecules can stimulate T cell proliferation [19]. Furthermore, cancer cell-derived exosomes contain gelatinolytic enzymes and other cell adhesion-related molecules to help tumor progression and metastasis [20]. Importantly, these cancer cell-derived exosomes are actively incorporated by MSCs in vitro and in vivo, in that the transfer of exosomal proteins and miRNAs acquire the physical and functional characteristics of tumor-supporting fibroblasts [21, 22]. For more details on the molecular cargos and extracellular signal transmission pathway of exosomes, the reader may refer to ExoCarta (http://www.exocarta.org) or EVpedia (http://evpedia.info), as well as the American Society for Exosomes and Microvesicles (http://www.asemv.org), for an in-depth exploration.

Table 1: Characterization of various extracellular vesicles.

Ultracentrifugation and a commercial kit rooted in polymer-based precipitation are the most well-established purification protocols [16]. Other conventional validated isolation methods described in the literature include ultrafiltration, chromatography, and affinity capture [23]. New protocols have been established in order to facilitate the large-scale and high-purity manufacture of exosomes. Microfluidic techniques [24] are based on electrochemical, electromechanical, viscoelastic [25], optical, nonoptical, and other principles, yet the isolation is a mixed population of small nanoparticles without further demonstration of their intracellular origin. Thus, we use the term exosomes in this review to refer to extracellular vesicles characterized by exosome-specific surface markers, regardless of the primitive appellations in the published data.

3. Cargos and Functions of MSC-Derived Exosomes

The abundance of cargos identified from MSC-derived exosomes attracts broad attention because of their therapeutic potential in cardiovascular disease, tissue (kidney, liver, skin, and cornea) repair, immune disease, tumor inhibition, and neurological disease (Figure 1). They function largely via the constant transfer of miRNAs and proteins, resulting in the alteration of a variety of activities in target cells via different pathways.

Figure 1: The main functions of MSC-derived exosomes. The external bilayer (green circle) is the membrane and the internal bilayer (white circle) is packed with various bioactive compounds.
3.1. Protein

Over 900 species of proteins have been collected from MSC-derived exosomes according to ExoCarta. With the exception of some common proteins involved in cell metabolism and the cytoskeleton, many proteins have been found in different tissue sources of MSC-derived exosomes. Proteomic studies by Kang’s group identified 103 proteins from neural stem cell-derived exosomes. For example, the presence of polymyositis/scleroderma autoantigen 2 (PM/Scl2), a highly specific nuclear autoantigen, indicates that exosomes may be involved in triggering autoimmunity. They also found an imparity between exosomes larger than the baseline (50 nm) and those of smaller morphology [26]. These findings may explain the phenomenon recently observed by Caponnetto et al. regarding size-dependent cellular uptake of exosomes by target cells [27]. Intriguingly, all enzymes involved in the ATP synthesis of glycolysis (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglucomutase (PGM), enolase (ENO), and pyruvate kinase m2 isoform (PKm2)), as well as the rate-limiting glycolytic enzyme phosphorylated PFKFB3 that upregulates phosphofructose kinase, were identified in MSC-derived exosomes. Furthermore, oxidative stress was reduced via peroxiredoxins and glutathione S-transferases in MSC-derived exosomes [28], which suggests that replenishing glycolytic enzymes to increase ATP production, as well as additional proteins to reduce oxidative stress through exosomal transportation, may help reduce cell death in myocardial ischemia/reperfusion injury. Comparable levels of VEGF, extracellular matrix metalloproteinase inducer (EMMPRIN), and MMP-9 have also been reported in MSC-derived exosomes.

These three proteins play a vital role in stimulating angiogenesis [29], which could be fundamental for tissue repair. Recent experimental evidence summarized by Burrello suggests that transcriptional factors, such as Nanog, octamer-binding transcription factor 4 (Oct-4), HoxB4, and Rex-1, play an important role in the immune system [30]. For example, HoxB4 has been shown to affect DC maturation and T-cell proliferation, differentiation, and activation through WNT signaling. Interestingly, membrane proteins and exosome-specific surface markers, such as CD81, CD63, and CD9, may affect the immune response by regulating cell adhesion, motility, activation, and signal transduction [31]. Several studies have also shown that exosomes derived from MSCs harbor cytokines and growth factors, such as TGFβ1, interleukin-6 (IL-6), IL-10, and hepatocyte growth factor (HGF), which have been proven to contribute to immunoregulation [30].

3.2. miRNAs

miRNAs consist of a class of small noncoding RNAs that regulate gene expression posttranscriptionally by targeting mRNAs to induce suppression of protein expression or cleavage [32]. Many miRNAs have been found in MSC-derived exosomes and are reportedly involved in both physiological and pathological processes such as organism development, epigenetic regulation, immunoregulation, tumorigenesis, and tumor progression. Notably, exosomes with membrane structure act as preservers and deliverers of miRNAs, transferring functional miRNAs into recipient cells. It has been reported that exosomal miR-23b, miR-451, miR-223, miR-24, miR-125b, miR-31, miR-214, and miR-122 [33, 34] may inhibit tumor growth and stimulate apoptosis through different pathways. For instance, miR-23b promotes dormancy in metastatic breast cancer cells via the suppression of the target gene MARCKS, which encodes a protein that promotes cell cycling and motility [34]. MiR-16, shuttled by MSC-derived exosomes, has also been found to suppress angiogenesis by downregulating VEGF expression in breast cancer cells [35].

Recently, let-7f, miR-145, miR-199a, and miR-221, which are released from umbilical MSC-derived exosomes, have been found to largely contribute to the suppression of hepatitis C virus (HCV) RNA replication [36]. Di Trapani’s group evaluated the immunomodulatory effects exerted by MSC-derived exosomes on unfractionated peripheral blood mononuclear cells and purified T, B, and NK cells. They observed that exosomes had higher levels of miRNAs compared to MSCs and could also induce inflammatory priming via increasing levels of miR-155 and miR-146, which are two miRNAs involved in the activation and inhibition of inflammatory reactions [37]. Similar immunosuppressive functions have also been reported in animal experiments by Cui et al. [38]. Exosomes from MSCs effectively increased the level of miR-21 in the brain of AD mice. Additionally, replenishment of miR-21 restored the cognitive deficits in APP/PS1 mice and prevented pathologic features by regulating inflammatory responses and restoring synaptic dysfunction [38]. Recent studies have also shown that aging is substantially controlled by hypothalamic stem cells, partially through the release of exosomal miRNAs [39]. However, contradictions regarding these outcomes remain. A quantitative analysis of exosomal miRNA abundance and stoichiometry by Chevillet’s group quantified both the number of exosomes and the number of miRNA molecules in replicate samples isolated from diverse sources. Regardless of the source, the study indicated that, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA, suggesting that most individual exosomes do not carry biologically significant numbers of miRNAs and are thus unlikely to be functional individually as vehicles for miRNA-based communication [40].

3.3. Others

In 2006, MSC-derived exosomes that could modulate the phenotype of target cells, supporting self-renewal of hematopoietic progenitors and multipotency by transfer of growth factors and mRNA, were first reported. For instance, exosomal SOX2 was found to initiate innate responses against microbial infection through neutrophil activation [41]. Although MSC-derived exosomes have the same morphology as exosomes from other cells and carry typical markers, they are quite different in regard to compartmentalization and protein and RNA composition. For example, studies have indicated that not all MSC-derived exosomes are equivalent [42]. Baglio’s group [6] characterized the small RNAome of exosomes released by early passage adipose-MSCs (ASC) and bone marrow-MSCs (BMSCs). They found a large discrepancy in the proportion of miRNAs in total small RNA content between cells (19–49%) and exosomes (2–5%), suggesting that the miRNAs in exosomes do not merely reflect the cellular content. Further studies regarding the overrepresentation of small RNA content-tRNAs revealed a similar outcome. The most abundant tRNA in ASC exosomes, tRNA GCC (Gly), represented only a small fraction (5%) of the total cellular tRNA. Importantly, the authors also determined that the striking differences in tRNA species seemed to be associated with the differentiation status of MSCs. Recent research has shown that the stability of exosome composition is susceptible to localized environmental conditions. For example, hypoxia and inflammatory signals, such as lipopolysaccharides, may be strong interference factors [43].

4. Exosomes as Drug Delivery Vehicles

Optimal features of drug delivery vehicles may be applied to improve carrier qualities, including cellular tropism, efficient therapeutic cargos, appropriate physicochemical properties, and sufficient immune tolerance. Among the many drug platforms, liposomes have been the preferred pharmaceutical vehicles for drug delivery. A wide range of liposome products have been approved for the treatment of diseases, including fungal infections, pain management, hepatitis A, influenza, and various types of cancer [44, 45]. In contrast to liposomes, exosomes are optimal for drug delivery because of their natural properties and plasticity with minor modifications. Here, we compare exosomes and liposomes and suggest that exosomes may be a promising star for drug delivery.

4.1. Exosomes versus Liposomes

Exosomes and liposomes are both coated with a phospholipid membrane. The membrane structure of exosomes is inlayed with multiple natural biomolecules, such as surface proteins and MHCs, while liposomes may be modified with targeting ligands or inert polymeric molecules such as oligosaccharides, glycoproteins, polysaccharides, and synthetic polymers [45]. The size of liposomes is in the range of 30 nm to several microns [46]. Smaller liposomes (as small as exosomes) display a prolonged circulation time compared to larger ones, but the capacity for optimal drug reservation and release profiles is partly lost. For more details regarding circulation time and biodistribution, readers can refer to other sources [46]. Regarding cellular interactions and uptake, liposomes can be equipped with targeting ligands, which can bind to receptors or other molecules that are specific or overexpressed by target cells for interactions and the intracellular delivery of drugs [46]. However, the drug delivery of liposomes is not efficient, since many modifications have been designed to minimize clearance and poisonousness. In general, liposomes accumulate in the macrophages of the liver and spleen after intravenous injection. Few liposomes are interspersed in other tissues, which may be due to the lack of immunocompatibility. On the other hand, exosomes are born with many features of an ideal drug delivery vehicle. For example, they exhibit lower toxicity compared to liposomes. In addition, they are well tolerated by the immune system, even across the blood-brain barrier, avoiding phagocytosis or degradation by macrophages [47]. Exosomes exhibit an innate targeting tendency. For instance, MSC-derived exosomes home preferentially to inflamed tissues and tumor tissues [48]. Furthermore, abundant bioactive materials within exosomes or on the surface provide primitive treatment potential, and there are abundant modification methods for membrane targeting and drug loading. Alvarez-Erviti et al. engineered dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide and loaded these modified exosomes with siRNAs by electroporation. These intravenously injected exosomes showed a strong knockdown of BACE1 (mRNA (60%) and protein (62%)), a therapeutic target of Alzheimer’s disease, in wild-type mice [49].

4.2. Exosomal Modification and Cargo Loading

To amplify the therapeutic effects, many studies try to modify and load various treatment factors into exosomes via various methods. To date, these methods can be classified into two categories: (1) loading after isolation and (2) loading exosomes during biogenesis.

The first approach has been applied to load chemotherapeutic agents, siRNAs, and miRNAs. To reduce immunogenicity and toxicity of doxorubicin, Tian’s group facilitated exosomal tumor targeting by engineering mouse immature dendritic cells (imDCs) to express a well-characterized exosomal membrane protein (Lamp2b) fused to a breast cancer-specific iRGD peptide (CRGDKGPDC). Chemotherapeutic agents were loaded via electroporation. The results showed an encapsulation efficiency of up to 20% and exosomal-delivered doxorubicin specific to breast cancer cells in vitro, leading to strong antiproliferative activity without overt toxicity after intravenous injection of BALB/c nude mice [50]. For nucleic acid, electroporation method has also been the first-rank used reported in several studies [49, 51]. Although these studies provided positive delivery outcomes, debates remain. Some studies indicate that siRNA encapsulation is an illusion caused by nonspecific aggregate formation, independent of the exosomes. In addition, no significant encapsulation of siRNA could be measured when aggregate formation was blocked [52]. Therefore, it is necessary to establish multiple protocols for loading exosomes with nucleic acid.

The second approach is based on transfection methods to package active proteins, nucleic acid, and other active molecules into exosomes, where cells are transfected with an engineered effector-expressing vector. Liu’s group used this method to load cells with opioid receptor Mu (MOR) siRNA in order to treat drug addiction via downregulating the expression of MOR, the primary target for opioid analgesics used clinically, including morphine, fentanyl, and methadone. This novel study provided a new strategy for the treatment of drug addiction [53]. Similarly, synthesized RNA oligonucleotides were transferred to MSCs in order to produce miR-143-rich exosomes, inhibiting the migratory potential of osteosarcoma cells [54]. Akt was transfected into umbilical cord-derived MSCs by using an adenovirus transfection system that improved cardiac function in animals treated with modified exosomes [55]. In addition, Pascucci reported that MSCs can acquire strong antitumor properties after incubation with paclitaxel (PTX), including the uptake of high drug doses followed by release into exosomes, inhibiting tumor growth activity. This method provides the possibility of using MSCs for the development of drugs with a higher cell-target specificity [56]. Sterzenbach reported the usage of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes [57]. They labeled an intracellular target protein with a WW tag, which was recognized by the L-domain motifs on Ndfip1, resulting in the loading of the target protein into exosomes.

For better tissue-targeting and an enhanced exosomal therapeutic effect, surface modification of exosomes was recently attempted by many groups using gene transfection techniques. The conventional method for surface protein loading was the expression of a genetic fusion between the targeted peptide and a protein that natively localized on the exosomal surface, such as Lamp2 [50]. Similarly, Ohno and colleagues engineered donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide, which efficiently delivered let-7a miRNA to epidermal growth factor receptor- (EGFR-) expressing breast cancer cells [58]. Furthermore, Tamura modified the exosomal surface by a simple mixing of original exosomes and cationized pullulan through an electrostatic interaction of both substances, thus targeting injured liver tissue and enhancing the therapeutic effects [59].

The fate of nucleic acid cargos in target cells remains controversial. For example, Kanada et al. suggests that exosomes cannot deliver functional nucleic acids to target cells by detecting differential fates of transfection-loaded biomolecules (plasmid DNA (pDNA), mRNA, and siRNA) delivered to target cells [60].

4.3. MSCs as an Ideal Source of Exosomes for Drug Delivery

Despite the fact that the properties of natural MSC-derived exosomes are disputed and distinctive of different origins, the use of MSC-derived exosomes has been confirmed for the cell type-specific targeting of drug delivery as a better alternative because of several features. First, exosomes do not elicit acute immune rejection, and there is no risk for tumor formation [61]. Second, MSCs are efficient mass producers of exosomes, which can be manufactured large scale in culture [62], providing support for individualized therapy. Third, the safety of exosomes has been confirmed in vivo by different animal models [63, 64]. To achieve cell-specific targeted drug delivery, several studies have tested donor cells, loading methods, and therapeutic cargos of MSC-derived exosomes. Bone marrow stem cells are typically used as the donor cells, and miRNAs are typically used for therapeutic cargos (Table 2). A phase II-III study has also been processed by a group in Egypt, who hypothesized that intravenous infusion of cell-free umbilical cord blood-derived MSC microvesicles may reduce the inflammatory state, thus improving the β-cell mass and glycemic control in patients with type 1 diabetes (T1DM). However, these outcomes remain controversial, particularly in reference to dose responses. The data reported in several studies is highly dependent on the drug-loading of exosomes, not the quantity of exosomes.

Table 2: Studies focusing on MSC-derived exosomes, their loading methods, therapeutic cargos, and biological function.

5. Exosomes for Cell-Free Therapy

Currently, the use of exosomes as early diagnostic tools for various types of cancer is underway. In addition, the use of exosomes as diagnostics for prostate cancer is undergoing FDA-approved tests. While complexities surrounding the therapeutic potential of exosomes continue to unravel, several clinical trials (Table 3, data from http://clinicaltrials.gov) have been completed or are underway in order to evaluate this therapeutic potential. In these trials, largely modified exosomes were used rather than native exosomes.

Table 3: Clinical trials of exosome–based therapies.

6. Conclusion and Perspective

The therapeutic potential of exosomes presents exciting new avenues for intervention in many diseases. The ability instinct to transport genetic messages and to protect the cargos to natural preferential recipient cells has drawn a rapid rise in attention. Therefore, specialized journals and websites have been established to disseminate this continuously unraveling information. While various clinical trials are underway to evaluate the safety and effectiveness of exosomes as therapeutic targets, many issues still remain. Questions regarding how clinical-grade exosomes can be produced in quantity and how various loading and isolation strategies impact the potency of exosome-based drug delivery remain unanswered. Therefore, there is an urgent need to closely examine the following aspects of exosomes: (1) natural therapeutic potential; (2) biogenesis mechanism; and (3) circulation kinetics and biodistribution. There is still a long road ahead, from promising phenomenological observations to clinical applications.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (81302242), Jilin Province Science and Technology Funds (20150204007YY and 20140204022YY), and Jilin Province Development and Reform Commission funds (2014G073 and 2016C046-2).

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Stem Cells Offer Hope for Hair Growth

UCLA researchers have discovered a new way to activate the stem cells in the hair follicle to make hair grow. The research may lead to new drugs that could promote hair growth for people with baldness or alopecia, which is hair loss associated with such factors as hormonal imbalance, stress, aging or chemotherapy. Hair follicle stem cells are long-lived cells in the hair follicle that are present in the skin and produce hair throughout a person’s lifetime. They are quiescent, meaning they are normally inactive, but they quickly activate during a new hair cycle, which is when new hair growth occurs. The quiescence of hair follicle stem cells is regulated by many factors. In certain cases, they fail to activate, which is what causes hair loss.

The study by Heather Christofk, PhD, and William Lowry, PhD, both of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, found that hair follicle stem cell metabolism is different from other cells of the skin. Cellular metabolism involves the breakdown of the nutrients needed for cells to divide, make energy and respond to their environment. The process of metabolism uses enzymes that alter these nutrients to produce metabolites. As hair follicle stem cells consume the nutrient glucose — a form of sugar — from the bloodstream, they process the glucose to eventually produce a metabolite called pyruvate. The cells then can send pyruvate to their mitochondria — the part of the cell that creates energy — or convert pyruvate into another metabolite called lactate.

The research team first blocked the production of lactate genetically in mice and showed that this prevented hair follicle stem cell activation. Conversely, in collaboration with the Rutter lab at the University of Utah, they increased lactate production genetically in the mice, which accelerated hair follicle stem cell activation, increasing the hair cycle.

The team identified two drugs that, when applied to the skin of mice, influenced hair follicle stem cells in distinct ways to promote lactate production. The first drug, called RCGD423, activates a cellular signaling pathway called JAK-Stat, which transmits information from outside the cell to the nucleus of the cell. The research showed that JAK-Stat activation leads to the increased production of lactate, and this in turn drives hair follicle stem cell activation and quicker hair growth. The other drug, called UK5099, blocks pyruvate from entering the mitochondria, which forces the production of lactate in the hair follicle stem cells and accelerates hair growth in mice.

“Lactate Dehydrogenase Activity Drives Hair Follicle Stem Cell Activation,” Nature Cell Biology, August 14, 2017

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Platelet-Rich Plasma Vs Stem Cell Therapy: Who’s Bad?

The entire field of orthopedics is looking for new regenerative technology that can save more patients more safely. Currently there are two contenders: Platelet-Rich Plasma and Stem Cell.
While PRP is the safest of the two, it’s really hard to dismiss the remarkable capabilities of stem cell therapy. In fact, I believe it’s the future of regenerative medicine. But not at the level it’s playing right now. Which is a totally different discussion we’ll save for another day.
The thing is… there are potential harm with stem cells. And unlike PRP, stem cell’s constituents are man-made, so things can go wrong. We’ll discuss the potential dark side of this therapy later in this article. However, I feel it’s important to highlight how good a treatment stem cell therapy is.
Quick Overview: Stem Cell Vs Platelet-Rich Plasma
Platelet-Rich Plasma is like water and nutrients that help restore (and sometimes accelerate) your body’s EXISTING healing mechanism. If your body is stuck with its healing, PRP can help. It releases growth factors and cytokines to kick start the healing. Stem cells on the other hand is not used to enhance healing, but to create new solutions to healing challenges. So it’s more for tissues that are totally lost.
Stem Cell Vs Platelet-Rich Plasma
With me? Before we proceed, let’s look at a little background of stem cells. We’ll stick to orthopedics for the sake of simplicity.
Orthopedic Stem Cell Therapy
Stem cells are naturally found in the human body and they are a fundamental part of the body’s normal healing process. Stem cells are known as ‘raw potential’ as they can be converted into any cell that the body needs. The body utilizes stem cells to substitute damaged and/or injured cells. This process allows natural healing and repair of the injured or damaged cells.
As the body gets older the amount of natural reserved stem cells starts to decline, which explains why the healing process is slower as the body gets older. Stem cell therapy resolves this shortage by injecting supplementary stem cells into the injured/damaged area of the body, which triggers the cell replacement, natural healing, and pain relief.
Stem cell therapy is a simple and quick procedure, taking about 15 minutes. Pain discomfort is often felt immediately, with the majority people reporting a significant improvement within one to two days.
With stem cell therapy the patient does not have to have any type of surgical procedure, local or general or downtime. Most of the patients experience a complete restoration of the damaged/ injured ligaments, tendons, and cartilage within about in 28 days. Stem cell therapy has been proven to be complexly safe, with no side effects reported in the US or in Europe.
The Difference Between Stem Cell Therapy and Platelet-Rich Plasma (PRP) Therapy
Often times, stem cell therapy and PRP can be confused because they have a lot in common during the healing process. The easiest way to tell the difference between the two, is PRP is removed from the patient’s own body, it goes through a scientific process and is them injected into the area being treated.
The cells used for stem cell therapy can come from a few different places; from an unviable embryo, and unviable fetal stem cells these stem cells are the most often used because the cells are unspecialized and can be made into specialized cells. As it sounds, preparing stem cells for therapy is a complex process. Stem cells are produced in a sophisticated labs by cell biologists and are typically grown over several weeks before it’s ready.
Plus, adult stem cells may be used, although it is not nearly as common yet because scientists are still working on ways to identify stem cells within the tissue of an adult human body.
Stem Cell Vs Platelet-Rich Plasma
So what’s the dark side of Stem Cell Therapy?
The obvious concern is that treatments with stem cells could be dangerous if not carefully controlled. I know we are all doing things for saving lives and helping people live longer, more happily, but the risks must also be considered.
Below are the 5 risks that stem cells carry. (which Platelet-Rich Plasma doesn’t.)
Risk of viruses: Since the stem cells are foreign bodies, if they happen to carry harmful microscopic agents, it’ll bring unnecessary complications. Especially those patients whose immune systems are weak, could be highly vulnerable diseases.
Uncontrolled growth: As I said before, stem cells are produced in a lab and grown over a period of several weeks. However, there is very tiny possibility the growth will continue uncontrolled after installing it into the patient. We pray it doesn’t happen.
Multi-tasking of cells: Stem cells are cultivated and grown into specialized cells that are designed to be doing just one thing and one thing only. But what if, in the long run, they also do other things that wasn’t in the original scope of things? Something to ponder.
That said, I still believe stem cells hold great promise. Now, I want to take this rest of the article to highlight a few of the common conditions that are found to be best for stem cell treatments.
Stem Cell Vs Platelet-Rich Plasma
Rheumatoid Arthritis
Rheumatoid Arthritis is caused by inflammation of the joints as a result of an autoimmune progression. The body’s immune system attacks the joints. Patients with Rheumatoid Arthritis suffer from mild to severe pain, constant fatigue, warm, and swollen joints. This type of chronic inflammation has the potential to easily damage the joints. Therefore, treatment is concentrated on decreasing the inflammation and slowing down the progress of the condition. Stem cell therapy provides a treatment alternative that takes advantage of the healing and anti-inflammatory effects.
Osteoarthritis
Osteoarthritis is joint inflammation caused by the deterioration of the cartilage that cause the bones to rub up against one another. Patients who suffer from osteoarthritis have pain, stiffness, and a decrease in their range of motion in their joints. Although, there is no cure for osteoarthritis, stem cell treatment focuses on reducing the pain reduction through medication, physical therapy, or occupational therapy. Stem cell therapy provides a treatment alternative that takes advantage of the healing and anti-inflammatory effects. While medication helps with the pain.
Shoulder Repair
Shoulder injuries such as rotator cuff tears and arthritis of the shoulder joint, as well as other types shoulder pain may be responsive to stem cell therapy. Stem cells goal is to renew damaged joints.
Stem Cell Treatment for Joint Repair
Hand and elbow problems caused by arthritis of the joints is a type of deteriorating joint disease that has disabled millions of people. Definite types of wrist and elbow joint issues including certain ligamentous injuries and tendon problems may not benefit from cell therapy. It is very important that the doctor evaluate each patient to see if stem cell therapy is a viable treatment for their patients.
Stem Cell Treatment for Knee
Knee arthritis is a type of deteriorating joint disease, which affects millions of people. Most people believe there only option for pain relief and better mobility is steroid injections or surgery, including total knee replacement surgery. However, that is not the case, many people benefit greatly from stem cell therapy. Specific types of knee issues such as, ligamentous injuries and substantial meniscal injuries may not be responsive to regenerative therapy (stem cell therapy). Each case must be carefully evaluated and the orthopedist will decide what options are best for the patient, in some cases, stem cell therapy is tried even if the patient is not exactly an ideal candidate, but trying is better than just scheduling surgery.
Stem Cell Treatment for Hip
Hip arthritis is similar to knee arthritis; millions of people suffer from hip problems. Patients usually try to delay the hip replacement surgery as long as they can and try other methods such as steroid injections, which for some people do help for a short period of time. However, long tern injects can damage the tissue near the hip. While fractured hips and certain kinds of hip injuries cannot be treated with stem cell therapy, surgery is the only available option left.
Stem Cell Treatment for Joint Repair
Problems with the hands and elbow joints usually respond well to stem cell therapy. If there are problems with the ligaments and tendons, then surgery may be necessary.
Degenerative joint diseases disable millions of people. While certain types of injuries are not a good match for stem cell therapy, there are several that are a good match. Before you prescribe surgery to repair damaged or injured joints consider about stem cell therapy, and if possible give it a try first.

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Platelet-Rich Plasma Stays Quietly Popular Despite Neglect

Fact: According to research, PRP treatments are one of the most in-demand treatments available in healthcare.
This is impressive considering the following.
PRP is not supported by the medical industry. No big pharma funding on extensive research or marketing. No medical associations lobbying to increase its awareness.
PRP is shunned by the insurance companies. No reimbursements from them. So getting patients to pay is difficult. Especially for a treatment that’s relatively “unproven” like this.
The cost of PRP treatments are actually rising. In 2006, you can get a PRP treatment for $450. Today it costs $800. The cheapest we’ve seen is $650. The prices are still robust as demand keeps up.
However, we believe the best of PRP is not even here yet. We’re just one breakthrough study away from exploding into mainstream hospitals and clinics. We see the biggest growth in Platelet-Rich Plasma happening in Asia.
Strongly based on fundamental healing theory
The growth can be attributed to PRP’s fundamental healing property. More platelets. More growth factors and cytokines. And therefore more healing. It’s as simple as that. And no one can argue this fact.
Our body’s natural healing mechanism operates with 150,000/ul-350,000/ul platelets in blood. Using Platelet-Rich Plasma means this number is amplified by 3X to 5X. How can this be not translated into better healing?
Believe it or not, the best orthopedic doctors use Platelet-Rich Plasma. And do so regularly.
PLATELET-RICH PLASMA TRENDS
PRP can be used to promote healing of injured tendons, ligaments, muscles, and joints, can be applied to various musculoskeletal problems. And they conduct regular studies to test it’s effectiveness.
One landmark study involved double-blind randomized controlled trials to see the effect of PRP on patients with chronic low back pain caused by torn discs. The study outcome says 60% of the patients felt significant improvements.
Some were cured. CURED!
Platelet-Rich Plasma Variants
So far, there are the following type of PRP variants.
Plasma Rich in Growth Factors (PRGF)
Plasma Rich in Platelets and Growth Factors (PRPGF)
Platelet-Rich Plasma (PRP); Platelet Poor Plasma (PPP)
Plasma Rich in Platelets and Rich in Leukocytes (LR-PRP)
Plasma Rich in Platelets and Poor in Leukocytes (LP-PRP)
Platelet-Rich Fibrin Matrix (PRFM)
All of them involve Plasmapherisis — the two stage centrifugation process to separate platelets from blood. However, what happen what happens after that can be different. And the industry hasn’t found it’s middle ground as to which variant to be standardized. We believe the confusion will clear up in 3-5 years.
PLATELET-RICH PLASMA TRENDS
No matter which variant you end up using, the bio-factors at play are the following:
Growth factors: TGF-B, PDGF, IGF-I,II,  FGF, EGF, VEGF, ECGF
Adhesive proteins: Fibrinogen, Fibronectin, Vitronectin, Thrombospondin-1
Clotting & Anti-Clotting factors: Proteins,  Antithrombin, Plasminogen, Proteases, Antiproteases
How Platelet-Rich Plasma Actually Work
Why is the treatment commonly used for wound healing and pain management? The answer is because the platelets’ main job is to aid coagulation, act as a biological glue and support stem or primary cell migration. In addition, it also helps in restoring hyaluronic acid and accelerates the synthesis of collagen and glycosaminoglycans and increases cartilage matrix.
Not only that, the platelets are delivered in a clot which means it can immediately act as a scaffold to enable the healing process. 95% of the bio-active proteins are released within 1 hour of injecting Platelet-Rich Plasma. The platelets continue to release growth factors for 7-10 days. Thus it’s recommended to re-inject PRP every 7 days.
PLATELET-RICH PLASMA TRENDS
Why are patients coughing up their hard earned money for this?
This reminds me of hundreds of thousands of PRP treatments paid from patient’s own pocket even though they’ve been paying for years to get covered by their respective insurance provider. In 2015, PRP costs were anywhere between $600 and $800 per site per treatment. And most patients go for repeated treatments. So why were they forking up their hard earned money if the treatment was not working? Weren’t there any better alternatives under the “coverage” of their insurance provider? The answer is 1) the treatment works. 2) there’s nothing else out there that’s as natural and side-effect-free as PRP.
Consider the case of osteoarthritis. 27 millions Americans are impacted by it. 33.6% of people older than 65 are victims. All of them experience gradual degeneration of cartilage and bones — they lose roughly 5% cartilage per year. Yet, our medical industry doesn’t have a fix to stop it.
However, when doctors started doing PRP treatments for their osteoarthritis patients, they found a large majority of them had no further cartilage loss.
To me, it means we should make PRP treatments the default first-line treatment for osteoarthritis across the country.
Another huge market is hair loss and cosmetic facial applications. I know there are many people who believe PRP doesn’t work for hair. Here’s what one of the Platelet-Rich Plasma studies found were the effect of the treatment on hair loss.
“Hair loss reduced and at 3 months it reached normal levels. Hair density reached a peak at 3 months (170.70 ± 37.81, P < 0.001). At 6 months and at 1 year, it was significantly increased, 156.25 ± 37.75 (P < 0.001) and 153.70 ± 39.92 (P < 0.001) respectively, comparing to baseline. Patients were satisfied with a mean result rating of 7.1 on a scale of 1-10. No remarkable adverse effects were noted.”
I’ll take that.
That’s me getting PRP for hair. ??
PLATELET-RICH PLASMA TRENDS
PRP market is expected to hit $126 million in 2016
That number looks paltry. But that’s an 180% increase over the 2009 figure of  $45 million.
Consider this. Just for osteoarthritis alone, if all the 27 million Americans receive 1 PRP shot a year at a conservative $400 per treatment, it would be a market of $10 billion. And that’s one condition out of the many that Platelet-Rich Plasma injections are proven to work.
Another condition that PRP is known to work very well is Tennis Elbow. It affects on average 1% to 3% of the overall population. That number is as high as 50% among tennis players.
Do the math.
Just getting Platelet-Rich Plasma covered by insurance will unleash the market big time and will help heal millions of patients naturally, more effectively.
Oh ya, that means the insurance companies will have to pay more. Why would they?
HOWEVER, if this treatment could reduce further expensive intervention like surgery then it may actually be a blessing for the insurance guys in terms of savings. One surgery avoided by a patient through right intervention through PRP treatments will save the insurance companies at least $25,000. Now, that’s a win-win for both patients and insurance.
I believe it’s a matter of time before insurance companies start realizing their folly of not supporting this treatment.
PLATELET-RICH PLASMA TRENDS
After all is said and done, it’s still “unproven”
The problem with PRP is that it can be used for just about everything, which is a good problem to have until health care officials (and insurance companies) start realizing that people are going to misuse it.
So it’s classified as unproven. The VAST scope of the treatment calls for urgent structure and guidelines. There are some 20+ conditions where researchers have found it “helps” in one way or another. It’s a daunting task to prove its efficiency in all the areas. Nevertheless, we’ll get there.
Though we’ll need a lot of funding for that.
And yes, we need to standardize the procedure. As well as come up with optimized protocols for each conditions. Someone need to take initiative on that. We’re counting on independent doctors and medical institutions. The big pharma won’t jump in because what’s in it for them, right?
It’s so simple, you’d be an idiot to not try it.
You only need a vacuum blood harvesting tube like what we offer here, a centrifuge with adapter for the tube, pipettes and 10ml ampules of 10% calcium chloride.
The only complexity comes from not following a standard PRP system. Because the final platelet count can depend on a variety of factors. Like initial volume of blood, the technique used and relative concentration of WBC and/or RBC. As well as on the patient’s side, there are factors such as age, growth factor and WBC content.
However, concentration-wise, there’s little confusion as once a sufficiently high range is reached, more doesn’t have any adverse or enhancing effect — it saturates at a certain point. So that’s the minimum. Once you reach that, you’re good. Although the outcome is not always guaranteed to be same, with the right number of platelets, platelet activation and cytokine release, you can get a consistency in your PRP offerings.
There’s still some uncertainty over the number of injections, the timing and delivery method of Platelet-Rich Plasma. But with wide-spread adoption, some kind of structure will emerge.
Let’s hope the first glimpses of it will arrive this year.
Do you know in 2015, the world saw approximately 1 million knee arthroplasties for osteoarthritis? At $25,000 apiece, $25 billion.
How many of these patients had the good fortune of their doctor recommending PRP early on?

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The Growth Factor Showdown: Plasma Vs Fibrin

Yep, it’s Platelet-Rich Plasma. There has been numerous speculations about which one among the latest Platelet-Rich family was the greatest—is it the plasma or the fibrin or even latest the A-fibrin? That confusion is somewhat over now.
Platelet-products are known to facilitate angiogenesis, hemostasis, osteogenesis, and bone growth. But see, the only reason plasma can do that is because of the growth factors it carries. Let’s review the specific roles of these growth factors in the healing process.
Growth Factors In Platelet-Rich Plasma
These are growth factors that are traditionally known to have played a vital healing role in PRP. If you’re seeing your patients get better as a result of that injection you gave, these are guys you need to thank for.
Platelet-Derived Growth Factor (PDGF): Regulates cell growth and division. Especially in blood vessels. In other words, this guy is the reason the blood vessels in our body reproduces.
Transforming Growth Factor Beta(TGF-b): Responsible for overall cell proliferation, differentiation, and other functions.
Fibroblast Growth Factor (FGF): Plays a vital role in the wound healing process and embryonic development. Also behind the proliferation and differentiation of certain specialized cells and tissues.
Vascular Endothelial Growth Factor: Responsible for vasculogenesis and angiogenesis. Restores oxygen supply in cells when inadequate. It also helps create new blood vessels after injury.
Keratinocyte Growth Factor (KGF): Found in the epithelialization-phase of wound healing. In other words, it causes the formation of epithelium immediately after a wound or injury occurs.
Connective Tissue Growth Factor: Major functions in cell adhesion, migration, proliferation, angiogenesis, skeletal development, and tissue wound repair.
These growth factors are what enables a Platelet-Rich product in tissue regeneration.
Platelet-Rich Plasma Rules
However, this new study suggests Platelet-Rich Plasma and it’s gelled cousin Platelet-Rich Fibrin both differ in the release of these growth factors which can significantly affect the healing outcome.
Here’s the takeaway:
“The advantage of PRP is the release of significantly higher proteins at earlier time points whereas PRF displayed a continual and steady release of growth factors over a 10-day period.”
Some argue that PRP enriched with large number of growth factors (a portion of it may even be excess) produce short-term effect and so is less desirable than a PRF whose release is slower and thus more beneficial in the long run.
That being said, PRF do have some advantage over PRP. Mainly:
It doesn’t need thrombin and anticoagulants.
It results in better healing due to its slow polymerization process.
And it helps in hemostasis.
How Platelet-Rich Plasma Differs From Platelet-Rich Fibrin
Platelet-Rich Plasma is a result of double spin method — a hard spin to separate red blood cells from everything everything else in the autologous (or whole) blood and a soft spin to separate the platelets and white blood cells. The result is Platelet-Rich Plasma (PRP), Platelet-Poor Plasma (PPP) and Red Blood Cells.
PRF is a newer method. Here after the first centrifugation, the middle layer is taken—which contains less platelets but more clotting factors. This gradually forms into a fibrin network and traps in the cytokines. It is then centrifuged in a PRF centrifuge resulting in PRF, a fibrin layer containing platelets and plasma.
What Matters In Healing
Obviously, when it comes to accelerating healing, immediate availability of growth factors and cytokines matter. So I believe PRP does a better job in this than PRF. Also the immediate release of growth factors for PRP means we can repeat the PRP injections for more healing factors just days after initial injection.
Platelet-derived products are in it’s infancy now. However, considering the huge potential benefits, there’s still a lot more research to be done. How about you? Which of these do you find beneficial?
If you’re a physician using any or both of these, do write to us and let us know of your experiences. Use the contact form here.

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