Experimental Hematology
Volume 38, Issue 7 , Pages 581-592, July 2010

Stem cell plasticity revisited: The continuum marrow model and phenotypic changes mediated by microvesicles

  • Peter J. Quesenberry

      Affiliations

    • Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI., USA
    • ,
  • Mark S. Dooner

      Affiliations

    • Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI., USA
    • ,
  • Jason M. Aliotta

      Affiliations

    • Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI., USA
    • Division of Pulmonary, Sleep and Critical Care Medicine, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI., USA
    • Corresponding Author InformationOffprint requests to: Jason M. Aliotta, M.D., Division of Hematology/Oncology, Rhode Island Hospital, 3rd Floor George Building, 593 Eddy Street, Providence, RI 02903

Received 27 February 2010; received in revised form 27 February 2010; accepted 31 March 2010. published online 12 April 2010.

Article Outline

The phenotype of marrow hematopoietic stem cells is determined by cell-cycle state and microvesicle entry into the stem cells. The stem cell population is continually changing based on cell-cycle transit and can only be defined on a population basis. Purification of marrow stem cells only addresses the heterogeneity of these populations. When whole marrow is studied, the long-term repopulating stem cells are in active cell cycle. However, with some variability, when highly purified stem cells are studied, the cells appear to be dormant. Thus, the study of purified stem cells is intrinsically misleading. Tissue-derived microvesicles enhanced by injury effect the phenotype of different cell classes. We propose that previously described stem cell plasticity is due to microvesicle modulation. We further propose a stem cell population model in which the individual cell phenotypes continually change, but the population phenotype is relatively stable. This, in turn, is modulated by microvesicle and microenvironmental influences.

 

We and others have observed that marrow cell populations are intrinsically heterogeneous and continually changing. This phenotypic lability extends to the capacity of marrow cells to assume the phenotype of other hematopoietic cells or nonhematopoietic cells and appears to be tightly linked to the cell-cycle status of the marrow stem cell.

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Continuum model and cell cycle: Intrahematopoietic plasticity 

All proliferating cell populations are intrinsically heterogeneous and must continually change phenotypes as they progress through cell cycle. Thus, a proliferating population can be defined on a population basis only; clonal studies will only address the degree of heterogeneity of a stem cell population. These concepts were elegantly addressed by Till, McCulloch, and Siminovitch in the 1960s, when they compared the nature of colony-forming unit spleen [1], the first described stem cell, to radioisotopes [2]. An isotope has a very predictable half-life. However, the individual nuclei that compose it have markedly varied half-lives, making them totally heterogeneous. This is a reasonable view of the nature of adult marrow stem cells; they can only be appropriately defined on a population basis.

A number of studies from our laboratory have shown that the phenotype of the lineage-negative rhodamine-low Hoechst-low (and, to a lesser extent, the lineage-negative Sca-1+) stem cell continuously changes, in a reversible fashion, with cell-cycle passage 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. Characteristics studied have included short and long-term engraftment into lethally irradiated mice, progenitor numbers, differentiation into granulocytes and megakaryocytes, expression of adhesion protein and cytokine receptor genes, global gene expression, expression of cell cycle genes, capacity to convert to pulmonary epithelial cells and, most recently, the capacity to take up microvesicles. These characteristics vary with cycle phase and are reversible (or at least continue to modulate). These observations led to a continuum theory of stem cell biology in which the phenotype of the adult marrow stem cell is continuously changing based, at least in part, on the cell-cycle position of the stem cell 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. The applicability of this model to normal steady-state hematopoiesis depends on the assumption that the adult marrow stem cell is an actively cycling cell.

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Cell-cycle status of marrow stem cells 

The extant literature on this point is discordant, with the general consensus being that the adult marrow stem cell is “dormant” or quiescent, but with some reports indicating that it is an actively cycling cell. The colony-forming unit spleen (CFU-S), the original clonal stem cell assay [1], was extensively studied and it was generally found to be relatively quiescent with S-phase values of ≤10% 29, 30, 31, 32, 33. A number of studies showed higher S-phase values for CFU-S, ranging from 16% to 48% 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45. Our own work showed varying results from no killing with hydroxyurea or tritiated thymidine to killing rates of up to 25% [45]. The work by Necas and Znojil [46] is particularly informative. They determined the number of CFU-S and the fraction synthesizing DNA in individual normal mice of several inbred strains and the data obtained during a period of 5 years was subjected to analysis of variance. Large differences were shown to exist in the number of CFU-S in the femoral bone marrow of individual mice measured on the same day. These differences were greater if measurements were performed on different days. The fraction of DNA synthesizing CFU-S was, on average, 30% in these normal mice, but the range of measurements on both the same and different days was 0% to 60%. The authors measured CFU-S from day 7 to day 12 and found similar results. This work led to a proposal that there may be “bursts” of CFU-S proliferation over time, not on a circadian basis, but rather stochastic in nature.

A major focus of more recent studies of marrow renewal stem cells has been on highly purified marrow stem cells. In general, marrow is depleted of differentiated cells using differentiated cell-specific antibodies to surface epitopes and magnetic bead separation. This is followed by staining of lineage-negative cells for stem cell−related surface antigens and separation by fluorescent-activated cell sorting 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67. Studies on purified stem cells have given different views of the cell-cycle status of the primitive marrow stem cells. Work by Bradford and colleagues [68], confirmed by Cheshier and colleagues [56] and Pang and colleagues [69], suggested that primitive stem cells were a continuously cycling population. Work on long-term hematopoietic stem cells (LT-HSC) done by Fleming and colleagues [70] suggested that >20% of these cells were in cell cycle at isolation. Cheshier and colleagues [56] proposed that the population is continuously in cycle and transits cycle fairly rapidly, with 50% of LT-HSC showing bromodeoxyuridine (BrdU) incorporation by 6 days. They estimated that 8% of stem cells entered cycle each day in in vivo experiments, while other work proposed a stem cell turnover time of 154 days. This latter study by Wilson and colleagues [71] actually produced in vivo BrdU data more consistent with a rapid turnover, but explained this by supposed BrdU toxicity to hematopoietic cells with secondary effects. However, carefully conducted experiments in the Cheshier studies [56] did not show any BrdU toxicity.

Studies on the marrow stem cell side population indicated that S/G2/M cells had the same long-term repopulating capacity as G0 cells [72]. Other work using a variety of approaches on different stem cell populations all indicate that a percentage were in S/G2/M at the time of interrogation 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 56, 68, 69, 70, 73. Our own studies (Quesenberry PJ, Johnson K, Dooner MS, unpublished data, 2010), employing Hoechst/Pyronin separations, have shown that the cell that gives long-term marrow repopulation in whole unseparated marrow is, in fact, actively cycling. Although, with a great deal of variation, the purified LT-HSC, as described by Christensen and colleagues,52 had a small percentage (to none) of stem cells in S/G2/M.

There are several important implications of these results. First, stem cells purified by antibody-epitope selection are not representative of the stem cells in whole marrow. The epitope-selected cells represent specific and relatively rare subsets of stem cells that exclude proliferating and other stem cells from consideration. Although the characteristics of a particular epitope-defined stem cells may exist at one point in time or cycle, these characteristics do not persist as that particular cell will continue to change its characteristics, eventually returning to its original phenotype. Thus, a G0 Lin−/Sca-1+/c-kit+/slam+ cell may be present at one time in G0, but later, perhaps a few hours into G1, its phenotype may be that of a megakaryocyte progenitor or even a monocyte. Fluxes of phenotypes are obligatory if the cell is in active cell cycle. The overall description of this situation is a stable stem cell population in which the individual entities (or cells) are continually changing, but the whole maintains its general aspect. This is very similar to the radioisotope situation described here. The challenge in the stem cell field is to define the total stem cell population, i.e., those cells that maintain a potential to assume the characteristics of a long-term multipotent engrafting stem cell. One might start with all cells that maintain a capacity for proliferation. This would only exclude anucleated erythrocytes and mature polymorphonuclear granulocytes. The true stem cell population might only be the lineage-negative cells, but this remains as speculation. We propose here that marrow hematopoietic repopulating stem cells are actively cycling and continuously changing. The classically recognized LT-HSC or LT-HSC−slam phenotypes are simply subpopulations of the true stem cell population. This population contains multiple stem/progenitor cells and possibly differentiated cell phenotypes previously placed in a hierarchy. Thus, there is impressive plasticity within the hematopoietic marrow system. A conceptual model is presented in Figure 1.

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  • Figure 1 

    Population model of stem cell phenotype. Numbers and circles represent different phenotypic cell classes. The concept here is that the phenotypes change at different points in cell cycle and eventually return to the original phenotype. For example, cell #1 is a long-term repopulating cell in G2/M/G0 and becomes a different cell in G1, a colony-forming unit megakaryocyte (CFU-Meg) in S phase, then returns to the original phenotype. In this model, the individual cell phenotype continuously changes while the population remains stable. CLP = common lymphoid progenitor; ETC = et cetera.

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Stem cell plasticity: The marrow to nonhematopoietic variety 

The capacity of murine marrow cells to form nonhematopoietic cells and tissues after transplantation into irradiated mice was termed stem cell plasticity. The classic proof of principle came from the studies on the fumarylacetoacetate hydroxylase−deficient mouse; a mouse afflicted with a fatal tyrosinemia [74]. This can be controlled with administration of the drug 2-(2-nitro-4-fluoromethylbenzoyl)-1, 3-cyclohexanedione. Repeated withdrawal of the drug provides both injury and selection. In fumarylacetoacetate hydroxylase−negative mice transplanted with β-galactosidase−positive transgenic marrow, and subjected to repeated withdrawal of 2-(2-nitro-4-fluoromethylbenzoyl)-1, 3-cyclohexanedione, large areas of diseased hepatic tissue were replaced with normal β-galactosidase−positive donor-derived hepatocytes. In addition, some mice were cured of this fatal disease. Furthermore, very convincing studies have shown that these marrow-to-hepatocyte conversions are due to cell fusion [75]. A large number of subsequent studies have shown that host tissue, usually under injury circumstances, can be partially replaced by cells derived from transplanted marrow. This led to the stem cell plasticity controversy, a rather meaningless exercise that we have addressed previously in a perspective titled “Ignoratio Elenchi” (irrelevant conclusions or red herrings) [76]. Proposals were made that for results in this area of investigation to be taken seriously, they had to be “robust,” they had to be on a clonal basis (which only shows heterogeneity), they had to show function (which was never adequately defined), and, the biggest red herring of all, they could not involve cell fusion. Why this latter point became an issue is unclear, but it unfortunately became a major negative feature of grant and manuscript reviews. There were a few “negative” studies that appeared to be designed to obtain negative results and that represented marginal science.

Nonetheless, there is now little question that after marrow transplantation, cells can be found in many different tissues, lung and liver being prominent here. These cells have defining characteristics of the specific tissue under consideration, but also carry markers indicating origin from the transplanted marrow cells. In some instances, cell fusion may have been involved, but in others it was not. A summary of some of this early work showing marrow conversions to liver, lung, and skeletal muscle is presented in Table 1.

Table 1. Marrow to muscle and lung conversions
TissueInjuryDonor cellsConversion result tissue cells (%)Reference
Skeletal muscleRadiation and exerciseGFP-marrow3.5 (peak)77
TBI/mdx mouseSpleen and marrow0.2 (approx)79
TBI/mdx mouseMarrow side population1−1079
TBI/cardiotoxin injury anterior tibialisGFP-marrow1-280
(+direct injection of lineage-negative marrow cells)Intra-arterial12.581
α-Sarcoglycan null dystrophic miceMesangioblast stem cells5082
Lung700−950 cGyGFP marrow, mononuclear cells or side population1−7 (mixed population, type I pneumocytes)83
NonirradiatedRosa MAPC3−584
(+250 cGy)Fr25/Lin−(10)85
1,050 cGyCytokine treated GFP marrow20% type II pneumocytes86
900 cGy, cardiotoxin or bleomycin lung injury with G-CSF mobilization (×2) 35 (peak)

GFP = green fluorescent protein; G-CSF = granulocyte colony-stimulating factor; MAPC = multipotent adult progenitor cell; mdx = dystrophin-deficient mouse; TBI = total body irradiation.

In continuing studies, virtually every tissue in the body has been found to be subject to marrow conversions or stem cell plasticity. There have been >30 articles on marrow-to-lung conversions and, although the percent conversions varied widely, all studies have demonstrated this. Many studies have also addressed whether cell fusion was the mechanism underlying the observed plasticity. A summary of some of these is presented in Table 2, Table 3.

Table 2. Fusion demonstrated in converted cells
Tissue/cellModel/detectionReference
HepatocyteFah+/+ from Fancc−/− into Fah−/− with withdrawal. 50% conversion rate. Purified repopulating cells were heterozygous Fah+/+ and Fanc−/−.74
HepatocyteFah+/+ from ROSA26 female marrow into male Fah−/−. Cytogenetic analysis of LacZ+ marrow-derived hepatocytes – most with Y chromosome. Karyotypes Fah+/+ 80XXXY or 120 XXXXYY.75
Purkinje neuronGFP to adult mice and both donor and host nuclei found, the Purkinje neurons were stable heterokaryons.87
Purkinje neuron, cardiomyocyte, hepatocyteUsed Cre/lox recombinase system to show that in marrow transplanted mice all detectable contributions of marrow to nonhematopoietic cell types arose through cell fusion.88
Skeletal muscleMurine cardiotoxin injury model male to female, female to male or Rosa β-galactosidase to GFP muscle fibers show both donor and recipient phenotypes. However, mononuclear satellite cells with donor markers suggest conversion to satellite cells occurs without fusion.80, 81

GFP = green fluorescent protein; Fah = fumaryl acetoacetate hydrolase; Fanc = Fanconi anemia complementation group; Fancc = Fanconi anemia complementation group, c protein; LacZ = β-galactosidase.

Table 3. Conversions without fusion
Tissue/cellModel/detectionReference
PancreasRosa-stop lox and GFP female hosts transplanted with insulin-dependent Cre-male marrow. No GFP+ donor cells in islets.89
HepatocyteHuman cord blood to irradiated NOD/SCID mouse. Human hepatocytes with positive protein and chromosome markers, no mouse chromosomes. Conversion rate 1−2%.90
HepatocyteHuman cord blood (USSC) into fetal sheep without injury. 20% conversion rate. Microdissected human hepatocytes had only human protein or PCR product.91
Endothelialc-kit+, Sca-1+, Lin− into irradiated mouse. Donor endothelia in portal vein. Normal ploidy. Also cord blood to mouse with new blood vessel formation in the eye-no fusion.92
Renal mesangial cellsMale GFP marrow to male mice resulted in numerous GFP+ mesangial cells. None had more than one Y chromosome.93
Epithelial cells in lung, skin, and liverCre/lox recombinase system. Transplant Z/EG marrow into Cre expressing mice. No mice expressed GFP indicated that fusion had not occurred.94
Skeletal muscleConverted mononucleated satellite cells precede muscle fiber fusion.78

GFP = green fluorescent protein; NOD/SCID = nonbese diabetic severe combined immunodeficient; PCR = polymerase chain reaction.

Ogawa and colleagues [95] and Lang and colleagues [96] have extended these studies by publishing observations that hematopoietic marrow cells were the origin of fibroblasts and myofibroblasts, which can be found in many tissues, including intestine, skin, liver, and lung. In addition, a number of the plasticity studies have shown function.

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Marrow to lung studies in plasticity 

We have focused on the capacity of transplanted murine marrow cells to convert to pulmonary epithelial cells. We initially studied the capacity of engrafted marrow cells to convert to pulmonary epithelial cells in a lethally irradiated mouse model [86]. In these studies, we saw a wide stochastic variation in conversion rates but always saw conversions. Using green fluorescent protein or the Y chromosome (in gender-mismatched transplants) to track transplanted cells, the percentage of bone marrow−derived CD45-negative and cytokeratin-positive or prosurfactant B−positive cells in the lungs transplanted mice varied from 0% in nonirradiated mice to 1.17% to 18.9% in irradiated mice. The variations seen in irradiated mice depended upon the dose of irradiation, with increasing conversions rates with increasing levels of host irradiation. This latter also correlated directly with bone marrow engraftment levels. Other variables that influenced plasticity were the marrow subpopulation infused. Our initial studies showed that the marrow cells that led to conversions were c-kit+, Sca-1+, and lineage-negative. c-kit−, Sca-1−, and lineage-positive cells did not significantly engraft in the lung. Treatment of engrafted host mice with granulocyte colony-stimulating factor also increased the conversion rates of marrow to lung cells, presumably on the basis of stem cell mobilization. Further studies indicated that treatment of the marrow cells prior to infusion with the cytokines interleukin (IL)-3, IL-6, IL-11, and steel factor markedly influenced marrow-to-lung conversions [97]. This correlated with cell-cycle progression of the marrow cells in vitro and peak conversion rates of green fluorescent protein−positive marrow cells to lung cells were seen at the G1/S interface. Here, we saw a threefold increase in cells assuming a nonhematopoietic or pulmonary epithelial cell phenotype. This increase was no longer seen in late S/G2. These data indicated that engrafted marrow cells were capable of converting to significant numbers of pulmonary epithelial cells in the irradiated mouse and suggested that radiation-induced lung injury might be important in this process. This work is summarized in Figure 2.

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  • Figure 2 

    Marrow conversion to epithelial lung cell. This shows conversion of a marrow stem cell phenotype to a pulmonary epithelial cell, which is affected by host irradiation, treatment of host or exogenous marrow cells with granulocyte colony-stimulating factor (G-CSF) and stem cell phenotype.

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Mechanisms underlying marrow conversions to pulmonary epithelial cells or, more accurately, the presence of lung cells with marrow markers after marrow transplantation: The role of microvesicles 

Microvesicle information transfer 

Our studies (which will be outlined in detail) have indicated to us that transfer of cell-derived microvesicles between cells may underlie much of the previously described stem cell plasticity. The exact nature of and nomenclature for microvesicles is still evolving. Particles derived from cells, especially injured cells, have been described repeatedly. Small membrane−enclosed vesicles from platelets or red blood cells were first considered to represent cellular junk and largely dismissed as having little biologic significance. Subsequently, membrane-bound particles have been described as originating from mast cells [98], dendritic cells [99], tumor cells [100], reticulocytes [101], epithelial cells [102], B cells [103], and neural cells [104]. In fact, it is now apparent that these vesicles probably can be derived from virtually all cell types in the body. Membrane-enclosed vesicles derived from a wide variety of cells have been shown to affect the phenotype of putative target cells under different conditions. Different terms have been used to describe these cellular-derived membrane-enclosed entities, including exosomes [105], microvesicles [106], ectosomes [107], membrane particles [108], exosome-like particles [109], and apoptotic vesicles [110]. Vesicles have been characterized by size, density in a sucrose gradient, electron microscopy, sedimentation by ultracentrifugation, lipid composition, main protein markers and intracellular origin [111]. Exosomes are 50 to 80 nm in diameter, endocytic in origin and released into the environment during fusion of multivesiclular bodies with plasma membranes. Microvesicles have been described as being 100 nm to 1 um in diameter and released from surface membranes during membrane blebbing in a calcium flux and calpain-dependent manner. As noted by Théry and colleagues [111], in practice, all vesicle preparations are heterogeneous with different protocols allowing enrichment of one type over another. We have studied vesicles sedimented at 100,000g by ultracentrifugation, which would include both exosomes and microvesicles as classically described, and have found that the mode of electron microscopic tissue preparation changed the morphology dramatically. Cup-shaped vesicles can be seen with one approach and irregularly-shaped and electron dense vesicles with another approach. We will use the generic term microvesicle to encompass these populations of vesicles, realizing the heterogeneity of most reported vesicle populations. The evolution of microvesicles from different cell populations is influenced by hypoxia, shear stress, irradiation, chemotherapy, cytokines, and different drugs, such as acetaminophen (hepatocytes). A particular focus recently has been on the capacity of microvesicles to influence the phenotype of neighboring cells in other tissues. They have been found to transfer CD41, integrins, and CXCR4 110, 112, 113, 114, as well as HIV and prions 115, 116 between cells. Embryonic stem cell−derived microvesicles have been reported to reprogram hematopoietic stem/progenitor cells via the horizontal transfer of messenger RNA (mRNA) and protein [117]. Similarly, tumor-derived microvesicles, which carry several surface determinants and mRNA, can transfer some of these determinants to monocytes [112]. Apoptotic bodies from irradiated Epstein-Barr virus−carrying cell lines have been shown to transfer DNA to a variety of cocultured cells by integrating copies of Epstein-Barr virus, resulting in expression of Epstein-Barr virus−encoded genes EBER and EBNAI in recipient cells at high copy number [118]. Extracts from T lymphocytes containing transcription factor complexes can induce fibroblasts to express lymphoid genes [119]. In addition, endothelial cells exposed to microvesicles derived from endothelial progenitor cells form capillary-like structures both in vitro and in vivo [120]. It is of particular interest that previously described endothelial progenitor cells may, in fact, represent mononuclear cells that have consumed platelet-derived microvesicles [121]. All of these studies indicate a capacity of microvesicles to alter the phenotype of “target” cells toward the phenotype of the microvesicle producing cell.

Microvesicles and marrow to lung conversions 

Jang and colleagues [122] cultured hematopoietic stem cells across from damaged liver cells, but separated from them by a cell impermeable membrane and demonstrated that the marrow cells expressed genes specific for hepatocyte, such as albumin. This was interpreted as humoral induction of differentiation. These findings prompted our own studies, which indicated that it might have represented microvesicle induction of phenotype change [123]. Accordingly, we studied marrow cells cultured across from murine lung cells, which had been exposed to 0, 500, or 1,200 cGy irradiation from 3 hours to 14 days previously. We then assessed the marrow cells for expression of pulmonary epithelial cell-specific mRNA. Our studies indicated that high levels of expression of clara cell−specific protein, surfactant C and surfactant B were seen when marrow cells were exposed for 48 hours or 7 days opposite murine lungs. The highest levels were seen when lungs from mice exposed to 500 cGy 5 days previously were cocultured with marrow. The basic culture system is shown in Figure 3.

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  • Figure 3 

    Marrow-lung coculture. Marrow cells were cocultured across from lung fragments but separated from them by a cell impermeable (0.4 μm) membrane for 2 or 7 days and expression of pulmonary epithelial genes in marrow cells determined by reverse transcription polymerase chain reaction (RT-PCR) analysis.

Further work here showed that cell-free−conditioned media from lung, irradiated or not, would induce pulmonary epithelial cell-specific mRNA production in marrow cells and that the inducing principle was present in the pellet of ultracentrifuged (100,000g) conditioned medium. The pellet contained large numbers of microvesicles, as defined by electron microscopy. There were numerous 100 to 250 nm membrane-bound vesicles; although, in different experiments, smaller vesicles were also seen. These microvesicles could be stained with the supravital membrane dye PKH26 (red fluorescence) and the supravital cytoplasmic dye carboxyfluorescein diacetate succinimyl ester (green fluorescence) and then separated and purified as red/green events by fluorescent-activated cell sorting. These fluorescent-labeled microvesicles were then incubated with marrow cells and a minority of the marrow cells took up the microvesicles. Marrow cells loaded with microvesicles are shown in Figure 4 along with electron microscopic images of these microvesicles.

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  • Figure 4 

    Lung-derived microvesicles. (A−D) shows a marrow cell with incorporated PKH26 and carboxyfluorescein diacetate succinimyl ester−labeled lung-derived microvesicles. (A) merged image; (B) DAPI filter; (C) Texas Red filter; (D) fluorescein isothiocyanate filter. (E) An electron micrograph of fluorescent-activated cell sorting-sorted lung-derived microvesicles. Red bar = 10 μm; black bar = 100 nm.

Further work isolating marrow cells that had taken up fluorescent microvesicles by fluorescent-activated cell sorting and then determining expression of pulmonary epithelial cell-specific mRNAs showed that only marrow cells that had taken up the microvesicles expressed the pulmonary epithelial cell-specific mRNA. Cocultured marrow cells were shown to express prosurfactant-B protein 21 days after a 7-day exposure to irradiated lung fragments. Functional effects of marrow cells cocultured with irradiated lung cells for 7 days were seen. These cells gave higher levels of prosurfactant-C−positive donor cells in host lungs after transplantation, as compared to marrow cells that had not been cocultured. Other investigators have also shown functional effects of microvesicle modulation on target cells. Deregibus and colleagues [120] showed modulation of vascular phenotypes by exposure to endothelial progenitor-derived microvesicles. They demonstrated promotion of endothelial cell survival, proliferation, and organization into capillary-like structures in vitro. In vivo, in severe immunodeficient mice, microvesicle-stimulated endothelial cells organized into patent vessels; this did not happen without microvesicle exposure.

Mechanisms of phenotype change 

Initially, we thought that the observed expression of pulmonary epithelial cell−specific mRNA in marrow cells taking up microvesicles was simply due to the transfer of mRNA in microvesicles to the target cells. We had demonstrated pulmonary epithelial cell−specific mRNA inside the microvesicles, showed that microvesicles entered marrow cells, and that only the marrow cells that contained microvesicles expressed pulmonary epithelial cell−specific mRNA. However, despite some early results suggesting that RNase exposure of microvesicles inhibited pulmonary epithelial cell−specific mRNA in target marrow cells, more recent work indicated that, in most instances, exposure of microvesicles to RNase actually increased expression of pulmonary epithelial cell−specific mRNA in target cells. We found 185 species of microRNA in these microvesicles, with 8 having potential lung-specific targets. Thus, these data could be explained by RNase degradation of inhibitory microRNA.

However, we also observed the persistence of pulmonary epithelial cell−specific mRNA expression in marrow cells after 3 weeks in cytokine-supported culture. This was inconsistent with a simple transfer of mRNA, because we would have expected the RNA to be degraded by this time. We addressed the issue of whether de novo transcription was involved in the observed pulmonary epithelial cell−specific mRNA elevations in target marrow cells. Studies with actinomycin D and α-amantin, both transcriptional inhibitors, showed predominantly increased expression of the pulmonary epithelial cell−specific mRNA in marrow cells that had been cultured with lung-derived microvesicles, suggesting complex transcriptional regulation [124]. In order to address this further, we employed rat/mouse hybrid cocultures. In these experiments, rat lung was cultured opposite mouse marrow and mouse marrow then evaluated for expression of surfactant C or B mRNA expression. Species-specific primers allowed us to determine whether the observed mRNA was of rat or mouse origin. In every case, the mRNA was of both origins, indicating that mRNA was transferred along with transcriptional agents that induced de novo surfactant mRNA production in cultured marrow cells. Thus, the mechanisms underlying the genetic phenotype change of target cells is complex, involving transfer of both mRNA and microRNA and of protein-based transcription factors. These phenomena appear to be universal and tissue-specific as we have shown that murine lung, brain, heart, and liver tissue will all transfer a tissue-specific phenotype, but not the phenotype of other tissues [124]. This concept is shown in Figure 5.

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  • Figure 5 

    Injury induction of microvesicles. Irradiation injures a nonhematopoietic cell that releases bioactive microvesicles containing protein, messenger RNA (mRNA) and microRNA. These microvesicles enter marrow cells and alter their phenotype to that of the cell of microvesicle origin.

We have presented a model of stem cell regulation termed the continuum model, in which the potential of marrow stem cells continually changes with cell-cycle transit. We have also shown that the marrow stem cell is a cycling cell. Studies with murine lung−derived microvesicles and murine marrow have now shown that the capacity to take up microvesicles also varies with cycle phase. Thus, phenotype modulation at the stem cell level involves both cell-cycle and microvesicle phenotype change. This model is presented in Figure 6.

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  • Figure 6 

    Effect of microvesicles on the stem cell population model. This indicates that microvesicles impose a different order of phenotypic change on stem cells progressing through a cell-cycle−related stem cell continuum. CLP = common lymphoid progenitor; ETC = et cetera.

One can envision both intrahematopoietic and extrahematopoietic cell systems as systems that have a continually changing potential that will only be expressed if there is an appropriate interrogation. In addition, entry of microvesicles into hematopoietic cells varies with cell-cycle phase and resets the potentials. One can envision this as represented in a modulogram (Fig. 7).

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  • Figure 7 

    Stem cell modulogram. Stem cells progressing though cycle continuously change individual cell phenotypes while maintaining the population phenotype. This is further modulated by microvesicle cell entry and the final cell fate determined by interactions with different microenvironments.

Cancer stem cells and microvesicles 

The microvesicle cell modulation also holds for cancer cells. Investigators have shown the movement of cancer phenotype to monocytes [112] and we recently developed data indicating that both human lung cancer and prostate cancer cells isolated at surgery from patients will move the tissue phenotype to normal human marrow cells 125, 126. This opens new strategies for the treatment of cancer. The similarities between cancer cells and normal stem cells also suggest that the concept of a definable cancer stem cell is probably not correct. Rather, there must be numerous cancer cell phenotypes with varying stem cell potential.

Stem cell plasticity explained by microvesicle cell phenotype modulation 

We would propose that most of the studies characterizing stem cell or marrow plasticity were, in fact, explained by microvesicle cell phenotype modulation. This could occur by tissue microvesicles altering the phenotype of marrow or blood cells to the phenotype of the microvesicle originating tissue. Conversely, blood or marrow cells could deliver microvesicles to damaged tissue, restoring the tissue but also delivering the phenotypic markers of the marrow cells. In this latter case, marrow cells would not convert to nonhematopoietic tissue cells, but they would dramatically alter the phenotype of these cells by microvesicle docking, cell entry, and genetic modulation. These concepts are presented in Figure 8.

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  • Figure 8 

    Concepts of stem cell plasticity. Panel 1 indicates that marrow-derived microvesicles may enter lung cells and induce marrow characteristics in the lung cells. Panel 2 indicates than lung-derived microvesicles may enter marrow cells and alter their phenotype toward that of a lung cell.

We consider this a more satisfactory explanation for the descriptions of stem cell plasticity, which might preferably be referred to as cellular phenotype modulation. One does not have to propose whole-cell fusion, dedifferentiation, or transdifferentiation to explain the described events with tissue cells showing markers of transplanted marrow.

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Conclusions 

Purification of stem cells is a failed concept; it only contributes information on heterogeneity. Purified stem cells are not representative of marrow stem cells in unseparated marrow populations. Regulation of marrow stem cells is on a cell-cycle−regulated continuum of potential, which is probably continually altered by exposure to tissue-derived microvesicles. These latter are increased in conditions of injury. The continuum model probably holds for cancer cells along with the concept that there will not be a specific cancer stem cell, but rather a continuously changing population of cancer cells with different potentials. Stem cell plasticity, both intrahematopoietic and extrahematopoietic, is mediated by tissue-derived microvesicles acting selectively on cells in different phases of cell cycle. It is a form of mini-multiple cellular fusions through microvesicles.

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Conflict of Interest Disclosure 

No financial interest/relationships with financial interest relating to the topic of this article have been declared.

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References 

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PII: S0301-472X(10)00143-8

doi:10.1016/j.exphem.2010.03.021

Experimental Hematology
Volume 38, Issue 7 , Pages 581-592, July 2010

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