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Pivotal role of paracrine effects in stem cell therapies in regenerative medicine: can we translate stem cell-secreted paracrine factors and microvesicles into better therapeutic strategies?

Abstract

Although regenerative medicine is searching for pluripotent stem cells that could be employed for therapy, various types of more differentiated adult stem and progenitor cells are in meantime being employed in clinical trials to regenerate damaged organs (for example, heart, kidney or neural tissues). It is striking that, for a variety of these cells, the currently observed final outcomes of cellular therapies are often similar. This fact and the lack of convincing documentation for donor–recipient chimerism in treated tissues in most of the studies indicates that a mechanism other than transdifferentiation of cells infused systemically into peripheral blood or injected directly into damaged organs may have an important role. In this review, we will discuss the role of (i) growth factors, cytokines, chemokines and bioactive lipids and (ii) microvesicles (MVs) released from cells employed as cellular therapeutics in regenerative medicine. In particular, stem cells are a rich source of these soluble factors and MVs released from their surface may deliver RNA and microRNA into damaged organs. Based on these phenomena, we suggest that paracrine effects make major contributions in most of the currently reported positive results in clinical trials employing adult stem cells. We will also present possibilities for how these paracrine mechanisms could be exploited in regenerative medicine to achieve better therapeutic outcomes. This approach may yield critical improvements in current cell therapies before true pluripotent stem cells isolated in sufficient quantities from adult tissues and successfully expanded ex vivo will be employed in the clinic.

Introduction

There is mounting evidence that stem cells secrete a variety of growth factors, cytokines, chemokines and bioactive lipids that regulate their biology in an autocrine/paracrine-manner and orchestrate interactions with the surrounding microenvironment.1, 2 These factors are secreted, in particular, from activated cells removed from their physiological niches (for example, aspirated from the bone marrow (BM)) or mobilized into circulation (for example, mobilized peripheral blood (mPB)).1, 2, 3 Therefore, different types of cells, including hematopoietic stem progenitor cells (HSPCs), multipotent stromal cells (MSCs), skeletal muscle myoblasts (SMMs), adipose tissue stem cells (ASCs), neural stem cells (NSCs) or cardiac stem cells (CSCs) that are employed in regenerative medicine to rescue damaged organs are potential sources of paracrine factors. These factors may (i) inhibit apoptosis of cells residing in the damaged organs, (ii) stimulate proliferation and (iii) promote vascularization of affected tissues to improve oxygen delivery and metabolic exchange. The most important factors secreted from stem cells include vascular endothelial growth factor (VEGF), stem cell factor (SCF), hepatocyte growth factor (HGF), insulin-like growth factor-1 and -2 (IGF-1, -2) and stromal-derived factor-1 (SDF-1).1, 2, 3, 4

In addition to soluble factors, activated stem cells also secrete microvesicles (MVs), which are small, spherical membrane fragments shed from the cell surface or secreted from the endosomal compartment and seem to have an important and underappreciated role in improving the function of damaged organs.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 A growing body of evidence suggests that MVs secreted, for example, from HSPCs, MSCs, ASCs, NSCs or CSCs employed in various treatment strategies efficiently inhibit apoptosis of cells residing in the damaged tissues, stimulate their proliferation and promote vascularization.5, 6, 7, 16, 17, 18 These pro-regenerative effects mediated by MVs can be explained by the fact that these small, spherical membrane fragments (i) are enriched in bioactive lipids (for example, sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P)), (ii) display several anti-apoptotic and pro-stimulatory growth factors or cytokines (for example, VEGF or SCF) on their surface and (iii) deliver mRNA, regulatory microRNA (miRNA) and proteins that improve overall cell function. In support of this notion, several species of miRNA that regulate cell survival and angiogenesis (for example, mir126 and mir130) have been identified in CD34+ HSPC-derived MVs.3

Such cell-derived paracrine signals may explain why, after applying various types of stem cells, the final therapeutic benefits are similar. For example, coronary infusion or local delivery of CD34+ or CD133+ HSPCs, MSCs, SMMs or CSCs following heart infarct often yield a similar improvement in left ventricular ejection fraction.4, 19, 20, 21 This argues against transdifferentiation of cells employed for therapy into cardiomyocytes and strongly supports a role for paracrine effects.

These observations should prompt investigators to develop novel therapeutic strategies that harness these mechanisms to ensure more efficient outcomes than current therapies. This could be achieved, for example, by modification of cells employed in regenerative medicine to become better sources of paracrine signals. On the other hand, the therapeutic application of large-scale, modified MVs, instead of whole cells, is emerging as an exciting new concept in regenerative medicine.

Regenerative medicine is searching for a holy grail

The field of regenerative medicine is searching for stem cells that can be safely and efficiently employed for regeneration of damaged organs.22, 23, 24 The ideal would be pluripotent stem cells, which, according to their definition, have a broad potential to differentiate into cells from all three germ layers (meso-, ecto- and endoderm). However, true pluripotent stem cells in adult tissues are extremely rare and are still waiting to be tested in the clinic. In the meantime, various types of stem cells isolated from adult tissues are being employed in several in vivo experiments 25, 26, 27, 28, 29, 30, 31 and clinical trials 19, 20, 21, 22, 23 aimed at regeneration of damaged organs (for example, myocardial infarction, liver damage, ischemic kidney failure or stroke). Such cells are isolated most frequently from hematopoietic tissues (such as BM, mPB or umbilical cord blood (HSPCs or MSCs), Figure 1), heart- (CSCs) or skeletal muscle biopsies (SMMs) or ASCs.

Figure 1
figure1

The potential role of cells isolated from BM, mPB and umbilical cord blood in regeneration of non-hematopoietic organs. The hematopoietic tissues are a relatively accessible source of cells that can be employed in regenerative medicine (for example, HSPCs or MSCs). We propose that the major beneficial effects of these cells in regeneration of damaged organs are based mostly on paracrine signals (for example, secretion of growth factors, cytokines, chemokines, bioactive lipids, and miRNA and RNA transfer by MVs). In addition, cells isolated from adult tissues may contain a small admixture of true pluripotent stem cells (for example, VSELs) that can differentiate into organ-specific cells. However, because of epigenetic changes in some of the imprinted genes, these cells have a locked cell cycle and remain quiescent. Without appropriate modulation of the expression of imprinted genes, their contribution to cells in damaged organs is not sufficient. Pluripotent cells must first be purified in therapeutically significant quantities from adult tissues and efficiently expanded ex vivo before they can be successfully employed in the clinic.

Interestingly, while some beneficial effects have been reported following cell-based therapies, there is no solid evidence, particularly in humans, that cells employed to regenerate damaged solid organs (for example, HSPCs, MSCs or ASCs) give rise to significant organ-specific cell populations (for example, functional cardiomyocytes in heart, hepatocytes in liver or tubular epithelium cells in kidney). Overall, the concept proposed a few years ago that stem cells (for example, HSPCs or MSCs) are plastic and may extensively transdifferentiate into cells from different germ layers lacks solid experimental support and was never satisfactorily confirmed in independent laboratories.32, 33, 34 As a result, the concept of stem cell plasticity or transdifferentiation has been challenged and some positive effects of stem cell therapies have been explained by alternative mechanisms (Table 1).

Table 1 Alternative explanations for the phenomenon of transdifferentiation or plasticity of BM, mPB, or UCB-derived HSPCs employed in regenerative medicine

First, it is possible that some of the stem cell plasticity data can be explained by the phenomenon of cell fusion.35 For example, the ‘new cardiomyocytes’ identified in damaged heart that express markers of cells employed for treatment (for example, HSPCs or MSCs) could be heterokaryons, the result of the fusion of infused cells with cardiomyocytes in the damaged heart. However, cell fusion is an extremely rare event that cannot fully account for the extensive positive transdifferentiation/plasticity data claimed in several reports.36

Second, cells employed for therapy derived, for example, from BM, mPB or umbilical cord blood, may, from the beginning, contain heterogeneous populations of stem cells (Figure 1). This simple fact was, unfortunately, not taken into careful consideration in the first optimistic reports that purported to demonstrate stem cell plasticity.25, 26, 27, 28, 29, 30, 31 We argued from the beginning that studies that did not address this possibility when employing donor-derived BM, mPB or umbilical cord blood cells for the regeneration of non-hematopoietic tissues (for example, heart or liver) may result in misleading interpretations.25, 26, 27, 28, 29, 30, 31 Cells isolated from hematopoietic tissues are enriched for several types of non-hematopoietic stem/progenitor cells, including endothelial progenitor cells, as well as a population of very small embryonic-like stem cells (VSELs).37 These latter stem cells are pluripotent and in appropriate experimental models may differentiate into various tissues.38 However, because of molecular control mechanisms involving somatic imprinted genes that protect them from unleashing proliferation, the differentiation of un-manipulated VSELs to cells in damaged tissues is very rare. The key to successful application of VSELs in regenerative medicine is reestablishment of their proper imprinting.39, 40

Finally, positive effects observed following cell therapies might be explained by the involvement of paracrine effects. As mentioned above, we envision that paracrine effects have a major role in current therapies in regenerative medicine in which stem cells or even more differentiated cells are employed to regenerate damaged organs.

Stem cells as a source of growth factors, cytokines, chemokines and bioactive lipids

It is logical that the repertoire of secreted, anti-apoptotic, proliferation-stimulating and pro-angiopoietic factors varies with the stem cell type to be employed for treatment. These paracrine factors have an important role in regeneration of damaged tissues and recent data show that conditioned media harvested from stem cells (for example, HSPCs or ASCs) can even replace intact cells in cellular therapies.41

In further support of this notion, purified normal human BM- and mPB-derived CD34+ HSPCs express mRNA for various growth factors, cytokines and chemokines, and many of these factors were detected by enzyme-linked immunosorbent assay in media conditioned by these cells.1, 2, 3, 4 Accordingly, we found mRNA transcripts for numerous growth factors (SCF, FLT3 ligand, fibroblast growth factor-2, VEGF, HGF, IGF-1 and thrombopoietin), cytokines (tumor necrosis factor-α, Fas-ligand, interferon-α, interleukin (IL)-1, and IL-16) and chemokines (macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted), monocyte chemotactic protein-2, monocyte chemotactic protein-3, monocyte chemotactic protein-4, IL-8, interferon gamma-induced protein-10, and platelet factor-4) and confirmed the presence of VEGF, HGF, fibroblast growth factor-2, SCF, FLT3 ligand, thrombopoietin, IL-16, IGF-1, transforming growth factor-β1, transforming growth factor-β2, RANTES, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, IL-8 and platelet factor-4 proteins in media conditioned by these cells by employing sensitive enzyme-linked immunosorbent assay assays. Moreover, in vitro functional studies revealed that media conditioned by CD34+ cells may inhibit apoptosis, stimulate proliferation and chemo-attract several types of cells, including endothelial cells.1, 2 These initial observations were recently confirmed in an elegant study by another group.3 Interestingly, CD34+ cells were also demonstrated to express SDF-1, which is an important homing factor for circulating stem cells, including endothelial progenitor cells.4

Like CD34+ HSPCs, MSCs also secrete several growth factors, cytokines and chemokines,42 which is easily explained, because MSCs support and regulate BM hematopoiesis. Furthermore, in addition to BM-derived MSCs, MSCs isolated from other sources, such as adipose tissue or umbilical cord blood, are also a rich source of various paracrine factors, which are released at much higher levels from stressed MSCs removed from their normal microenvironment, as seen, for example, during isolation.

Interestingly, in a recent work that employed ASCs to regenerate a mouse model of lung tissue microvascular injury, it was demonstrated that conditioned media isolated from these cells had a similar protective effect as intact ASCs.41 This strongly argues against transdifferentiation of ASCs and supports a major involvement of paracrine effects.

The secretion of soluble factors from other types of cells employed in regenerative medicine (for example, CSCs, NSCs and SMMs) awaits careful evaluation to better explain the therapeutic effects of these cell types.

Stem cells as a source of MVs

Both prokaryotic and eukaryotic cells communicate and exchange information by employing different cell–cell contact mechanisms involving secreted peptides, bioactive lipids, nucleotides, as well as interactions mediated by sets of specialized adhesion molecules and their ligands. However, growing attention is now being focused on cell-to-cell communication that involves mentioned above MVs,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 a mechanism that for many years has been largely overlooked. Mounting evidence demonstrates that several cell types employed in regenerative medicine for therapy of damaged organs, such as HSPCs, MSCs, SMMs, ASCs, CSCs and NSCs, are a rich source of MVs. Moreover, recent data show that MVs can even replace intact cells to improve the function of damaged organs in several tissue injury models.6, 7, 18

MVs are shed from the cell surface of normal healthy or damaged cells during membrane blebbing and ‘hijack’ both membrane components and the engulfed cytoplasmic contents.5, 6, 7, 8 Shedding of membrane-derived MVs is a physiological phenomenon that accompanies cell activation and growth. Interestingly, rapidly proliferating cells tend to secrete more MVs than slowly growing ones. Generally, the number of MVs shed from cells, including stem cells, increases upon (i) cell activation, (ii) hypoxia or irradiation, (iii) oxidative injury, (iv) exposure to proteins from the activated complement cascade and (v) exposure to shear force stress.9, 10, 11, 12, 13, 14, 15 Any of these conditions could be employed to increase the release of MVs from therapeutic cells.

Another source of MVs is the intracellular endosomal membrane compartment. These MVs, termed exosomes, are usually released from cells as secretory granules during the process of exocytosis.9, 10 Although MVs released from the surface membranes during membrane blebbing are relatively large (0.1–1 μm), exosomes are much smaller (30–100 nm) and appear more homogeneous in size. Furthermore, as soluble factors secreted from the Golgi apparatus are transported to the cell surface in exosomes, their secretion is accompanied by the release of exosomes.9, 10 Therefore, in conditioned media harvested from the cells, both soluble factors and MVs are always simultaneously present.

There is no doubt that the biological significance of MVs has for many years been largely overlooked, and MVs have been regarded, like apoptotic bodies, as mere cellular fragments or debris. However, it is already acknowledged that MVs are secreted or shed by healthy and not dying cells, are much smaller in size than apoptotic bodies, and do not contain fragments of nuclei loaded with nuclear DNA, which is a common feature of apoptotic bodies. MVs not only contain numerous proteins and lipids similar to those present in the membranes of the cells from which they originate, but because MV membranes engulf some cytoplasm during membrane blebbing, they may also contain intracellular proteins,17 mRNA,17 regulatory miRNA,18 and even intact organelles, such as mitochondria.43 In this transfer of mRNA or proteins, MVs act as a naturally engineered liposome. Interestingly, it has been demonstrated that mRNA and miRNA molecules are somehow preferentially enriched within MVs by a mechanism that involves docking proteins. Evidence has accumulated that cells under steady-state conditions tend to store mRNA and miRNA for later utilization under stress conditions. This explains why they can release these molecules into the extracellular space ‘encapsulated’ within MVs.

A novel role for MV-derived paracrine signals in preventing apoptosis and promoting proliferation of cells in damaged tissues

Stem cell-derived MVs may affect cell function in damaged organs by horizontal transfer of proteins, mRNA and miRNA.7, 17, 44 These biologically active components of MVs depend on the cell of origin. However, cellular components that are commonly shared between cells, for example, bioactive lipid components of cell membranes, including S1P, C1P and lysophosphatidylic acid, are highly enriched in all types of MVs. In particular, as S1P and C1P inhibit cell apoptosis and stimulate angiogenesis,45, 46 they can be envisioned as important paracrine factors delivered by all types of MVs.

In addition to the release of soluble factors, the shedding of MVs by cells employed for therapy explains many of the beneficial effects observed following stem cell therapy in regenerative medicine. In particular, horizontal transfer of mRNA and miRNA by MVs has an important role in understanding their pro-regenerative properties. From an historical point of view, the phenomenon of horizontal transfer of mRNA between cells was first demonstrated in a model of expansion of murine HSPCs in the presence of murine embryonic stem cell (ESC)-derived MVs.17 As reported in that study, murine ESC-derived MVs in serum-free cultures significantly enhanced survival, improved expansion of murine HSPCs and upregulated the expression of early pluripotency markers (Oct-4, Nanog and Rex-1) and early hematopoietic stem cells markers (Scl, HoxB4 and GATA2) in these cells. Further molecular analysis revealed that ESC-derived MVs are selectively highly enriched in mRNA for several pluripotency transcription factors compared with parental ESCs and that these mRNAs are delivered/shuttled to HSPCs and translated into the corresponding proteins. These positive biological effects of ESC-derived MVs on HSPCs were inhibited after heat inactivation or pre-treatment with RNAse, indicating a major involvement of protein and mRNA components in the observed phenomena.17

This novel mechanism has been subsequently confirmed in several other elegant studies. For example, it has been reported that rat BM-derived HSPCs changed their phenotype in co-cultures with murine lung tissue-derived MVs,18 which was demonstrated in vitro in a co-culture system in which BM cells and lung cells were separated by an MV-permeable, but intact cell-impermeable, membrane. The MV-transferred murine mRNAs encoding pulmonary epithelial cell-specific surfactants B and C were detected in the rat cells using species-specific primers. Furthermore, a similar mechanism of transfer of organ-specific mRNA into BM cells has been demonstrated for MVs derived from brain, heart and liver, suggesting that MV-mediated cellular phenotype change is a universal phenomenon.6 All this clearly supports the conclusion that transfer of both mRNA and miRNA by MVs may affect the phenotype and biology of target cells.

Based on this mechanism, proteins, mRNA and miRNA from cells employed for therapy (for example, bone marrow mononuclear cells, HSPCs, ASCs or MSCs) could be delivered by MVs to the cells in damaged organs,6, 7, 18, 47 giving the false impression of transdifferentiation and occurrence of donor–recipient chimerism. On the other hand, this mechanism may have beneficial effects in recovery and regeneration of damaged tissues, because MVs may deliver important survival and angiopoietic signals that improve the function of damaged organs.1, 2, 3, 4, 5, 48

As shown in Figure 2, this underappreciated mechanism may explain many of the positive effects of stem cell therapies currently employed in cardiology, nephrology and neurology. More specifically, we propose that these beneficial effects rely mostly on paracrine secretion of growth factors, cytokines and chemokines, and, most importantly, on secretion of pro-regenerative signals delivered by MVs derived from cells employed for therapy.

Figure 2
figure2

Using a heart infarct model, two possible scenarios illustrating the beneficial effects of stem cell therapies in regenerative medicine. Left panel (first scenario). Cells employed for therapy (for example, HSCs or MSCs) may theoretically transdifferentiate into cardiomyocytes. However, if this occurs at all, it is a very rare and random phenomenon and is not well substantiated by current experimental data. Right panel (second scenario). Cells employed for therapy (for example, HSCs or MSCs) do not transdifferentiate into cardiomyocytes, but secrete several paracrine factors and shed MVs that inhibit apoptosis in damaged cardiomyocytes, promoting their proliferation and stimulating angiogenesis. Evidence is accumulating that this is a major effect in currently employed stem cell therapies.

While for many years unrecognized, the paracrine effects of cells employed in stem cell therapy are emerging as important mechanisms in stimulating regeneration. Moreover, new potential applications of MVs are emerging based on recent data showing that MVs may replace whole cells for therapy. In support of this possibility, MSC-derived MVs were found to have the same beneficial effect in protecting the kidney against ischemia-reperfusion-induced acute and chronic kidney injury as intact MSCs.18 The investigators observed that a single administration of MVs immediately after kidney ischemia reperfusion injury protected experimental animals from acute kidney failure by inhibiting apoptosis and stimulating tubular epithelial cell proliferation.18 This phenomenon was strongly dependent on mRNA being present in these MVs, as pre-treatment of MVs with RNAse abrogated this effect. Taken together with previous work from this group in which a similar protective effect was observed after infusion of MSCs, these reports suggest that MSCs and MSC-derived MVs show comparable therapeutic effects.18

In a similar study, it was demonstrated that human liver stem cell (LSC)-derived MVs were able to accelerate regeneration of liver in partially hepatectomized rats in which 70% of the liver parenchyma had been removed,49 while in in vitro co-cultures, human LSC-derived MVs stimulated proliferation and induced apoptotic resistance in human and rat hepatocytes, which required internalization of MVs in an α4-integrin-dependent manner. Interestingly, MVs pre-treated with RNase did not show any beneficial effects, which suggest the importance of an mRNA- and miRNA-dependent transfer mechanism to overexpress appropriate anti-apoptotic genes in the target cells. In strong support of this interpretation, both microarray analysis and quantitative reverse transcriptase-PCR demonstrated that human LSC-derived MVs are able to transfer mRNA species for genes involved in the control of transcription, translation, proliferation and apoptosis. More important, when these MVs were administered in vivo into hepatectomized rats, they accelerated the morphological and functional recovery of liver, and this pro-regenerative effect associated with an increase in hepatocyte proliferation was abolished when MVs were pre-treated with RNase.49

These experiments imply that MVs and their mRNA and miRNA cargo are important and long-underappreciated players in cellular therapies involving HSPCs and MSCs. This also shows that MVs are an important source of several mRNA- and miRNA-encoded pro-proliferation and anti-apoptotic factors. Nevertheless, the mechanisms that govern the selectivity and enrichment of MVs for appropriate mRNA and miRNA species require further study.

MVs as a potent pro-angiopoietic factor

Improvement in vascularization of the damaged organ (for example, myocardium after heart infarct) is an important step in the regenerative process. Damaged cells secrete several pro-angiopoietic factors, such as SDF-1 and VEGF, which are upregulated in the damaged tissues in a hypoxia-inducible factor-1 alpha-dependent manner.50 In addition, several factors, such as bioactive lipids (for example, S1P or C1P) that possess strong pro-angiopoietic potential,45, 46 are released in a soluble form from the stressed cells or are secreted in MVs. All these factors promote sprouting of endothelial cells from existing vessels, as well as chemoattract endothelial progenitor cells, which circulate in PB and are mobilized in response to organ injury. In addition important source of pro-angiopoietic MVs are stem cells (for example, HSPCs, MSCs and CSCs) that are infused into damaged organ in an attempt to promote regeneration.

In further support of these observations, it has been shown that MVs are truly endowed with strong pro-angiopoietic properties. We have previously reported that bone marrow mononuclear cell-derived MVs directly chemoattract endothelial cells,1, 49 which has been subsequently confirmed for MVs derived from various cell types. Therefore, MVs, regardless from which cells they originate, are very strong chemoattractants for endothelial cells and have been shown to promote formation of endothelial ‘tubes’ in vitro.49 In a recent elegant study, MVs collected from the conditioned media of CD34+ HSPCs isolated from mPB cells replicated the angiogenic activity of CD34+ cells by increasing endothelial cell viability, proliferation and tube formation in Matrigel assays.4 More importantly, in in vivo experiments, the same CD34+ HSPC-derived MVs stimulated angiogenesis in both Matrigel plug and corneal assays. In control experiments, MVs from PB mononuclear cells depleted of CD34+ HSPCs had no angiogenic activity, which demonstrates the uniqueness of stem cell-derived MVs compared with those derived from more differentiated cells.4

Based on these findings, stem cells delivered to the damaged organ lead to release of S1P- and C1P-enriched MVs that are endowed with strong pro-angiopoietic properties. Furthermore, as MVs are enriched for mRNA and regulatory miRNA, further studies are needed to identify, which MV-delivered RNA species promote angiogenesis.

Toward harnessing stem cell paracrine effects in regenerative medicine

A growing body of evidence suggests that paracrine effects of cells employed as therapeutics in regenerative medicine could be more efficiently exploited to optimize cell-based therapies. This could be achieved by ex vivo manipulation of cells to enhance secretion of pro-regenerative factors and the development of novel therapeutic strategies in which large-scale, ex vivo-generated, modified MVs replace intact cells.

Increase in release of pro-regenerative factors

As autocrine secretion of various soluble factors, as well as MVs, by stem cells can be increased during the stress response, one can envision different approaches to augmenting this phenomenon. Theoretically, this could be accomplished by (i) exposure of cells to hypoxia before infusion and delivery to the injured organ, (ii) transduction of these cells by expression vectors that increase secretion of pro-angiopoietic factors (for example, VEGF or fibroblast growth factor-2), (iii) modulation of expression of miRNAs that regulate transcription of anti-apoptotic or pro-angiopoietic genes and (iv) exposure of these cells to complement cascade cleavage fragments or cytokines that promote autocrine release of appropriate paracrine peptides or bioactive lipids.

Development of engineered MVs for regenerative medicine therapies

Based on the fact that MVs have similar beneficial effects in regenerative therapy as the intact cells from which they are derived,6, 7, 18 it should be possible to produce MVs on a large scale and even to modify their composition. Several possibilities for how to modify MVs are shown in Figure 3. Overall, MVs for application in regenerative medicine could be isolated from appropriate generator cells (for example, MSCs) expanded ex vivo. Release of MVs by founder cells could be enhanced by proper manipulation of culture conditions (for example, hypoxia), and custom-engineered MVs more suitable for therapy could be produced by genetic modification of the founder cells.

Figure 3
figure3

Different approaches to generating more efficient pro-regenerative MVs in vitro. MVs could be harvested from large-scale in vitro cultures of MV-producing cell lines. Such cell lines may be modified to obtain MVs that (i) do not express HLA antigens, (ii) are enriched in growth factors, cytokines and chemokines that promote regeneration of damaged organs, (iii) are enriched in mRNA and regulatory miRNA facilitating regeneration of damaged tissues and/or promoting angiogenesis and (iv) display molecules that direct them to, and subsequently retain them in, damaged tissues.

First, as depicted in Figure 3, it should be possible to expand MV-producing cell lines that lack genes encoding histocompatibility antigens. This approach would minimalize the possibility of cross-immunization with donor HLA antigens. Second, MV producer cell lines could be transduced with genes that overexpress on the cell surface (i) peptides that protect target cells in damaged organs from apoptosis and stimulate proliferation of residual remaining cell populations (for example, SCF or Notch ligands) or (ii) factors that effectively induce angiogenesis (for example, VEGF, fibroblast growth factor-2 or SDF-1). Third, we speculate that MVs derived from cells in hypoxic conditions would be enriched in mRNAs and miRNAs that promote angiogenesis. Furthermore, producer cell lines could be enriched for mRNA and regulatory miRNA species that, after delivery to the damaged tissues, would promote regeneration. Finally, we envision that MV producer cell lines could be enriched for molecules that facilitate their tropism to the damaged organ and subsequently promote retention of MVs in the damaged tissues. In the example mentioned above, human LSC-derived MVs stimulated proliferation and induced apoptotic resistance in human and rat hepatocytes, which required their internalization in an α4-integrin-dependent manner.49

MV-based therapies also open up new possibilities for clinical applications of induced pluripotent stem cells (iPSCs). As in vivo application of iPSCs is still limited by the high risk of teratoma formation by these cells,22, 24 MVs from patient-derived iPSCs could be employed as a novel generation of therapeutics to rescue damaged organs and tissues. Based on this possibility, we envision that patient-derived iPSCs could be employed as MV-producing cells. Moreover, taking advantage of the recently proposed epigenetic memory of cells employed for generation of iPSCs, one can also envision that, for example, MVs from keratinocyte-derived iPSCs would preferentially affect regeneration of damaged skin (for example, after burns), or MVs isolated from supernatants of cardiomyocyte-derived iPSCs would have advantages in regeneration of damaged myocardium.

Conclusions

It is evident that by applying various types of stem cells to damaged organs, for example, to treat heart infarct, the final outcomes are often similar regardless of the cell type employed. To explain this paradox, strong evidence has accumulated that soluble autocrine factors and MVs are abundantly secreted by stem cells infused locally or systemically to rescue damaged tissues.7, 8, 17, 18, 49 In several elegant studies, it has been demonstrated that infusion of conditioned media from cells41 or delivery of MVs18, 49 has the same pro-regenerative potential as infusion of intact cells that are the source of these paracrine factors.

Based on these data, the rescue of damaged organ or tissue following infusion of cells, which was previously attributed to stem cell plasticity or transdifferentiation, is better explained by the paracrine effects of growth factors, cytokines, chemokines and bioactive lipids secreted from the infused or locally delivered stem cells, as well as by secretion of pro-regenerative MVs by these cells.

These remarkable paracrine properties of cellular therapeutics should have an impact in the development of new strategies in regenerative medicine in which stem cells would be (1) engineered to overexpress greater levels of anti-apoptotic and pro-angiopoietic factors (growth factors, cytokines, surface molecules, mRNA and miRNA) and (2) MVs would be harvested from large-scale in vitro cultures of these MV-producing cells. Such custom-engineered stem cells and ‘super MVs’ could both inhibit apoptosis of target cells and promote neovascularization of damaged tissues as a new class of cell-derived therapeutics in regenerative medicine.

These bold new strategies could be developed to improve current clinical trial outcomes using adult cells. However, the ultimate goal of regenerative medicine is to employ pluripotent stem cells that have broad differentiation potential for regeneration. Such cells (for example, VSELs)37, 40 have to be purified in therapeutically significant quantities from adult tissues and efficiently expanded ex vivo before they can be employed in the clinic.

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Acknowledgements

This work was supported by NIH Grant R01 DK074720, the Stella and Henry Endowment, and the European Union structural funds (Innovative Economy Operational Program POIG.01.01.02-00-109/09-00) to MZR.

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Correspondence to M Z Ratajczak.

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Ratajczak, M., Kucia, M., Jadczyk, T. et al. Pivotal role of paracrine effects in stem cell therapies in regenerative medicine: can we translate stem cell-secreted paracrine factors and microvesicles into better therapeutic strategies?. Leukemia 26, 1166–1173 (2012). https://doi.org/10.1038/leu.2011.389

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Keywords

  • stem cell therapies
  • paracrine effects
  • microvesicles
  • exosomes
  • regenerative medicine

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