The immunobiological development of human bone marrow mesenchymal stem cells in the course of neuronal differentiation

The central nervous system (CNS) has been referred to as the "immunological privileged site". However, it is now clear that the privileged status of the CNS is a result of a balance between immune privilege and effective response. In vitro, human bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into neurons. Based on this biological attribute we gain the possibility by means of using MSCs as the donors to develop a future cell therapy in clinical application. But using MSCs as donor cells inevitably raises the question as to whether these donor cells would be immunogenic, and if so, would they be rejected after transplantation. To investigate this, human MSCs were cultured in vitro and induced to differentiate along neuronal lineage. The expression of human leukocyte antigen (HLA) class I and class II molecules and the co-stimulatory protein CD80 were increased on the surface of MSCs in the course of neuronal differentiation. But neither of the co-stimulatory proteins, CD40 or CD86, was expressed. After IFN-gamma exposure, the expression of the HLA molecules was further enhanced, but the co-stimulatory proteins were unaffected. MSCs that had been differentiated along neuronal lineage were not capable of inducing the proliferation of peripheral blood lymphocytes (PBLs). Even after IFN-gamma exposure, PBLs remained unresponsive. Furthermore, MSCs differentiated along neuronal lineage suppressed the proliferation of PBLs induced by allogeneic PBLs and mitogens. The mechanisms involved in the immunosuppression may be related to the effect of soluble factors and cell-cell interactions of neuronal differentiated MSCs and PBLs. From the above data we suggested that the low immunogenicity and immunomodulatory function of MSCs in the course of neuronal differentiation in vitro, which will be helpful to further investigation in order to establish the new way for future medical application.     

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.     

Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts

Osteocytes are the most abundant cells in bone and there is increasing evidence that they control bone remodeling via direct cell-to-cell contacts and by soluble factors. In the present study, we have used the MLO-Y4 cell line to study the effect of osteocytes on the proliferation, differentiation and bone-forming capacity of bone marrow mesenchymal stem cells (MSC). Conditioned media (CM) from osteocytic MLO-Y4 and osteoblastic MC3T3-E1 cell lines were collected and added on mouse bone marrow cultures, in which MSC were induced to osteoblasts. There was a significant increase in alkaline phosphatase activity and osteocalcin expression in the presence of MLO-Y4 CM. No such stimulus could be observed with MC3T3-E1 CM. There was almost 4-fold increase in bone formation and up to 2-fold increase in the proliferation of MSC with MLO-Y4 CM. The highly proliferating bone marrow cells were negative for ALP and OCN, suggesting that they could represent early osteoblast precursors. MLO-Y4 CM did not enhance the viability of mature osteoblasts nor protected them of apoptosis. This is the first study to describe soluble signals between osteocytes and osteoblasts and there most likely are several still unidentified or unknown factors in osteocyte CM. We conclude that osteocytes have an active stimulatory role in controlling bone formation.     

Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction

Bone marrow comprises heterogeneous cell populations, of which certain progenitors have demonstrated the ability to differentiate into multiple mesenchymal cell lineages. This study demonstrates the bone marrow stromal cells (BMSCs) with intrinsic plasticity to differentiate into hepatocyte-like phenotypes under in vitro induction of hepatocyte growth factor (HGF). BMSCs isolated from rat femurs and tibias were cultured and passaged 3-4 times in the presence of HGF. Cells were harvested on days 0, 10, and 20 and subjected to examination of any hepatocyte characteristics by flow cytometry, RT-PCR, Western blot, and immunocytochemistry. Expression of albumin and alpha-fetoprotein at both mRNA and protein levels was detectable on day 10. By contrast, c-Met mRNA was significantly decreased in BMSC in the course of HGF induction. Here BMSC was shown to differentiate into hepatocyte-like phenotypes given the HGF induction, as an alternative source for adult stem cell transplantation in liver repair.     

Galanin is highly expressed in bone marrow mesenchymal stem cells and facilitates migration of cells both in vitro and in vivo

Galanin peptide has recently been found to be highly abundant in early embryonic mouse mesenchyme, while galanin and its receptors are expressed in embryonic mouse stem cells. Bone marrow mesenchymal stem cells (BMMSCs) represent the primary source for adult stem cell therapy. In this study we examined the abundance of galanin and its receptors in BMMSCs and evaluated its possible function. Galanin mRNA and protein were highly expressed in BMMSCs cultures up to four passages, while among the three galanin receptor subtypes (GalR1, GalR2, and GalR3) only GalR2 and to a lesser extent GalR3 were expressed. Using chemotaxis and wound assays we found that galanin protein increased the migration of BMMSCs. Furthermore, increased serum galanin levels in a galanin transgenic model enhanced the mobilization (homing) of injected BMMSCs in vivo. These data suggest a role for galanin in BMMSC migration, probably through activation of the GalR2 receptor.     

Recovery of neurological function of ischemic stroke by application of conditioned medium of bone marrow mesenchymal stem cells derived from normal and cerebral ischemia rats

Background

Several lines of evidence have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSC) release bioactive factors and provide neuroprotection for CNS injury. However, it remains elusive whether BM-MSC derived from healthy donors or stroke patients provides equal therapeutic potential. The present work aims to characterize BM-MSC prepared from normal healthy rats (NormBM-MSC) and cerebral ischemia rats (IschBM-MSC), and examine the effects of their conditioned medium (Cm) on ischemic stroke animal model.

Results

Isolated NormBM-MSC or IschBM-MSC formed fibroblastic like morphology and expressed CD29, CD90 and CD44 but failed to express the hematopoietic marker CD34. The number of colony formation of BM-MSC was more abundant in IschBM-MSC than in NormBM-MSC. This is in contrast to the amount of Ficoll-fractionated mononuclear cells from normal donor and ischemic rats. The effect of cm of BM-MSC was further examined in cultures and in middle cerebral artery occlusion (MCAo) animal model. Both NormBM-MSC Cm and IschBM-MSC Cm effectively increased neuronal connection and survival in mixed neuron-glial cultures. In vivo, intravenous infusion of NormBM-MSC Cm and IschBM-MSC Cm after stroke onset remarkably improved functional recovery. Furthermore, NormBM-MSC Cm and IschBM-MSC Cm increased neurogenesis and attenuated microglia/ macrophage infiltration in MCAo rat brains.

Conclusions

Our data suggest equal effectiveness of BM-MSC Cm derived from ischemic animals or from a normal population. Our results thus revealed the potential of BM-MSC Cm on treatment of ischemic stroke.