The HOXB4 homeoprotein promotes the ex vivo enrichment of functional human embryonic stem cell-derived NK cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34(+)CD45RA(+) precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells.     

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.     

Hepatic stellate cells modulate the differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells

Differentiation of stem cells is tightly regulated by the microenvironment which is mainly composed of nonparenchymal cells. Herein, we investigated effect of hepatic stellate cells (HSCs) in different states on mesenchymal stem cells (MSCs) differentiation. Rat HSCs were isolated and stayed quiescent within 5 days. Primary HSCs were activated by being in vitro cultured for 7 days or cocultured with Kupffer cells for 5 days. MSCs were cocultured with HSCs of different states. Expression of hepatic lineage markers was analyzed by RT-PCR and immunofluorescence. Glycogen deposition was detected by periodic acid-schiff staining. MSCs cocultured with HSC-T6 or Kupffer cell activated HSCs were morphologically transformed into hepatocyte-like cells. Hepatic-specific marker albumin was expressed in 78.3% of the differentiated MSCs 2 weeks after initiation of coculture. In addition, the differentiated MSCs also expressed alpha-fetoprotein, cytokeratin-18, glutamine synthetase and phosphoenolpyruvate carboxykinase. Glycogen deposition was detectable in 55.4% of the differentiated MSCs 6 weeks after initiation of coculture. However, the quiescent HSCs or culture activated HSCs did not exert the ability to modulate the differentiation of MSCs. Moreover, Kupffer cell activated HSCs rather than culture activated HSCs expressed hepatocyte growth factor mRNA. We draw the conclusion that fully activated HSCs could modulate MSCs differentiation into hepatocyte-like cells.     

Mesenchymal stem cells: characteristics and clinical applications

Mesenchymal stem cells (MSCs) are bone marrow populating cells, different from hematopoietic stem cells, which possess an extensive proliferative potential and ability to differentiate into various cell types, including: osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes and neurons. MSCs play a key role in the maintenance of bone marrow homeostasis and regulate the maturation of both hematopoietic and non-hematopoietic cells. The cells are characterized by the expression of numerous surface antigens, but none of them appears to be exclusively expressed on MSCs. Apart from bone marrow, MSCs are located in other tissues, like: adipose tissue, peripheral blood, cord blood, liver and fetal tissues. MSCs have been shown to be powerful tools in gene therapies, and can be effectively transduced with viral vectors containing a therapeutic gene, as well as with cDNA for specific proteins, expression of which is desired in a patient. Due to such characteristics, the number of clinical trials based on the use of MSCs increase. These cells have been successfully employed in graft versus host disease (GvHD) treatment, heart regeneration after infarct, cartilage and bone repair, skin wounds healing, neuronal regeneration and many others. Of special importance is their use in the treatment of osteogenesis imperfecta (OI), which appeared to be the only reasonable therapeutic strategy. MSCs seem to represent a future powerful tool in regenerative medicine, therefore they are particularly important in medical research.     

Human umbilical cord Wharton's jelly mesenchymal stem cells do not transform to tumor-associated fibroblasts in the presence of breast and ovarian cancer cells unlike bone marrow mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hBMMSCs) were shown to transform into tumor-associated fibroblasts (TAFs) when in the vicinity of breast cancer tumors and played an important role in tumor enhancement and metastasis. In early human development MSCs migrating from the yolk sac and aorta-gonad-mesonephros (AGM) via the umbilical cord to the placenta and back to the fetal bone marrow were shown to get trapped in the gelatinous Wharton's jelly of the umbilical cord. The common origin of the Wharton's jelly MSCs and the finally homed hBMMSCs prompted us to evaluate whether hWJSCs are also involved in TAF transformation. hWJSCs and hBMMSCs were grown in the presence of breast and ovarian cancer cell conditioned medium (MDA-TCM, TOV-TCM) for 30 days. No changes were observed in the hWJSCs but the hBMMSCs transformed from short to thin long fibroblasts, their proliferation rates increased and CD marker expression decreased. The transformed hBMMSCs showed positive staining for the tumor-associated markers FSP, VEGF, EGF, and Tn-C. Real-time PCR and multiplex luminex bead analysis showed upregulation of TAF-related genes (FSP, FAP, Tn-C, Tsp-1, EGF, bFGF, IL-6, α-SMA, VEGF, and TGF-β) for hBMMSCs with low expression for hWJSCs. The luciferase assay showed that hWJSCs previously exposed to MDA-TCM or TOV-TCM had no stimulatory growth effect on luciferase-tagged MDA or TOV cells unlike hBMMSCs. The results confirmed that hWJSCs do not transform to the TAF phenotype and may therefore not be associated with enhanced growth of solid tumors making them a safe MSC for cell based therapies.     

Mesenchymal progenitor cells localize within hematopoietic sites throughout ontogeny

Mesenchymal stem cells (MSCs) have great clinical potential for the replacement and regeneration of diseased or damaged tissue. They are especially important in the production of the hematopoietic microenvironment, which regulates the maintenance and differentiation of hematopoietic stem cells (HSCs). In the adult, MSCs and their differentiating progeny are found predominantly in the bone marrow (BM). However, it is as yet unknown in which embryonic tissues MSCs reside and whether there is a localized association of these cells within hematopoietic sites during development. To investigate the embryonic origins of these cells, we performed anatomical mapping and frequency analysis of mesenchymal progenitors at several stages of mouse ontogeny. We report here the presence of mesenchymal progenitors, with the potential to differentiate into cells of the osteogenic, adipogenic and chondrogenic lineages, in most of the sites harboring hematopoietic cells. They first appear in the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence. However, at this developmental stage, their presence is independent of HSC activity. They increase numerically during development to a plateau level found in adult BM. Additionally, mesenchymal progenitors are found in the embryonic circulation. Taken together, these data show a co-localization of mesenchymal progenitor/stem cells to the major hematopoietic territories, suggesting that, as development proceeds, mesenchymal progenitors expand within these potent hematopoietic sites.     

Growth factor receptor expression during in vitro differentiation of partially purified populations containing murine stem cells

We have investigated, by semiquantitative RT-PCR, the kinetics of activation of hematopoietic receptors and differentiation markers in partially purified murine hematopoietic stem cells (HSC) induced to differentiate in serum-free culture with combinations of growth factor (GF). The combinations of GF used sustained either multilineage [stem cell factor (SCF) + interleukin 3 (IL-3)], or erythroid [SCF + IL-3 + erythropoietin (Epo)] or myeloid [SCF + IL-3 + granulocyte colony-stimulating factor (G-CSF)] differentiation. The GF receptor genes investigated were the α and β subunits of the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin receptor, the G-CSF receptor, and c-Fms, the receptor for macrophage colony-stimulating factor (M-CSF). The expression of Gata1 and α- and β-globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, which include partitioning of lineage-negative bone marrow cells with the mitochondrial dye Rhodamine 123 (Rho) into Rho-dull (≥17% of which reconstitute long-term hematopoiesis in recipient mice) and into Rho-bright (which are as capable as Rho-dull of multilineage differentiation but do not permanently reconstitute the host). The following pattern of expression was observed: the α subunit of the IL-3 receptor clearly was expressed in both Rho-bright and Rho-dull cells at the outset, and its expression did not change over time in culture. The β subunits of the IL-3 and GM-CSF receptor, the α subunit of the GM-CSF receptor, the Epo and G-CSF receptors and Fms barely were expressed in purified Rho-bright and Rho-dull cells, but their expression increased in cells cultured both in erythroid and in myeloid GF combinations. Gata1 was expressed maximally in Rho-bright cells but was below the level of detection in Rho-dull cells. Rho-dull cells expressed Gata1 when cultured both in erythroid and in myeloid GF combinations. In contrast, α- and β-globin, which also were not expressed in the purified cells, were induced only in cells stimulated with Epo. These results indicate that the genes for all the GF receptors investigated (with the exception of the α subunit of the IL-3 receptor) are expressed at low levels, if any, in purified Rho-bright or Rho-dull cells, but are expressed in their progeny cultured either in erythroid or myeloid GF combinations. The expression of the Epo receptor,in particular, is activated both in erythroid (α- and β-globin positive) and in myeloid (α- and β-globin negative) cells. Therefore, activation of the expression of the Epo receptor gene and activation of the erythroid differentiation program are two independent events in normal hematopoiesis. J. Cell. Physiol. 171:343–356, 1997. © 1997 Wiley-Liss, Inc.     

Nanofiber-expanded stem cells mitigate liver fibrosis: Experimental study

ObjectivesThis study examines a pretreatment strategy to strengthen the hepatic lineage divergence of mesenchymal stem cells (MSCs).Design and methodsBMSCs were expanded in the presence or absence of nanofiber (NF) and treated with growth factors (GF) prior to transplantation. Thioacetamide (TA) was used for liver fibrosis induction and transplantation of NF-expanded BMSCs was compared biochemically and histologically to the cells expanded without NF scaffold.ResultsThe ultraweb NF caused better proliferation and characterization of MSCs. MSCs transplantation significantly improved liver functions, increased hepatic HGF and Bcl-2 levels, whereas decreased serum fibronectin, hepatic TNF-α and TGF-β1 levels. Hepatic HNF4α, FOXa2, CYP7a1 genes expression were enhanced while β-5-Tub and AFP genes expression were depressed. Histological study documented these results. Differentiated NF-MSCs showed pronounced enhancement of the aforementioned parameters as compared to differentiated MSCs in the absence of NF.Conclusionpretreatment with growth factors in the presence of NF augment homing, repopulation and hepatic differentiation abilities of MSCs and proves to be a promising approach for the treatment of liver fibrosis.     

Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are adult pluripotent cells that are considered to be an important resource for human cell-based therapies. Understanding the clinical potential of MSCs may require their use in preclinical large-animal models, such as pigs. The objectives of the present study were 1) to establish porcine MSC (pMSC) cultures; 2) to optimize in vitro pMSC culture conditions, 3) to investigate whether pMSCs are amenable to genetic manipulation, and 4) to determine pMSC reprogramming potential using somatic cell nuclear transfer (SCNT). The pMSCs isolated from bone marrow grew, attached to plastic with a fibroblast-like morphology, and expressed the mesenchymal surface marker THY1 but not the hematopoietic marker ITGAM. Furthermore, pMSCs underwent lipogenic, chondrogenic, and osteogenic differentiation when exposed to specific inducing conditions. The pMSCs grew well in a variety of media, and proliferative capacity was enhanced by culture under low oxygen atmosphere. Transient transduction of pMSCs and isogenic skin fibroblasts (SFs) with a human adenovirus carrying the gene for green fluorescent protein (GFP; Ad5-F35eGFP) resulted in more pMSCs expressing GFP compared with SFs. Cell lines with stable genetic modifications and extended expression of transgene were obtained when pMSCs were transfected with a plasmid containing the GFP gene. Infection of pMSC and SF cell lines by an adeno-associated virus resulted in approximately 12% transgenic cells, which formed transgenic clonal lines after propagation as single cells. The pMSCs can be expanded in vitro and used as nuclear donors to produce SCNT embryos. Thus, pMSCs are an attractive cell type for large-animal autologous and allogenic cell therapy models and for SCNT transgenesis.     

Nestin Positive Bone Marrow Derived Cells Responded to Injury Mobilize into Peripheral Circulation and Participate in Skin Defect Healing

Exogenously infused mesenchymal stem cells (MSCs) are thought to migrate to injury site through peripheral blood stream and participate in tissue repair. However, whether and how endogenous bone marrow MSCs mobilized to circulating and targeted to tissue injury has raised some controversy, and related studies were restricted by the difficulty of MSCs identifying in vivo. Nestin, a kind of intermediate filament protein initially identified in neuroepithelial stem cells, was recently reported as a credible criteria for MSCs in bone marrow. In this study, we used a green fluorescent protein (GFP) labeled bone marrow replacement model to trace the nestin positive bone marrow derived cells (BMDCs) of skin defected-mice. We found that after skin injured, numbers of nestin⁺ cells in peripheral blood and bone marrow both increased. A remarkable concentration of nestin⁺ BMDCs around skin wound was detected, while few of these cells could be observed in uninjured skin or other organs. This recruitment effect could not be promoted by granulocyte colony-stimulating factor (G-CSF), suggests a different mobilization mechanism from ones G-CSF takes effect on hematopoietic cells. Our results proposed nestin⁺ BMDCs as mobilized candidates in skin injury repair, which provide a new insight of endogenous MSCs therapy.     

The Additive Effect of Mesenchymal Stem Cells and Bone Morphogenetic Protein 2 on γ-Irradiated Bone Marrow in Mice

Irradiation from γ-rays can cause severe damage to bone marrow and hematopoietic tissues. Presently, the most effective method available to treat severe hematopoietic injury is a bone marrow transplant (BMT). Allogeneic BMT is a difficult technique to perform due to the differences in human leukocyte antigen proteins between the donor and recipient, with acute graft-versus-host disease being a major complication of the technique. This limits the widespread applicability of allogeneic BMT. To develop a novel treatment for acute hematopoietic damage, we transplanted bone marrow derived mesenchymal stem cells (MSCs) into recipient mice and treated them with recombinant human bone morphogenetic protein 2 (rhBMP2) to investigate whether MSCs and rhBMP2 could additively promote the restoration of hematopoietic function. MSCs are vital components of the hematopoietic microenvironment that supports hematopoiesis, and bone morphogenic protein is a key factor in hematopoiesis. The 30-day survival rate as well as the numbers of nucleated cells, bone marrow colony-forming unit-granulocyte macrophages, spleen colony-forming units and peripheral blood cells were enumerated. The results showed that, after γ-irradiation and transplantation, MSCs and rhBMP2 additively promoted and improved hematopoietic restoration and function in vivo and in vitro. This additive effect of MSCs and rhBMP2 may one day provide a novel means of treating acute hematopoietic damage.     

Adult bone-marrow-derived mesenchymal stem cells contribute to wound healing of skin appendages

Adult bone-marrow-derived mesenchymal stem cells (MSCs) are well-established as having the capacity to differentiate into cells with mesodermal, ectodermal, and endodermal characteristics and can leave their niche to home toward and engraft within foreign tissues. To investigate whether adult MSCs contribute to the repair of skin appendages after injury, BrdU-labeled MSCs were co-cultured with heat-shocked confluent sweat gland cells (SGCs) in vitro and later intravenously injected into full-thickness skin wounds in rats. When adult MSCs were co-cultured with heat-shocked SGCs, a subset of adult MSCs differentiated into SGCs, the percentage of differentiation being enhanced by epidermal growth factor and the injured microenviroment, but weakened by PD98059. The ERK (extracellular signal-regulated kinase) pathway, especially pERK, was involved in the phenotype conversion of human MSCs into human SGC. Labeled MSCs were noted in hair follicles, sebaceous glands, blood vessels, and dermis in full-thickness wounds, and the incorporated cells in hair follicles and sebaceous glands were also positive for pan-cytokeratin. After wound healing, some labeled MSCs returned to the bone marrow, whereas other were retained in the dermis. We conclude that adult MSCs have the capacity to dock at specific sites, to contribute to wound healing of skin appendages, and to home toward marrow, and that engraftment of bone-marrow-derived cells is a functional event.This work was supported in part by the National Basic Science and Development Program (973 Program and 2005CB522603) and the National Natural Science Foundation of China (30230370 and 30500194).     

Co-culture of human CD34+ cells with mesenchymal stem cells increases the survival of CD34+ cells against the 5-aza-deoxycytidine- or trichostatin A-induced cell death

It has been suggested that epigenetic regulation plays an important role in maintaining the stemness and lineage differentiation of hematopoietic stem cells (HSCs), 5-aza-deoxycytidine (aza-D) and Trichostatin A (TSA) being candidate additives for HSC ex vivo expansion. Although they have potent activity to maintain the stemness, they can also cause serious cell death. This study examined the effects of mesenchymal stem cells (MSCs) on the maintenance of CD34+ cells driven by aza-D and TSA in culture with the combined cytokines of thrombopoietin, flt-3 ligand, stem cell factor, interleukin-3, and interleukin-6. In cultures without MSCs, although aza-D and TSA retained the CD34 frequency 4 to 8 times more than in the cytokines alone, a large portion of cells underwent apoptotic cell death. Consequently, CD34+ cell expansion could not be achieved in any condition without MSCs. In cultures with MSCs, the total cell number was higher in aza-D or TSA than in any conditions in the cultures without MSCs. The CD34 frequency was also similar to the level in the cultures in aza-D or TSA without the MSCs. These results suggest that a co-culture of CD34+ cells with the MSCs might not simply deliver the proliferation signals but also stemness and survival signals, and overlap the action of epigenetic regulators.