Very Small Embryonic-Like (VSEL) Stem Cells: Purification from Adult Organs,Characterization, and Biological Significance

In this review, we discuss current views of the bone marrow (BM) stem cell (SC) compartment and present data showing that BM contains heterogeneous populations of hematopoietic (H)SCs and non-HSCs. These cells are variously described in the literature as: endothelial progenitor cells (EPCs); mesenchymal (M)SCs; multipotent adult progenitor cells (MAPCs); marrow-isolated adult multilineage inducible (MIAMI) cells; and multipotent adult (MA)SCs. In some cases, it is likely that similar or overlapping populations of primitive SCs in the BM detected using various experimental strategies were assigned different names. Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4(+)) small SCs from the murine BM that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs. Our data indicate that VSELs may differentiate into cells from all three germ layers.     

Wharton's jelly-derived cells are a primitive stromal cell population

Here, the literature was reviewed to evaluate whether a population of mesenchymal stromal cells derived from Wharton's jelly cells (WJCs) is a primitive stromal population. A clear case can be made for WJCs as a stromal population since they display the characteristics of MSCs as defined by the International Society for Cellular Therapy; for example, they grow as adherent cells with mesenchymal morphology, they are self-renewing, they express cell surface markers displayed by MSCs, and they may be differentiated into bone, cartilage, adipose, muscle, and neural cells. Like other stromal cells, WJCs support the expansion of other stem cells, such as hematopoietic stem cells, are well-tolerated by the immune system, and they have the ability to home to tumors. In contrast to bone marrow MSCs, WJCs have greater expansion capability, faster growth in vitro, and may synthesize different cytokines. WJCs are therapeutic in several different pre-clinical animal models of human disease such as neurodegenerative disease, cancer, heart disease, etc. The preclinical work suggests that the WJCs are therapeutic via trophic rescue and immune modulation. In summary, WJCs meet the definition of MSCs. Since WJCs expand faster and to a greater extent than adult-derived MSCs, these findings suggest that WJCs are a primitive stromal cell population with therapeutic potential. Further work is needed to determine whether WJCs engraft long-term and display self-renewal and multipotency in vivo and, as such, demonstrate whether Wharton's jelly cells are a true stem cell population.     

骨髓间充质干细胞对致敏小鼠造血干/祖细胞移植植入的影响

目的: 前期的研究已经证实致敏小鼠造血干/祖细胞移植植入失败率高。本研究拟通过骨髓间充质干细胞(MSCs)进行干预,观察能否提高造血干、祖细胞移植的植入率。方法: 应用贴壁培养法体外培养正常小鼠骨髓MSCs,并分为6个实验组,包括实验组1:d11 MSCs干预的致敏组;实验组2: d0 MSCs干预的致敏组;实验组3:d11和d0 2次MSCs干预的致敏组;实验组4: 无MSCs干预的致敏小鼠对照组;实验组5:无MSCs干预的正常小鼠(非致敏小鼠)移植对照组;实验组6:无MSCs干预的正常小鼠不移植对照组。观察指标包括生存分析、移植效果分析(血象改变、骨髓细胞恢复及嵌合分析等)和移植物抗宿主病(GVHD)检测,最终评估MSCs干预对各实验组异基因造血干/祖细胞移植植入率的影响效果。结果: 与对照组(实验组4、5、6)比较,MSCs干预(实验组1、2、3)在2次异基因脾细胞注射法致敏的动物模型进行异基因造血干/祖细胞移植时,未能促进骨髓造血干/祖细胞移植的植入,也未能延长致敏动物移植后的生存时间。结论: 体内应用1×10⁶ MSCs干预,未能促进2次异基因1×10⁶ C57BL/6小鼠脾细胞输注法建立的重度致敏模型异基因造血干/祖细胞移植的植入。     

Mesenchymal stem cell-organized bone marrow elements: an alternative hematopoietic progenitor resource

Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent postnatal stem cells that have been used for the treatment of bone defects and graft-versus-host diseases in clinics. In this study, we found that subcutaneously transplanted human BMMSCs are capable of organizing hematopoietic progenitors of recipient origin. These hematopoietic cells expressed multiple lineages of hematopoietic cell associated markers and were able to rescue lethally irradiated mice, with successful engraftment in the recipient, suggesting a potential bone marrow (BM) resource for stem cell therapies. Furthermore, we found that platelet-derived growth factor (PDGF) promotes the formation of BMMSC-generated BM niches through upregulation of beta-catenin, implying that the PDGF pathway contributes to the formation of ectopic BM. These results indicate that the BMMSC-organized BM niche system represents a unique hematopoietic progenitor resource possessing potential clinical value.     

Very small embryonic-like stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy

Very small embryonic-like stem cells (VSELs) are possibly lost during cord blood banking and bone marrow (BM) processing for autologus stem cell therapy mainly because of their small size. The present study was conducted on human umbilical cord blood (UCB, n=6) and discarded red blood cells (RBC) fraction obtained after separation of mononuclear cells from human BM (n=6), to test this hypothesis. The results show that VSELs, which are pluripotent stem cells with maximum regenerative potential, settle along with the RBCs during Ficoll-Hypaque density separation. These cells are very small in size (3-5 μm), have high nucleo-cytoplasmic ratio, and express nuclear Oct-4, cell surface protein SSEA-4, and other pluripotent markers such as Nanog, Sox-2, Rex-1, and Tert as indicated by immunolocalization and quantitative polymerase chain reaction (Q-PCR) studies. Interestingly, a distinct population of slightly larger, round hematopoietic stem cells (HSCs) with cytoplasmic Oct-4 were detected in the "buffy" coat, which usually gets banked or used during autologus stem cell therapy. Immunohistochemical studies on the umbilical cord tissue (UCT) sections (n=3) showed the presence of nuclear Oct-4-positive VSELs and many fibroblast-like mesenchymal stem cells (MSCs) with cytoplasmic Oct-4. These VSELs with nuclear Oct-4, detected in UCB, UCT, and discarded RBC fraction obtained after BM processing, may persist throughout life, maintain tissue homeostasis, and undergo asymmetric cell division to self-renew as well as produce larger progenitor stem cells, viz. HSCs or MSCs, which follow differentiation trajectories depending on the somatic niche. Hence, it can be concluded that the true stem cells in adult body tissues are the VSELs, whereas the HSCs and MSCs are actually progenitor stem cells that arise by asymmetric cell division of VSELs. The results of the present study may help explain low efficacy reported during adult autologous stem cell trials, wherein unknowingly progenitor stem cells are injected rather than the pluripotent stem cells with maximum regenerative potential.     

Fibroblast growth factor-2 maintains a niche-dependent population of self-renewing highly potent non-adherent mesenchymal progenitors through FGFR2c

Bone marrow (BM) mesenchymal stem/stromal cells (MSC) are a heterogeneous population of multipotent progenitors currently under investigation for a variety of applications in regenerative medicine. While self-renewal of stem cells in different tissues has been demonstrated to be regulated by specialized microenvironments called niches, it is still unclear whether a self-renewing niche also exists for MSC. Here, we show that primary human BM cultures contain a population of intrinsically non-adherent mesenchymal progenitors (NAMP) with features of more primitive progenitors than the initially adhering colony-forming units-fibroblast (CFU-f). In fact, NAMP could generate an adherent progeny: (a) enriched with early mesenchymal populations (CD146+, SSEA-1+, and SSEA-4+); (b) with significantly greater proliferation and multilineage differentiation potential in vitro; and (c) capable of threefold greater bone formation in vivo than the corresponding CFU-f. Upon serial replating, NAMP were able to regenerate and expand in suspension as non-adherent clonogenic progenitors, while also giving rise to an adherent progeny. This took place at the cost of a gradual loss of proliferative potential, shown by a reduction in colony size, which could be completely prevented when NAMP were expanded on the initially adhering BM fraction. Mechanistically, we found that NAMP crucially depend on fibroblast growth factor (FGF)-2 signaling through FGFR2c for their survival and expansion. Furthermore, NAMP maintenance depends at least in part on humoral signals distinct from FGF-2. In conclusion, our data show a niche/progenitor organization in vitro, in which the BM adherent fraction provides a self-renewing microenvironment for primitive NAMP.     

Granulocyte-colony stimulating factor-mobilized mesenchymal stem cells: A new resource for rapid engraftment in hematopoietic stem cell transplantation

The bone marrow (BM) microenvironment plays an important role in regulating hematopoietic stem cell self-renewal and differentiation. Mesenchymal stem cells (MSCs), which constitute approximately 0.01-0.0001% of the nucleated cells in the adult human BM, are an important component of the BM stroma that supports hematopoiesis. The BM stroma system is often damaged in patients who have undergone high-dose chemotherapy and/or radiation treatment. Thus, the BM stroma should be reconstructed during hematopoietic stem cell transplantation (HSCT). Granulocyte-colony stimulating factor (G-CSF) is a potent hematopoietic cytokine that regulates neutrophil generation within the BM by modulating the mobility, proliferation and maturation of neutrophil progenitor cells. The results from our study here show that G-CSF markedly increased the number of donor-derived MSCs in the BM and the peripheral blood. Engraftment was faster in HSCTs with bone marrow that was treated with G-CSF (G-BM) or with G-BM- and G-CSF-treated peripheral blood stem cells compared to stead-state bone marrow (SS-BM). Based on these findings, we hypothesize that G-CSF-mobilized treatment of MSCs may accelerate engraftment in HSCT.     

AGM区来源的基质细胞促进造血干细胞扩增的体外实验研究

目的： 探讨主动脉-性腺-中肾（aorta-gonad-mesonephros，AGM）来源的基质细胞对造血干细胞（HSC）增殖的促进作用，为探寻HSC的体外扩增方法奠定实验基础。 方法： 分别从孕11 d BALB/c小鼠胚胎AGM区及6周龄小鼠骨髓分离、培养基质细胞，流式细胞仪等对基质细胞进行鉴定；利用小鼠胚胎干细胞（ESC）向造血细胞定向分化的模型，结合高增殖潜能集落（HPP-CFC）、原始细胞集落（BL-CFC）形成实验及流式细胞仪分析CD34+、CD34+Sca-1+细胞比例，对比研究AGM及骨髓基质细胞对ESC来源的HSC的扩增作用。 结果： 小鼠AGM和骨髓基质细胞在形态及表型上基本相似，均符合基质细胞的特征。AGM和骨髓基质细胞均可促进ESC来源的HPP-CFC的形成，但AGM基质细胞还可促进ESC来源的 BL-CFC的形成；流式细胞仪检测发现：在骨髓基质细胞支持下，CD34+细胞增加了3-4倍，但CD34+/Sca-1+却无明显增加；而在AGM基质细胞支持下CD34+、CD34+Sca-1+细胞均明显增加了4-5倍。 结论： AGM基质细胞在有效扩增小鼠HSC同时，能很好地维持HSC自我更新及多向分化的潜能。     

Granulocyte-colony-stimulating factor stimulation of bone marrow mesenchymal stromal cells promotes CD34+ cell migration via a matrix metalloproteinase-2-dependent mechanism

Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models.     

Clinical-scale expansion of a mixed population of bone-marrow-derived stem and progenitor cells for potential use in bone-tissue regeneration

Preclinical and clinical studies have demonstrated the ability of bone marrow derived stem and progenitor cells to regenerate many tissues, including bone. Methods to expand or enrich progenitors from bone marrow are common; however, these methods include many steps not amenable to clinical use. A closed automated cell production culture system was developed for clinical-scale ex vivo production of bone marrow-derived stem and progenitor cells for hematopoietic reconstitution. The current study tested the ability of this bioreactor system to produce progenitor cells, termed tissue repair cells (TRC), possessing osteogenic potential. Three TRC formulations were evaluated: (a) cells cultured without exogenous cytokines (TRC); (b) cells cultured with exogenous cytokines (TRC-C); and (c) an adherent subset of TRC-C (TRC-C(Ad)). Starting human bone marrow mononuclear cells (BM MNC) and TRC products were characterized for the expression of cell surface markers, in vitro colony forming ability, and in vivo osteogenic potential. Results showed significant expansion of mesenchymal progenitors (CD90+, CD105+, and CD166+) in each TRC formulation. In vivo bone formation, measured by histology, was highest in the TRC group, followed by TRC-C(Ad) and TRC-C. The TRC product outperformed starting BM MNC and had equivalent bone forming potential to purified MSCs at the same cell dose. Post hoc analysis revealed that the presence of CD90+, CD105+, and CD166+ correlated strongly with in vivo bone formation scores (r(2) > .95). These results demonstrate that this bioreactor system can be used to generate, in a single step, a population of progenitor cells with potent osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.     

Thrombopoietin, a direct stimulator of viability and multilineage growth of primitive bone marrow progenitor cells

Thrombopoietin (TPO), the ligand for c-mpl, has recently been demonstrated to be the primary regulator of megakaryocytopoiesis and platelet production. In addition, several studies have demonstrated that c-mpl is expressed on hematopoietic cell populations highly enriched in primitive progenitor cells. Here we summarize and discuss recent studies from our laboratory, as well as others, demonstrating that TPO has effects on primitive hematopoietic progenitor cells. When acting alone, TPO stimulates little or no growth, but promotes viability and suppresses apoptosis of murine multipotent (Lin- Sca-1+) bone marrow progenitor cells in vitro. In addition, TPO directly and potently synergizes with other early acting cytokines (kit ligand, flt3 ligand and interleukin 3) to promote multilineage growth of the same progenitor cell population. Although it remains to be established whether TPO also acts on the long-term reconstituting pluripotent stem cells, these studies combined with progenitor cell studies in c-mpl-deficient mice, suggest that TPO, in addition to its key role in platelet production, might also have an important impact on early hematopoiesis.     

Why should mesenchymal stem cells (MSCs) cure autoimmune diseases?

The adult stem/progenitor cells from bone marrow and other tissues referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) display a significant therapeutic plasticity as reflected by their ability to enhance tissue repair and influence the immune response both in vitro and in vivo. In this review we will focus on the paradigmatic preclinical experience achieved testing MSCs in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. We will emphasize how the paradigm changed over time from the original prediction that MSCs would enhance tissue repair through their transdifferentiation into somatic cells to the current paradigm that they can produce therapeutic benefits without engraftment into the injured tissues. The data will be reviewed in terms of the potentials of MSCs for therapy of autoimmune diseases.     

Neuroprotective Effect of Mesenchymal and Neural Stem and Progenitor Cells on Sensorimotor Recovery after Brain Injury

We studied the effect of systemic administration of multipotent stem cells on impaired neurological status in rats with brain injury. It was found that transplantation of multipotent mesenchymal stromal cells of the bone marrow or human neural stem and progenitor cells to rats with local brain injury promoted recovery of the brain control over locomotor function and proprioceptive sensitivity of forelegs. The dynamics of neurological recovery was similar after transplantation of fetal neural stem and progenitor cells and multipotent mesenchymal stromal cells. Transplantation of cell cultures improved survival of experimental animals. It should be noted that administration of neural stem and progenitor cells prevented animal death not only in the acute traumatic period, but also in delayed periods.     

The concept of mesenchymal stem cells

In this chapter we examine whether criteria usually defining adult tissue stem cells apply to mesenchymal stem cells (MSCs) that give rise to cells of the skeletal connective tissues. MSCs appear to constitute a heterogeneous population of undifferentiated and committed, lineage-primed cells, capable of: homing upon engraftment to a number of growth microenvironments, extensive proliferation, producing large numbers of differentiated progeny, and functional tissue repair after injury. In addition, MSCs are extensively distributed throughout tissues, and bone marrow MSCs provide the stromal component of the niche of hematopoietic stem cells. The capacity of apparently differentiated mesenchymal cells to shift their differentiation pathway with changing microenvironmental conditions (known as differentiation plasticity) may be due to de-differentiation and reprogramming in MSCs. Because they present several features setting them apart from other stem cells, MSCs may constitute another paradigm for stem cell systems, where self-renewal and hierarchy are no longer essential, but where plasticity is the major characteristic.     

Adipocyte differentiation of human marrow mesenchymal stem cells reduces the supporting capacity for hematopoietic progenitors but not for severe combined immunodeficiency repopulating cells

Bone marrow stromal cells provide a microenvironment for hematopoiesis. Adipocytes are the major stromal cell phenotype in bone marrow, but their function in hematopoiesis is poorly understood. In this study, we compared the hematopoietic-supporting capacity of adipocytes and their progenitor, mesenchymal stem cells (MSCs), by culturing human cord blood (CB) CD34+CD38- hematopoietic progenitor cells (HPCs) on a layer of adipocytes or MSCs. CB CD34+CD38- cells cultured on MSCs generated higher proportions of CD34+CD38- HPCs and colony-forming cells than those cultures on a layer of adipocytes, indicating an inferior hematopoietic support by adipocytes. However, CB CD34+CD38- HPCs cultured on MSCs and adipocytes were equally capable of reconstituting human hematopoiesis in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. These findings show that differentiation of MSCs into adipocytes is accompanied by the loss of capacity to support mature HPCs, but not transplantable SCID-repopulating cells.     

Wharton’s Jelly Mesenchymal Stromal Cells as a Feeder Layer for the Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells: a Review

In recent years, umbilical cord blood (UCB) has been widely used as an alternative source to bone marrow (BM) for transplantation of hematopoietic stem and progenitor cells (HSPCs) in a variety of hematological and non-hematological disorders. Nevertheless, the insufficient number of UCB-HSPCs for graft represents a major challenge. HSPCs ex vivo expansion prior to transplantation is a valid strategy to overcome this limit. Several attempts to optimize the expansion conditions have been reported, including the use of mesenchymal stromal cells (MSCs) as feeder layer. Wharton’s Jelly (WJ), the main component of umbilical cord (UC) matrix, is especially rich in MSCs, which are considered ideal candidates for feeder layer in co-culture systems. In fact, they can be easily harvested and grow robustly in culture, producing a confluent monolayer in a short time. Similarly to bone marrow-mesenchymal stromal cells (BM-MSCs), WJ-derived MSCs (WJ-MSCs) have been used to support hematopoiesis in vitro and in vivo. Here, we review the rationale for using MSCs, particularly WJ-MSCs, as a feeder layer for UCB-HSPCs ex vivo expansion. In addition, we report the main findings attesting the use of these MSCs as a support in hematopoiesis.     

Bone Marrow Mesenchymal Stem Cells: Biological Properties and Their Role in Hematopoiesis and Hematopoietic Stem Cell Transplantation

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that are present in practically all tissues as a specialized population of mural cells/pericytes that lie on the abluminal side of blood vessels. Originally identified within the bone marrow (BM) stroma, not only do they provide microenvironmental support for hematopoietic stem cells (HSCs), but can also differentiate into various mesodermal lineages. MSCs can easily be isolated from the BM and subsequently expand in vitro and in addition they exhibit intriguing immunomodulatory properties, thereby emerging as attractive candidates for various therapeutic applications. This review addresses the concept of BM MSCs via a hematologist’s point of view. In this context it discusses the stem cell properties that have been attributed to BM MSCs, as compared to those of the prototypic hematopoietic stem cell model and then gives a brief overview of the in vitro and vivo features of the former, emphasizing on their immunoregulatory properties and their hematopoiesis-supporting role. In addition, the qualitative and quantitative characteristics of BM MSCs within the context of a defective microenvironment, such as the one characterizing Myelodysplastic Syndromes are described and the potential involvement of these cells in the pathophysiology of the disease is discussed. Finally, emerging clinical applications of BM MSCs in the field of hematopoietic stem cell transplantation are reviewed and potential hazards from MSC use are outlined.     

Peripheral Blood Stem Cells: Phenotypic Diversity and Potential Clinical Applications

A small proportion of cells in peripheral blood are actually pluripotent stem cells. These peripheral blood stem cells (PBSCs) are thought to be heterogeneous and could be exploited for a variety of clinical applications. The exact number of distinct populations is unknown. It is likely that individual PBSC populations detected by different experimental strategies are similar or overlapping but have been assigned different names. In this mini review, we divide PBSCs into seven groups: hematopoietic stem cells (HSCs), CD34- stem cells, CD14+ stem cells, mesenchymal stem cells (MSCs), very small embryonic-like (VSEL) stem cells, endothelial progenitor cells (EPCs), and other pluripotent stem cells. We review the major characteristics of these stem/progenitor cell populations and their potential applications in ophthalmology.     

Bone-marrow-derived mesenchymal stem cells as a target for cytomegalovirus infection: implications for hematopoiesis, self-renewal and differentiation potential

Mesenchymal stem cells (MSCs) in bone marrow (BM) regulate the differentiation and proliferation of adjacent hematopoietic precursor cells and contribute to the regeneration of mesenchymal tissues, including bone, cartilage, fat and connective tissue. BM is an important site for the pathogenesis of human cytomegalovirus (HCMV) where the virus establishes latency in hematopoietic progenitors and can transmit after reactivation to neighboring cells. Here we demonstrate that BM-MSCs are permissive to productive HCMV infection, and that HCMV alters the function of MSCs: (i) by changing the repertoire of cell surface molecules in BM-MSCs, HCMV modifies the pattern of interaction between BM-MSCs and hematopoietic cells; (ii) HCMV infection of BM-MSCs undergoing adipogenic or osteogenic differentiation impaired the process of differentiation. Our results suggest that by altering BM-MSC biology, HCMV may contribute to the development of various diseases.     

Polarization of human hematopoietic progenitors during contact with multipotent mesenchymal stromal cells: effects on proliferation and clonogenicity

Establishment of a defined cell culture system that facilitates ex vivo expansion of isolated hematopoietic stem and progenitor cells (HSPCs) is a crucial issue in hematology and stem cell transplantation. Here we have evaluated the capacity of primary human multipotent mesenchymal stromal cells (MSCs) to support the ex vivo expansion of peripheral CD34(+)-enriched HSPCs. We observed that HSPCs co-cultured on MSCs showed a substantially higher total expansion rate compared to those growing without. Moreover, in addition to the expansion of CD34(+)CD133(+) and CD34(+)CD133(-) cells, a third population of CD133(+)CD34(-) stem cells became detectable after expansion. Direct contact between HSPCs and the feeder layer appears beneficial for the expansion of HSPCs harboring CD133(+) phenotype, i.e., CD34(+)CD133(+) and CD133(+)CD34(-), in contrast to CD34(+)CD133(-) cells. Interestingly, electron microscopy and immunofluorescence analyses revealed that adherent HSPCs display various morphologies; they are either round with, in some cases, the appearance of a microvillar pole or exhibit several distinct types of plasma membrane protrusions such as lamellipodium and magnupodium. CD133 is selectively concentrated therein, whereas CD34 is randomly distributed over the entire surface of HSPCs. Together, this co-culture offers a unique experimental system to further characterize the biology and role of markers of rare stem cell populations.