Differentiation capacity of stromal fibroblast-like cells from human bone marrow, adipose tissue, hair follicle dermal papilla and derma

We compared the morphology and differentiation capacity of human stromal cells derived from bone marrow (BMSC), adipose tissue (ATSC), hair follicle dermal papilla (DPC) and dermal fibroblasts (DFb). All cells have fibroblast-like morphology. ATSC and DPC cells expressed stem cell the surface markers CD105, CD49d, and STRO-1, which were revealed immunocytochemically. CD49d was not found on BMSC. The low expression of CD49d and STRO-1 was registered in the DFb population. ATSC, BMSC, and DPC have similar capacities for adipo- and osteogenic differentiation. These cells, cultured in appropriate induction media, alter the phenotype and synthesize specific proteins. However, the expression of differentiation in the DPC population is lower than in ATSC and BMSC cultures. We propose that these cell populations have primitive progenitor cells with properties of mesenchymal stem cells.     

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

A population of serum deprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures.     

Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells

Bone marrow stromal cells (BMSC) can differentiate into various cell types including myocytes, which may be valuable in cellular therapy of myocardial infarction. In an attempt to increase the myogenic commitment of BMSC, we investigated the extent of conversion induced by the demethylation agent 5-azacytidine. BMSC isolated from the adult rat tibia were exposed in culture to 5microM 5-azacytidine for 24h, 1 day after seeding. The treatment was repeated at weekly intervals and the expression of muscle-specific proteins and genes was assessed. The results revealed that cultured cells lost the native expression of osteocalcin and alkaline phosphatase as a function of time and began to express connexin 43. Exposure to 5-azacytidine of BMSC induced, at 14 days, a myocyte-resembling phenotype that included the expression of muscle-specific proteins (sarcomeric alpha-actin, troponin T, desmin, alpha-actinin, and GATA-4) and genes (GATA-4, myoD, desmin, and alpha-actinin), numerous mitochondria and myofilaments; however, the latter did not form sarcomeres. Although some of these myogenic markers also appeared in untreated cells, exposure to 5-azacytidine induced an enhanced response of calcium channels, as well as a threefold increase in desmin and myoD gene expression and a twofold increase in alpha-actinin gene and protein expression above the control values. In conclusion, the results demonstrate a promoting effect of 5-azacytidine on the expression of muscle-specific proteins and genes in BMSC in culture. Notably, the myogenic differentiation takes place over a short period of time. Priming of mesenchymal cells to cardiomyogenic differentiation may have significant applications in cellular approaches to ameliorate muscle loss after myocardial ischemia.     

Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.     

Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors

Bone marrow stromal cells (BMSC) have attracted interest through their possible use for cell therapy in neurological diseases. Recent reports demonstrated that these cells are able to migrate and have potential for neuronal differentiation after transplantation into brain parenchyma. The objective of this work was determine whether rat BMSC express NGF and GDNF, in order to study its potential application for treatment of neurodegenerative diseases. BMSC were harvested from male rats and cultured in DMEM supplemented with 20% fetal bovine serum. At passage 6 the total RNA was isolated using TriZol reactive. RT-PCRs to evaluate the expression of NGF and GDNF using specific primers were carried out. Our results indicate that rat BMSC have potential to produce NGF and GDNF. We have not found any report in favor of GDNF or NGF production from rat BMSC.     

Gene expression profile of human bone marrow stromal cells determined by restriction fragment differential display analysis

Using an in vitro osteogenic culture system, we carried out a restriction fragment differential display (RFDD-PCR) to identify genes expressed by these cells in their undifferentiated stage and not expressed, or expressed at a lower level, in a closely related but distinct cell type: bone marrow stromal cells (BMSC)-derived osteoblasts (BDO). Forty-seven candidate regulated genes, selected by RFDD, were analyzed by RT-PCR analysis in three cell clones and in primary cultures from seven different donors. A subset of three genes were confirmed as upregulated in BMSC relative to BDO in every primary culture and cloned population examined: betaIG-h3, IGFbp3, and LOXL2. Their differential expression was confirmed by Northern analysis and the corresponding proteins were detected by immunolocalization in BMSC.     

MT1-MMP down-regulates the glucose 6-phosphate transporter expression in marrow stromal cells: a molecular link between pro-MMP-2 activation, chemotaxis, and cell survival

Bone marrow-derived stromal cells (BMSC) are avidly recruited by experimental vascularizing tumors, which implies that they must respond to tumor-derived growth factor cues. In fact, BMSC chemotaxis and cell survival are regulated, in part, by the membrane type-1 matrix metalloproteinase (MT1-MMP), an MMP also involved in pro-MMP-2 activation and in degradation of the extracellular matrix (ECM). Given that impaired chemotaxis was recently observed in bone marrow cells isolated from a glucose 6-phosphate transporter-deficient (G6PT-/-) mouse model, we sought to investigate the potential MT1-MMP/G6PT signaling axis in BMSC. We show that MT1-MMP-mediated activation of pro-MMP-2 by concanavalin A (ConA) correlated with an increase in the sub-G1 cell cycle phase as well as with cell necrosis, indicative of a decrease in BMSC survival. BMSC isolated from Egr-1-/- mouse or MT1-MMP gene silencing in BMSC with small interfering RNA (siMT1-MMP) antagonized both the ConA-mediated activation of pro-MMP-2 and the induction of cell necrosis. Overexpression of recombinant full-length MT1-MMP triggered necrosis and this was signaled through the cytoplasmic domain of MT1-MMP. ConA inhibited both the gene and protein expression of G6PT, while overexpression of recombinant G6PT inhibited MT1-MMP-mediated pro-MMP-2 activation but could not rescue BMSC from ConA-induced cell necrosis. Cell chemotaxis in response to the tumorigenic growth factor sphingosine 1-phosphate was significantly abrogated in siMT1-MMP BMSC and in chlorogenic acid-treated BMSC. Altogether, we provide evidence for an MT1-MMP/G6PT signaling axis that regulates BMSC survival, ECM degradation, and mobilization. This may lead to optimized clinical applications that use BMSC as a platform for the systemic delivery of therapeutic or anti-cancer recombinant proteins in vivo.     

The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.     

Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds

In this work, we investigated whether osteoinductive constructs can be generated by isolation and expansion of sheep bone marrow stromal cells (BMSC) directly within three-dimensional (3D) ceramic scaffolds, bypassing the typical phase of monolayer (2D) expansion prior to scaffold loading. Nucleated cells from sheep bone marrow aspirate were seeded into 3D ceramic scaffolds either by static loading or under perfusion flow and maintained in culture for up to 14 days. The resulting constructs were exposed to enzymatic treatment to assess the number and lineage of extracted cells, or implanted subcutaneously in nude mice to test their capacity to induce bone formation. As a control, BMSC expanded in monolayer for 14 days were also seeded into the scaffolds and implanted. BMSC could be isolated and expanded directly in the 3D ceramic scaffolds, although they proliferated slower than in 2D. Upon ectopic implantation, the resulting constructs formed a higher amount of bone tissue than constructs loaded with the same number of 2D-expanded cells. Constructs cultivated for 14 days generated significantly more bone tissue than those cultured for 3 days. No differences in bone formation were found between samples seeded by static loading or under perfusion. In conclusion, the culture of bone marrow nucleated cells directly on 3D ceramic scaffolds represents a promising approach to expand BMSC and streamline the engineering of osteoinductive grafts.     

Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.     

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy

Background aimsTransplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model.MethodsASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10⁵ cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed.ResultsASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group.ConclusionsCompared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.     

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.     

STRO-1, HOP-26 (CD63), CD49a and SB-10 (CD166) as markers of primitive human marrow stromal cells and their more differentiated progeny: a comparative investigation in vitro

There is widespread interest in the use of bone marrow stromal cells (BMSC) for tissue reconstruction and repair and for gene therapy. BMSC represent the differentiated progeny of CFU-F, which however comprise a developmentally heterogeneous population as is reflected in the cellular heterogeneity of the cell populations to which they give rise. We have compared the efficacy of monoclonal antibodies recognising a series of stromal antigens, viz. STRO-1, HOP-26, CD49a and SB-10/CD166, as tools for the enrichment of CFU-F prior to culture and as developmental markers for culture-expanded BMSC. In freshly isolated bone marrow mononuclear cells (BMMNC), the proportion of antigen-positive cells was 27%, 46%, 5% and 19% for STRO-1, HOP-26, CD49a and CD166, respectively. All CD49a⁺ cells co-expressed STRO-1. The degree of CFU-F enrichment obtained with anti-CD49a (~18-fold) by a one-pass immunoselection strategy was significantly greater than that of all other antibodies tested. BMSC expressed higher levels of all antigens investigated (except for HOP-26) compared with BMMNC. Expression of STRO-1 and CD49a remained restricted to a subset of BMSC, whereas all BMSC were SB-10/CD166 positive. Treatment with dexamethasone (10 nM), which promotes the differentiation and further maturation of cells of the osteogenic lineage in this cell culture system, increased the expression of CD49a and HOP-26. The CD49a⁺ and HOP-26⁺ fractions of BMSC were further subdivided by dual-labelling with anti-STRO-1 and B4–78 (an antibody recognising the B/L/K isoform of the enzyme alkaline phosphatase), respectively. By using a variety of criteria, the HOP-26 antigen was identified as CD63, a member of the tetraspanin family of proteins thought to modulate integrin compartmentalisation and signalling.K.S., S.W., C.M.J. and J.A.L. gratefully acknowledge the financial support of the University Bath, the Arthritis Research Campaign and the Wellcome Trust     

Expression of red cell membrane proteins in erythroid precursor cells

Specific antibodies to human glycophorin A and spectrin were used to study the expression of these membrane proteins in normal and pathologic human bone marrow. In immunofluorescence experiments spectrin and glycophorin A are found in 50–60% of the nucleated cells in normal bone marrow. These two proteins are expressed at all stages of red cell differentiation and can be traced at least to the earliest morphologically recognizable nucleated red cell precursor, the proerythroblast; the two proteins are specific for cells of the red cell series and are not found to be expressed in lymphocytic, granulocytic cells or platelets. These conclusions were drawn from studies on bone marrow in patients with a temporary block in erythropoiesis at the level of stem cells or of the pronormoblast. Bone marrow from these individuals either lacked all nucleated cells stainable for glycophorin A and spectrin or contained only pronormoblasts. Similar findings were obtained on spleen cells from mice which were made severely anemic by multiple injections with N-acetyl-phenylhydrazine. Antibodies to a sialoglycoprotein isolated from mouse red cell membranes stain 70–80% of all cells in the spleen of anemic animals, while only 1–2% of such cells are seen in the spleen of normal animals. Spectrin and glycophorin A could be labeled metabolically and isolated using specific antibodies. The human tumor cell line K562 expresses both membrane proteins, but induction experiments with various agents thus far have failed to change their expression.     

Replacement of mouse embryonic fibroblasts with bone marrow stromal cells for use in establishing and maintaining embryonic stem cells in mice

We have investigated the use of BMSC (bone marrow stromal cell) as a feeder cell for improving culture efficiency of ESC (embryonic stem cell). B6CBAF1 blastocysts or ESC stored after their establishment were seeded on to a feeder layer of either SCA-1+/CD45-/CD11b- BMSC or MEF (mouse embryonic fibroblast). Feeder cell activity in promoting ESC establishment from the blastocysts and in supporting ESC maintenance did not differ significantly between BMSC and MEF feeders. However, the highest efficiency of colony formation after culturing of inner cell mass cells of blastocysts was observed with the BMSC line that secreted the largest amount of LIF (leukaemia inhibitory factor). Exogenous LIF was essential for the ESC establishment on BMSC feeder, but not for ESC maintenance. Neither change in stem cell-specific gene expression nor increase in stem cell aneuploidy was detected after the use of BMSC feeder. We conclude that BMSC can be utilized as the feeder of ESC, which improves culture efficiency.