Hypoxic conditioned medium derived from bone marrow mesenchymal stromal cells protects against ischemic stroke in rats

In recent years, studies have shown that the secretome of bone marrow mesenchymal stromal cells (BMSCs) contains many growth factors, cytokines, and antioxidants, which may provide novel approaches to treat ischemic diseases. Furthermore, the secretome may be modulated by hypoxic preconditioning. We hypothesized that conditioned medium (CM) derived from BMSCs plays a crucial role in reducing tissue damage and improving neurological recovery after ischemic stroke and that hypoxic preconditioning of BMSCs robustly improves these activities. Rats were subjected to ischemic stroke by middle cerebral artery occlusion and then intravenously administered hypoxic CM, normoxic CM, or Dulbecco modified Eagle medium (DMEM, control). Cytokine antibody arrays and label-free quantitative proteomics analysis were used to compare the differences between hypoxic CM and normoxic CM. Injection of normoxic CM significantly reduced the infarct area and improved neurological recovery after stroke compared with administering DMEM. These outcomes may be associated with the attenuation of apoptosis and promotion of angiogenesis. Hypoxic preconditioning significantly enhanced these therapeutic effects. Fourteen proteins were significantly increased in hypoxic CM compared with normoxic CM as measured by cytokine arrays. The label-free quantitative proteomics analysis revealed 163 proteins that were differentially expressed between the two groups, including 107 upregulated proteins and 56 downregulated proteins. Collectively, our results demonstrate that hypoxic CM protected brain tissue from ischemic injury and promoted functional recovery after stroke in rats and that hypoxic CM may be the basis of a potential therapy for stroke patients.     

Characterization of reticulofibroblastoid colonies (CFU-RF) derived from bone marrow and long-term marrow culture monolayers

The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.     

Amelioration of streptozotocin-induced diabetes in mice with cells derived from human marrow stromal cells

Background

Pluri-potent bone marrow stromal cells (MSCs) provide an attractive opportunity to generate unlimited glucose-responsive insulin-producing cells for the treatment of diabetes. We explored the potential for human MSCs (hMSCs) to be differentiated into glucose-responsive cells through a non-viral genetic reprogramming approach.

Methods and Findings

Two hMSC lines were transfected with three genes: PDX-1, NeuroD1 and Ngn3 without subsequent selection, followed by differentiation induction in vitro and transplantation into diabetic mice. Human MSCs expressed mRNAs of the archetypal stem cell markers: Sox2, Oct4, Nanog and CD34, and the endocrine cell markers: PDX-1, NeuroD1, Ngn3, and Nkx6.1. Following gene transfection and differentiation induction, hMSCs expressed insulin in vitro, but were not glucose regulated. After transplantation, hMSCs differentiated further and ∼12.5% of the grafted cells expressed insulin. The graft bearing kidneys contained mRNA of insulin and other key genes required for the functions of beta cells. Mice transplanted with manipulated hMSCs showed reduced blood glucose levels (from 18.9+/−0.75 to 7.63+/−1.63 mM). 13 of the 16 mice became normoglycaemic (6.9+/−0.64 mM), despite the failure to detect the expression of SUR1, a K⁺-ATP channel component required for regulation of insulin secretion.

Conclusions

Our data confirm that hMSCs can be induced to express insulin sufficient to reduce blood glucose in a diabetic mouse model. Our triple gene approach has created cells that seem less glucose responsive in vitro but which become more efficient after transplantation. The maturation process requires further study, particularly the in vivo factors influencing the differentiation, in order to scale up for clinical purposes.     

Secretome analysis of human bone marrow derived mesenchymal stromal cells

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis for future comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.     

The immunosuppressive capacity of human mesenchymal stromal cells derived from amnion and bone marrow

Mesenchymal stromal cells derived from amnion (AM-MSCs) can be easily obtained in large quantity by less invasive method in comparison to bone marrow-derived MSCs (BM-MSCs). However, the biological and immunosuppressive properties of AM-MSCs are still poorly characterized. Previous studies demonstrated that BM-MSCs expressed indoleamine 2,3-dioxygenase (IDO) to suppress T-cell responses. This study was designed to address whether IDO contributes to the immunosuppressive function of AM-MSCs. MSCs isolated from amnion were cultured in complete medium similar to BM-MSCs. After culture, AM-MSCs exhibited spindle shape morphology and expressed MSC markers similar to that of BM-MSCs. In addition, AM-MSCs were able to differentiate into adipocytes and osteoblasts. Fascinatingly, AM-MSCs and BM-MSCs exhibited comparable degree of immunosuppressive effect when they were co-cultured with activated T-cells. In addition, IDO secreted by AM-MSCs was responsible for induction of immunosuppressive activities in the same manner as BM-MSCs. Taken together; the results of the present study demonstrate that while AM-MSCs and BM-MSCs show similar immunosuppressive effect, AM-MSCs may have additional advantage over the BM-MSCs in terms of availability. Therefore, AM-MSCs might be considered a potential source for therapeutic applications especially for treatment of immune related diseases.     

Human mesenchymal progenitor cells derived from alveolar bone and human bone marrow stromal cells: a comparative study

The aim of the present study was to evaluate the potential of intraoral harvested alveolar bone as an alternative source of multipotent mesenchymal stromal cells for future applications in oral and maxillofacial tissue engineering. Explant cultures were established from 20 alveolar bone samples harvested from the oblique line immediately before wisdom tooth removal. Morphology and proliferation characteristics of the in vitro expanded cells, referred to as human alveolar bone-derived cells (hABDCs), were studied using phase-contrast microscopy. Immunocytochemical analysis of their surface marker expression was conducted using monoclonal antibodies defining mesenchymal stromal cells. To evaluate their multilineage differentiation potential, hABDCs were induced to differentiate along the osteogenic, adipogenic, and chondrogenic lineage and compared to bone marrow mesenchymal stromal cells (hBMSCs) on mRNA and protein levels applying RT-PCR and cytochemical staining methods. hABDCs showed typical morphological characteristics comparable to those of hBMSCs such as being mononuclear, fibroblast-like, spindle-shaped, and plastic adherent. Immunophenotypically, cells were positive for CD105, CD90, and CD73 while negative for CD45, CD34, CD14, CD79α, and HLA-DR surface molecules, indicating an antigen expression pattern considered typical for multipotent mesenchymal stromal cells. As evidenced by RT-PCR and cytochemistry, hABDCs showed multilineage differentiation and similar chondrogenic and osteogenic differentiation potentials when compared to hBMSCs. Our findings demonstrate that human alveolar bone contains mesenchymal progenitor cells that can be isolated and expanded in vitro and are capable of trilineage differentiation, providing a reservoir of multipotent mesenchymal cells from an easily accessible tissue source.     

Purmorphamine enhances osteogenic activity of human osteoblasts derived from bone marrow mesenchymal cells

Purmorphamine is a novel small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells, but there has been no evaluation of its effect on human cells to date. The aim of this study was to investigate the induction of osteogenic activity by purmorphamine in human osteoblasts differentiated from bone marrow mesenchymal cells. Cells were cultured in 24-well plates at a density of 2x10(4)/well in medium containing 1, 2 or 3 microM purmorphamine, or vehicle. At 7, 14 and 21 days, cell proliferation, viability, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Purmorphamine did not affect cell proliferation or viability, but increased ALP activity and bone-like nodule formation. These results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine.     

Establishment and characterization of a new murine cell line (SR-4987) derived from marrow stromal cells

A new murine cell line designated as SR-4987 was established by treating a long-term bone marrow culture with the supernatant from Y-1 cells which actively produce viral C-particles (MuLV). The line showed a fibrolbast-like morphology and its mesodermal origin was confirmed by immunocytochemical staining. Flow cytometric analysis of DNA index evidenced a tetraploid number of chromosomes whereas cell cycle analysis showed 34.8% of cells in S phase and 60.7% in G1.In vitro growth studies demonstrated a population doubling time of 14.7h, a good plating efficiency (52.3%) and a very poor agar clonogenic capacity (0.6%). SR-4987 was tumorigenic only in syngeneic mice in which sarcomas were induced. The line produced M-CSF in the culture supermatant whereas G-CSF, IL-3 and GM-CSF were not detected. Studies are in progress to assess the production of other cytokines and to verify if same autocrine growth factor is involved in the control of SR-4987 proliferation. Our line provides a further model of stromal cells for studying the interaction between hemopoietic progenitors and their micro-environment, as well as to study factors produced by stromal cells acting as modulators of proliferation and differentiation of related cell populations.     

Multilineage potential of adult human mesenchymal stromal cells derived from bone marrow of patients with polycytaemia vera

Human mesenchymal stromal cells were isolated from the bone marrow of patients with polycyteamia vera (the myeloproliferative disorder) with the aim to characterize the properties of the mesenchymal stromal cells originating from the pathologically affected bone marrow. Their in vitro growth and potential to differentiate were determined. Isolated mesenchymal stromal cells were able to differentiate into three mesenchymal lineages under appropriate cultivation conditions.     

Seasonal fluctuations in the activity of stromal precursor cells of human bone marrow

The results of the cloning of fibroblastic colony-forming units (CFU-F) from the bone marrow of normal sites of the spongy bones were analysed in 250 orthopaedic patients. It has been shown that the activity of CFU-F was changing during a year. The number of negative results of CFU-F's cloning were 33%, 60% and 50% in March, April and October respectively. The absolute values of CFU-F cloning were lower in March and April than in other months. The seasonal changes in the activity of CFU-F in human bone marrow should be taken into consideration when studying the physiology and pathology of the bone and hemopoietic system, and in clinical practice.     

Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells

Human induced pluripotent stem cells (HiPSCs) appear to be highly similar to human embryonic stem cells (HESCs). Using two genetic lineage-tracing systems, we demonstrate the generation of iPSC lines from human pancreatic islet beta cells. These reprogrammed cells acquired markers of pluripotent cells and differentiated into the three embryonic germ layers. However, the beta cell-derived iPSCs (BiPSCs) maintained open chromatin structure at key beta-cell genes, together with?a unique DNA methylation signature that distinguishes them from other PSCs. BiPSCs also demonstrated an increased ability to differentiate into insulin-producing cells both in?vitro and in?vivo, compared with ESCs and isogenic non-beta iPSCs. Our results suggest that the epigenetic memory may predispose?BiPSCs to differentiate more readily into insulin producing cells. These findings demonstrate that HiPSC phenotype may be influenced by their cells of origin, and suggest that their skewed differentiation potential may be advantageous for cell replacement therapy.     

Bortezomib enhances the osteogenic differentiation capacity of human mesenchymal stromal cells derived from bone marrow and placental tissues

Bortezomib (BZB) is a chemotherapeutic agent approved for treating multiple myeloma (MM) patients. In addition, there are several reports showing that bortezomib can induce murine mesenchymal stem cells (MSCs) to undergo osteogenic differentiation and increase bone formation in vivo. MSCs are the multipotent stem cells that have capacity to differentiate into several mesodermal derivatives including osteoblasts. Nowadays, MSCs mostly bone marrow derived have been considered as a valuable source of cell for tissue replacement therapy. In this study, the effect of bortezomib on the osteogenic differentiation of human MSCs derived from both bone marrow (BM-MSCs) and postnatal sources such as placenta (PL-MSCs) were investigated. The degree of osteogenic differentiation of BM-MSCs and PL-MSCs after bortezomib treatment was assessed by alkaline phosphatase (ALP) activity, matrix mineralization by Alizarin Red S staining and the expression profiles of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP. The results showed that 1 nM and 2 nM BZB can induce osteogenic differentiation of BM-MSCs and PL-MSCs as demonstrated by increased ALP activity, increased matrix mineralization and up-regulation of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP as compared to controls. The enhancement of osteogenic differentiation of MSCs by bortezomib may lead to the potential therapeutic applications in human diseases especially patients with osteopenia.     

ES cells derived from cloned embryos in monkey--a jump toward human therapeutic cloning

Therapeutic cloning refers to the derivation of embryonic stem cells （ntESC） from embryos derived from somatic cell nuclear transfer （SCNT） also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus （DNA） removed. The reconstructed oocyte can be activated to divide and develop into an embryo. The process that allows this to happen is termed nuclear reprogramming, and is defined as the mechanism through which a differentiated cell de-differentiates or returns to a totipotent state （capable of giving rise to any cell type, including extra-embryonic） and directs embryonic development [1]. Cells from blastocyst stage cloned embryos can be used to generate ntESC lines. Such cell lines can differentiate into any adult cell type, and have tremendous potential for patient-specific disease therapy [2].