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.     

骨髓间充质干细胞对脐血CD34+细胞分化为巨核细胞的影响

目的：探讨骨髓间充质干细胞对脐血CD34＋细胞诱导分化为巨核细胞的影响。方法：骨髓间充质干细胞培养采用低糖型DMEM培养基,待细胞满度达到约80%后加入脐血CD34＋细胞在一定的培养体系中进行实验,同时以无骨髓间充质干细胞的相应培养体系作为对照,培养14 d后观察结果。实验中共观察了两种不同的培养体系：基础培养液、基础培养液＋白细胞介素-11（IL-11）。其中基础培养液为含血小板生成素（TPO）、白细胞介素-3（IL-3）、干细胞因子（SCF）的低糖型DMEM。培养后单个核细胞数采用细胞计数仪分析,CD41＋细胞和血小板检测采用流式细胞仪,血小板功能评价采用凝血酶诱导的血小板凝集实验。结果：与相应的对照组比较,骨髓间充质干细胞实验组单个核细胞数增加不明显（P〉0.05）,而CD41＋细胞和血小板数量有明显的增加（P〈0.05）。显微镜下和流式细胞仪上均可观察到凝血酶诱导的血小板凝集现象。结论：骨髓间充质干细胞在实验培养体系中可以促进脐血中CD34＋细胞诱导分化为巨核细胞。     

Human bone marrow-derived mesenchymal stem cells differentiate into epidermal-like cells in vitro

Human bone marrow-derived mesenchymal stem cells (hMSCs) are a population of pluripotent cells. They can differentiate into different embryonic layer cells as osteoblasts, adipocytes, chondrocytes, myoblasts, neurocytes, etc. However, there are only few reports with regard to differentiate hMSCs into epidermal cells in vitro. In this study, we want to investigate the feasibility of inducing hMSCs into epidermal-like cells under specific medium in vitro. hMSCs in specific inducing medium expressed the early markers of epidermal cell lineage, P63, cytokeratin19 (CK19), the late differentiated marker, the pan-cytokeratin, and another early marker, the beta1-integrin, which up-regulated remarkably in inducing medium. Their morphologies were changed from spindle-like fibroblastic appearances to oblate or irregular shapes under phase contrast microscopy. The hemidesmosome structure was found using the transmission electron microscope. All these data suggested that, under certain conditions, hMSCs have the potential to differentiate into epidermal-like cells. It will be of great accordance in the study of the multipotential property of hMSCs.     

CD34+VEGFR‐3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34⁺VEGFR‐3⁺ EPCs were isolated from mononuclear cells of human cord blood by fluorescence‐activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7–10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF‐C for 2 weeks, CD34⁺VEGFR‐3⁺ EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5′‐nucleotidase, LYVE‐1 and Prox‐1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary‐like structures in three‐dimensional collagen gel and Matrigel. VEGF‐C enhanced VEGFR‐3 mRNA expression. After interfering with VEGFR‐3 siRNA, the effects of VEGF‐C were diminished. These results demonstrate that there is a population of CD34⁺VEGFR‐3⁺ EPCs with lymphatic potential in human cord blood. VEGF‐C/VEGFR‐3 signalling pathway mediates differentiation of CD34⁺VEGFR‐3⁺ EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood‐derived CD34⁺VEGFR‐3⁺ EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.     

Human CD4+CD25high regulatory T cells modulate myeloid but not plasmacytoid dendritic cells activation

Human CD4(+)CD25(+) regulatory T cells (Treg) play an essential role in the prevention of autoimmune diseases. However, the mechanisms of immune suppression and the spectrum of cells they target in vivo remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they have been shown to inhibit the Ag-presenting functions of macrophage- and monocyte-derived dendritic cells (DC). We have now studied the maturation of human blood-derived myeloid DC and plasmacytoid DC activated with TLR ligands in the presence of Treg. Preactivated Treg suppressed strongly TLR-triggered myeloid DC maturation, as judged by the blocking of costimulatory molecule up-regulation and the inhibition of proinflammatory cytokines secretion that resulted in poor Ag presentation capacity. Although IL-10 played a prominent role in inhibiting cytokines secretion, suppression of phenotypic maturation required cell-cell contact and was independent of TGF-beta and CTLA-4. In contrast, the acquisition of maturation markers and production of cytokines by plasmacytoid DC triggered with TLR ligands were insensitive to regulatory T cells. Therefore, human Treg may enlist myeloid, but not plasmacytoid DC for the initiation and the amplification of tolerance in vivo by restraining their maturation after TLR stimulation.     

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

Tumor skewing of CD34+ cell differentiation from a dendritic cell pathway into endothelial cells

Patients and animals bearing tumors have increased levels of CD34⁺ progenitor cells, which are capable of developing into dendritic cells. However, addition of medium conditioned by murine Lewis lung carcinoma cells increases the cellularity of the CD34⁺ cell cultures and redirects their differentiation into endothelial cells. The resulting cells resemble endothelial cells phenotypically as well as functionally by their capacity to reorganize into cord structures. Mechanisms by which tumors induced the increased cellularity and skewing toward endothelial cells were examined. Tumor-derived VEGF contributed to the increase in cellularity, but not to the redirection of differentiation. Differentiation into endothelial cells was blocked with sTie-2, suggesting tumor-derived angiopoietins in skewing differentiation. These studies show the capacity of tumors to skew progenitor cell development toward endothelial cells and define the mediators that contribute to endothelial cell development.     

Human adult skeletal muscle stem cells differentiate into cardiomyocyte phenotype in vitro

Cell transplantation to repair or regenerate injured myocardium is a new frontier in the treatment of cardiovascular disease. Most studies on stem cell transplantation therapy in both experimental heart infarct and in phase-I human clinical trials have focused on the use of undifferentiated stem cells. Based on our previous observations demonstrating the presence of multipotent progenitor cells in human adult skeletal muscle, in this study we investigated the capacity of these progenitors to differentiate into cardiomyocytes. Here we show an efficient protocol for the cardiomyogenic differentiation of human adult skeletal muscle stem cells in vitro. We found that treatment with Retinoic Acid directed cardiomyogenic differentiation of skeletal muscle stem cells in vitro. After Retinoic Acid treatment, cells expressed cardiomyocyte markers and acquired spontaneous contraction. Functional assays exhibited cardiac-like response to increased extracellular calcium. When cocultured with mouse cardiomyocytes, Retinoic Acid-treated skeletal muscle stem cells expressed connexin43 and when transplanted into ischemic heart were detectable even 5 weeks after injection. Based on these results, we can conclude that human adult skeletal muscle stem cells, if opportunely treated, can transdifferentiate into cells of cardiac lineage and once injected into infarcted heart can integrate, survive in cardiac tissue and improve the cardiac function.     

Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron‐like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule‐associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/β‐tubulin III). It also increased expression of brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine‐beta‐hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1–12 days. DA steadily increased after treatment with salidroside for 1–6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons.     

Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process

The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.     

Human CD34+ stem cells produce bone nodules in vivo

Abstract.   Objectives : The aim of this study was to select and provide enough stem cells for quick transplantation in bone engineering procedures, avoiding any in vitro expansion step. Materials and Methods : Dental germ pulp, collected from 25 healthy subjects aged 13–20 years, were subjected to magnetic-activated cell sorting to select a CD34⁺ stem cell population capable of differentiating into pre-osteoblasts. These cells were allowed to adhere to an absorbable polylactic–coglycolic acid scaffold for 30 min, without any prior expansion, and the CD34⁺ cell-colonized scaffolds were then transplanted into immunocompromised rats, subcutaneously. Results : After 60 days, analysis of recovered transplants revealed that they were formed of nodules of bone, of the same dimensions as the original scaffold. Bone-specific proteins were detected by immunofluorescence, within the nodules, and X-ray diffraction patterns revealed characteristic features of bone. In addition, presence of platelet endothelial cell adhesion molecule and von Willebrand factor immunoreactivity were suggestive of neo-angiogenesis and neovasculogenesis taking place within nodules. Importantly, these vessels were HLA-1⁺ and, thus, clearly human in origin. Conclusions : This study indicates that CD34⁺ cells obtained from dental pulp can be used for engineering bone, without the need for prior culture expanding procedures. Using autologous stem cells, this schedule could be used to provide the basis for bone regenerative surgery, with limited sacrifice of tissue, low morbidity at the collection site, and significant reduction in time needed for clinical recovery.     

Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells

The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl-binding receptors (Prl-R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage-specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit-granulocyte (CFU-G) and burst forming unit-erythroid (BFU-E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl-binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR-7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR-7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl-R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage-specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl. and hemopoietic factors. © 1995 Wiley-Liss, Inc.     

Mesodermal progenitor cells (MPCs) differentiate into mesenchymal stromal cells (MSCs) by activation of Wnt5/calmodulin signalling pathway

Background

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4⁺CD105⁺CD90^neg phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4^negCD105⁺CD90^bright and MSCA-1⁺), in the primary cultures, resulted lower than 2%.

Methodology/Principal Finding

We demonstrate that MPCs differentiate to MSCs through an SSEA-4⁺CD105⁺CD90^bright early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4^neg). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID₅₀ = 0.5 µM, p<0.01), while endothelial differentiation was unaffected.

Conclusion

The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.     

Human umbilical cord-derived mesenchymal stem cells differentiate into epidermal-like cells using a novel co-culture technique

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) isolated from human umbilical Wharton’s Jelly are a population of primitive and pluripotent cells. In specific conditions, hUCMSCs can differentiate into various cells, including adipocytes, osteoblasts, chondrocytes, neurocytes, and endothelial cells. However, few studies have assessed their differentiation into epidermal cells in vitro. To assess the potential of hUCMSCs to differentiate into epidermal cells, a microporous membrane-based indirect co-culture system was developed in this study. Epidermal stem cells (ESCs) were seeded on the bottom of the microporous membrane, and hUCMSCs were seeded on the top of the microporous membrane. Cell morphology was assessed by phase contrast microscopy, and the expression of early markers of epidermal cell lineage, P63, cytokeratin19 (CK19), and β1-integrin, was determined by immunofluorescence, Western blot, and quantitative real-time PCR (Q-PCR) analyses. hUCMSC morphology changed from spindle-like to oblate or irregular with indirect co-culture with ESCs; they also expressed greater levels P63, CK19, and β1-integrin mRNA and protein compared to the controls (p < 0.01). As compared to normal co-cultures, indirect co-culture expressed significantly greater CK19 protein (p < 0.01). Thus, hUCMSCs may have the capability to differentiate into the epidermal lineage in vitro, which may be accomplished through this indirect co-culture model.     

Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro

It was recently reported that pluripotent mesenchymal stem cells (MSCs) in rodent bone marrow (BM) have the capacity to generate insulin-producing cells (IPCs) in vitro. However, little is known about this capacity in human BM-MSCs. We developed a nongenetic method to induce human BM-MSCs to transdifferentiate into IPCs both phenotypically and functionally. BM-MSCs from 12 human donors were sequentially cultured in specially defined conditions. Their differentiation extent toward β-cell phenotype was evaluated systemically. Specifically, after induction human BM-MSCs formed spheroid islet-like clusters containing IPCs, which was further confirmed by dithizone (DTZ) staining and electron microscopy. These IPCs expressed multiple genes related to the development or function of pancreatic β cells (including NKX6.1, ISL-1, Beta2/Neurod, Glut2, Pax6, nestin, PDX-1, ngn3, insulin and glucagon). The coexpression of insulin and c-peptide was observed in IPCs by immunofluorescence. Moreover, they were able to release insulin in a glucose-dependent manner and ameliorate the diabetic conditions of streptozotocin (STZ)-treated nude mice. These results indicate that human BM-MSCs might be an available candidate to overcome limitations of islet transplantation.