Human Multipotent Stromal Cells Research Articles

Periostin conditions the matrix to generate a niche for islet regeneration.

Islet replacement via the Edmonton Protocol offers proof of concept that transient insulin independence can be achieved in patients with diabetes (1–4). However, the need for life-long immunosuppression, an extreme shortage of human islets, along with graft failure due to rejection and continued autoimmunity, limits the widespread application of this approach. Although recent progress has been made in the derivation of “ -like” cells from human pluripotent stem cell sources (embryonic stem cells or induced pluripotent stem cells) (5–10), inducing the replication of surviving -cells in situ (11, 12) and/or stimulating new islet formation from progenitor cells within the pancreas (islet neogenesis) (13–16) represents the most direct way to increase -cell mass. Studies in both rodents and humans suggest endogenous islet repair mechanisms may remain intact during diabetes and can be activated by cellor pharmacologically-mediated stimuli. Evidence of ongoing -cell regeneration has been observed in type 1 diabetes (T1D) patients at early onset (17) or many years after diagnosis (18). Indeed, recent analysis of T1D patients with disease duration of more than 50 years (medalists) has uncovered sustained c-peptide and proliferating -cells within the pancreas in the face of ongoing autoimmunity (19). Moreover, the increase in -cell mass in response to pregnancy suggests that the human endocrine pancreas has the capacity to regenerate if we can understand how to “tip the balance” in favor of islet regeneration vs destruction during diabetes. Interestingly, increased -cell replication was first suggested as the mechanism for increased -cell mass during pregnancy, but a recent autopsy study of human pancreases during pregnancy showed an increased proportion of small islets and increased number of insulin cells in the ducts but no change in -cell replication or apoptosis frequency (20). Nonetheless, novel strategies to optimize islet regeneration, either by stimulating -cell replication or by harnessing endogenous islet neogenic processes within the diabetic pancreas, remain under intense investigation for the treatment of diabetes. In initial publications documenting endogenous islet regeneration by stem cells, transplantation of bone marrow-derived c-kit cells reduced hyperglycemia in mice with streptozotocin (STZ)-induced -cell deletion (21). Importantly, transplanted stem cells did not acquire insulin expression as previously thought. In contrast, donor cells surrounded regenerating islets or engrafted in ductal regions and stimulated proliferation within recipient islets via undetermined paracrine activities (21). Subsequently, other preclinical and early clinical reports have shown that both human hematopoietic (22–26) or multipotent stromal cell (25–28), also known as mesenchymal stem cell (MSC) lineages, can enhance islet regeneration. Furthermore, islet regeneration and immune protection may be achieved by coinfusion of murine hematopoietic cells with MSC in autoimmune non-obese diabetic mice (29). Although this concept termed “stem cell-stimulated islet regeneration” has emerged as a central process for pancreas repair, either by stimulating -cell proliferation (11, 24– 26) or by initiating new islet formation from ductal or islet-derived precursors (14–16, 25, 26), the molecular signaling mechanisms by which distinct stem and progenitor cell types mediate islet recovery are not well understood. More importantly, recent controversy of the role of betatrophin in islet regeneration (30, 31) has underscored

Leukocyte transmigration into tissue-engineered constructs is influenced by endothelial cells through Toll-like receptor signaling.

IntroductionInflammation plays a crucial role in tissue regeneration, wound healing, and the success of tissue-engineered constructs. The aim of this study was to investigate the influence of human umbilical vein endothelial cells (ECs) on leukocyte transmigration when co-cultured with primary human bone marrow-derived multipotent stromal cells (MSCs).MethodsMSCs with and without ECs were cultured in poly (L-lactide-co-1, 5-dioxepan-2-one) (poly (LLA-co-DXO)) scaffolds for 1 week in vitro in a bioreactor system, after which they were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. After 1 and 3 weeks, scaffolds were retrieved, and the mRNA expression of interleukin 1-beta (IL-1β), IL-6, IL-10, hypoxia-inducible factor 1-alpha (HIF-1α), HIF-1β, and mammalian target of rapamycin was examined by real-time reverse transcription-polymerase chain reaction. Furthermore, immunofluorescent staining was performed for IL-1β, IL-6, neutrophils, and CD11b. In addition, Western blotting was done for IL-1β and IL-6. Leukocyte transmigration genes and genes in Toll-like receptor pathways, expressed by MSCs cultured in vitro with or without ECs, were further investigated with a microarray dataset.ResultsIn vitro, genes involved in leukocyte transmigration and Toll-like receptor pathways were clearly influenced by the addition of ECs. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) and cadherin-5 (CDH5), both genes involved in leukocyte transmigration, were expressed significantly higher in the MSC/EC group.In vivo, the MSC/EC group showed higher mRNA expression of hypoxia-inducible factors HIF-1α and HIF-1β. The mRNA expression of anti-inflammatory cytokine IL-10 showed no significant difference, whereas the mRNA and protein expression of pro-inflammatory cytokines IL-1β and IL-6 were lower in the MSC/EC group. The quantitative analysis of immunofluorescent staining revealed a significant difference in the number of neutrophils migrating into constructs, with the highest density found in the MSC/EC group. The number of macrophages positive for IL-6 and CD11b was significantly reduced in the MSC/EC group.ConclusionsThe recruitment of leukocytes into tissue-engineered constructs with MSCs is strongly influenced by the addition of ECs via activation of leukocyte transmigration and Toll-like receptor pathways.

Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth.

Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF)-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs), but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

Interleukin-1 beta enhances human multipotent mesenchymal stromal cell proliferative potential and their ability to maintain hematopoietic precursor cells

Multipotent mesenchymal stromal cells (MMSCs) have been demonstrated to produce mature stromal cells and maintain hematopoietic progenitor cells (HPC). It was previously demonstrated that interleukin-1 beta (IL-1 beta) stimulates the growth of the stromal microenvironment in vivo.The aim of this study was to investigate the effect of IL-1 beta treatment of human MMSCs on their proliferative potential, gene expression, immunomodulating properties, and their ability to support HPCs in vitro.Human bone marrow-derived MMSCs were cultivated in standard conditions or with IL-1 beta. The cumulative cell production was assessed for five passages. After withdrawal of IL-1 beta, MMSC clonal efficiency was investigated, and the maintenance of HPCs on top of MMSCs layers was estimated using cobblestone area forming cell (CAFC) and long-term culture initiating cell (LTC-IC) assays. The effect of untreated MMSCs or MMSCs pretreated with IL-1 beta on lymphocyte proliferation was studied by CFSE staining. The relative expression level of various genes by MMSCs was analyzed using RT-qPCR.The administration of IL-1 beta elevated MMSCs clonal efficiency and total cell production but did not affect lymphocyte proliferation. MMSCs pretreatment with IL-1 beta enhanced their ability to maintain HPCs, as detected by CAFC assay, and it altered the expression levels of genes participating in HPC regulation by stromal cells, e.g., adhesion molecules (ICAM1) and growth factors (SDF1).This study revealed the ability of IL-1 beta to stimulate MMSCs proliferation and enhance their potential to maintain HPCs. MMSCs are considered a stromal niche component in vitro. The combined in vitro and previous in vivo data suggest that IL-1 beta is a systemic regulator of the stromal microenvironment.

Decellularized Cartilage-Derived Matrix as Substrate for Endochondral Bone Regeneration

Following an endochondral approach to bone regeneration, multipotent stromal cells (MSCs) can be cultured on a scaffold to create a cartilaginous callus that is subsequently remodeled into bone. An attractive scaffold material for cartilage regeneration that has recently regained attention is decellularized cartilage-derived matrix (CDM). Since this material has shown potential for cartilage regeneration, we hypothesized that CDM could be a potent material for endochondral bone regeneration. In addition, since decellularized matrices are known to harbor bioactive cues for tissue formation, we evaluated the need for seeded MSCs in CDM scaffolds. In this study, ectopic bone formation in rats was evaluated for CDM scaffolds seeded with human MSCs and compared with unseeded controls. The MSC-seeded samples were preconditioned in chondrogenic medium for 37 days. After 8 weeks of subcutaneous implantation, the extent of mineralization was significantly higher in the MSC-seeded constructs versus unseeded controls. The mineralized areas corresponded to bone formation with bone marrow cavities. In addition, rat-specific bone formation was confirmed by collagen type I immunohistochemistry. Finally, fluorochrome incorporation at 3 and 6 weeks revealed that the bone formation had an inwardly directed progression. Taken together, our results show that decellularized CDM is a promising biomaterial for endochondral bone regeneration when combined with MSCs at ectopic locations. Modification of current decellularization protocols may lead to enhanced functionality of CDM scaffolds, potentially offering the prospect of generation of cell-free off-the-shelf bone regenerative substitutes.

Rapidly self-renewing human multipotent marrow stromal cells (hMSC) express sialyl Lewis X and actively adhere to arterial endothelium in a chick embryo model system.

BackgroundThere have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing.MethodsSialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC.ResultshMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC.ConclusionsThe results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route.

Gene markers of cellular aging in human multipotent stromal cells in culture.

IntroductionHuman multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined.MethodsCommercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology.ResultsThe phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7.ConclusionsOur results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development of assays to test the quality of MSCs before clinical use.

The marine sponge-derived inorganic polymers, biosilica and polyphosphate, as morphogenetically active matrices/scaffolds for the differentiation of human multipotent stromal cells: potential application in 3D printing and distraction osteogenesis.

The two marine inorganic polymers, biosilica (BS), enzymatically synthesized from ortho-silicate, and polyphosphate (polyP), a likewise enzymatically synthesized polymer consisting of 10 to >100 phosphate residues linked by high-energy phosphoanhydride bonds, have previously been shown to display a morphogenetic effect on osteoblasts. In the present study, the effect of these polymers on the differential differentiation of human multipotent stromal cells (hMSC), mesenchymal stem cells, that had been encapsulated into beads of the biocompatible plant polymer alginate, was studied. The differentiation of the hMSCs in the alginate beads was directed either to the osteogenic cell lineage by exposure to an osteogenic medium (mineralization activation cocktail; differentiation into osteoblasts) or to the chondrogenic cell lineage by incubating in chondrocyte differentiation medium (triggering chondrocyte maturation). Both biosilica and polyP, applied as Ca2+ salts, were found to induce an increased mineralization in osteogenic cells; these inorganic polymers display also morphogenetic potential. The effects were substantiated by gene expression studies, which revealed that biosilica and polyP strongly and significantly increase the expression of bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase (ALP) in osteogenic cells, which was significantly more pronounced in osteogenic versus chondrogenic cells. A differential effect of the two polymers was seen on the expression of the two collagen types, I and II. While collagen Type I is highly expressed in osteogenic cells, but not in chondrogenic cells after exposure to biosilica or polyP, the upregulation of the steady-state level of collagen Type II transcripts in chondrogenic cells is comparably stronger than in osteogenic cells. It is concluded that the two polymers, biosilica and polyP, are morphogenetically active additives for the otherwise biologically inert alginate polymer. It is proposed that alginate, supplemented with polyP and/or biosilica, is a suitable biomaterial that promotes the growth and differentiation of hMSCs and might be beneficial for application in 3D tissue printing of hMSCs and for the delivery of hMSCs in fractures, surgically created during distraction osteogenesis.

Therapeutic Effect of Mesenchymal Multipotent Stromal Cells on Memory in Animals with Alzheimer-Type Neurodegeneration

Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

Human placental multipotent mesenchymal stromal cells modulate trophoblast migration via Rap1 activation

IntroductionLittle is known about the interaction between human placental multipotent mesenchymal stromal cell (hPMSC) and trophoblast. We hypothesize that hPMSCs produce hepatocyte growth factor (HGF) which may interact with trophoblasts and regulate their migration during placentation. MethodshPMSCs were isolated from term placentas and conditioned medium was collected after 2 days of culture in hypoxic (<1% O2) or control (20% O2) conditions. Selective agonist and inhibitor or siRNA for protein kinase A (PKA) or Rap1 were combined with Rap1-GTP pull down assays, flow cytometry, integrin β1 activation assays and adhesion and migration studies to investigate HGF signaling effects in trophoblasts. The hPMSC abundance and HGF level in preeclamptic placentas were compared with gestational age-matched controls. ResultsHGF was expressed by hPMSCs and was decreased in hypoxia. HGF induced cAMP production and Rap1 activation in trophoblasts, which in turn activated integrin β1. The HGF and PKA activator 6-Bnz-cAMP induced Rap1 activation with increased trophoblast adhesion and migration. The alterations were inhibited by PKA inhibitor H89 or Rap1 siRNA. HGF and cAMP expression were reduced in preeclamptic placentas. hPMSC number was decreased in preeclamptic placenta compared to controls (0.68 ± 0.1% vs. 1.32 ± 0.5%; P = 0.026). hPMSC conditioned medium enhanced trophoblast migration which was inhibited by c-Met blocking antibody, but migration was reduced by conditioned medium from hPMSC cultured in hypoxia. ConclusionshPMSCs secrete HGF and increase trophoblast cAMP production. The cAMP effector PKA modulates adhesion and migration of trophoblast via signaling to Rap1 and integrin β1.

Multipotent mesenchymal stromal cells protect against kidney injury

Multipotent mesenchymal stromal cells protect against kidney injury

Multipotent stromal cells skew monocytes towards an anti-inflammatory interleukin-10-producing phenotype by production of interleukin-6

Multipotent stromal cells have immunomodulatory capacities and have been used in transplantation and autoimmune diseases. One of the effects of multipotent stromal cells involves the inhibition of dendritic cell differentiation. Since interleukin-6 and interleukin-10 are known to play a role in inhibiting immature dendritic cell differentiation, we hypothesized that these cytokines may also mediate the inhibitory effect of human multipotent stromal cells in immature dendritic cell differentiation. In order to test this hypothesis monocytes were cultured with interleukin-4 and granulocyte-monocyte colony-stimulating factor in the presence or absence of culture-expanded bone marrow-derived multipotent stromal cells. Neutralization and cytokine-depletion strategies were applied to reveal the cellular source and effect of interleukin-6 and interleukin-10. Addition of multipotent stromal cells to monocyte cultures significantly reduced the generation of immature dendritic cells (CD14(-)CD1a(+)) and resulted in the generation of CD14(+)CD1a(-) cells that displayed a significantly reduced immunostimulatory effect. We found that culture supernatants of co-cultures of multipotent stromal cells and monocytes contained higher concentrations of interleukin-6 and interleukin-10. Multipotent stromal cells produced interleukin-6 and neutralizing this interleukin-6 reversed the inhibitory effect of the multipotent cells. Interleukin-10 was not produced by multipotent stromal cells, but exclusively by monocytes after exposure to multipotent stromal cell-produced interleukin-6. In conclusion, through constitutive production of interleukin-6, multipotent stromal cells prevent the differentiation of monocytes towards antigen-presenting immunogenic cells and skew differentiation towards an anti-inflammatory interleukin-10-producing cell type.

The Globoseries Glycosphingolipid SSEA-4 Is a Marker of Bone Marrow-Derived Clonal Multipotent Stromal Cells In Vitro and In Vivo

The therapeutic potential of multipotent stromal cells (MSC) may be enhanced by the identification of markers that allow their discrimination and enumeration both in vivo and in vitro. Here, we investigated the ability of embryonic stem cell-associated glycosphingolipids to isolate human MSC from both whole-bone-marrow (BM) and stromal cell cultures. Only SSEA-4 was consistently expressed on cells within the CD45loCD105hi marrow fraction and could be used to isolate cells with the capacity to give rise to stromal cultures containing MSC. Human stromal cultures, generated in either the presence or absence of serum, contained heterogeneous cell populations discriminated by the quantity of SSEA-4 epitopes detected on their surface. A low level of surface SSEA-4 (SSEA-4lo) correlated with undetectable levels of the α2,3-sialyltransferase-II enzyme required to synthesize SSEA-4; a reduced proliferative potential; and the loss of fat-, bone-, and cartilage-forming cells during long-term culture. In vitro, single cells with the capacity to generate multipotent stromal cultures were detected exclusively in the SSEA-4hi fraction. Our data demonstrate that a high level of surface epitopes for SSEA-4 provides a definitive marker of MSC from human BM.

Improved proteomic profiling of the cell surface of culture-expanded human bone marrow multipotent stromal cells

A comprehensive analysis of the membrane proteome is essential to explain the biology of multipotent stromal cells and identify reliable protein biomarkers for the isolation as well as tracking of cells during differentiation and maturation. However, proteomic analysis of membrane proteins is challenging and they are noticeably under-represented in numerous proteomic studies. Here we introduce new approach, which includes high pressure-assisted membrane protein extraction, protein fractionation by gel-eluted liquid fraction entrapment electrophoresis (GELFREE), and combined use of liquid chromatography MALDI and ESI tandem mass spectrometry. This report presents the first comprehensive proteomic analysis of membrane proteome of undifferentiated and culture-expanded human bone marrow multipotent stromal cells (hBM-MSC) obtained from different human donors. Gene ontology mapping using the Ingenuity Pathway Analysis and DAVID programs revealed the largest membrane proteomic dataset for hBM-MSC reported to date. Collectively, the new workflow enabled us to identify at least two-fold more membrane proteins compared to published results on hBM-MSC. A total of 84 CDs were identified including 14 CDs identified for the first time. This dataset can serve as a basis for further exploration of self-renewal, differentiation and characterization of hBM-MSC.

Ischemia Is the Prime but Not the Only Cause of Human Multipotent Stromal Cell Death in Tissue-Engineered Constructs In Vivo

Local tissue ischemia is a prime cause responsible for the massive cell death in tissue-engineered (TE) constructs observed postimplantation. To assess the impact of ischemia on the death of implanted human multipotent stromal cells (hMSCs), which have great potential for repairing damaged tissues, we hereby investigated the in vivo temporal and spatial fate of human Luc-GFP-labeled MSCs within fibrin gel/coral scaffolds subcutaneously implanted in nude mice. In vivo bioluminescence imaging monitoring and histological analyses of the constructs tested confirmed the irremediable death of hMSCs over 30 days postimplantation. The kinetics of expression of three hypoxic/ischemic markers (HIF-1α, LDH-A, and BNIP3) was also monitored. Our results provided evidence that hMSCs located within the core of implanted constructs died faster and predominantly and strongly expressed the aforementioned ischemic markers. In contrast, cells located in the outer regions of TE constructs were reperfused by neovascularization and were still viable (as evidenced by their ex-vivo proliferative potential) at day 15 postimplantation. These results support the explanation that in the central part of the constructs tested, death of hMSCs was due to ischemia, whereas in the periphery of these constructs, cell death was due to another mechanism that needs to be elucidated.

Toll-Like Receptor 3 and Suppressor of Cytokine Signaling Proteins Regulate CXCR4 and CXCR7 Expression in Bone Marrow-Derived Human Multipotent Stromal Cells

BackgroundThe use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to “danger” signals – by-products of damaged, infected or inflamed tissues – via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC.Methodology/Principal FindingsWe demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization.Conclusion/SignificanceThese results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals.

Spontaneous Formation of Tumorigenic Hybrids between Breast Cancer and Multipotent Stromal Cells Is a Source of Tumor Heterogeneity

Breast cancer progression involves cancer cell heterogeneity, with generation of invasive/metastatic breast cancer cells within populations of nonmetastatic cells of the primary tumor. Sequential genetic mutations, epithelial-to-mesenchymal transition, interaction with local stroma, and formation of hybrids between cancer cells and normal bone marrow-derived cells have been advocated as tumor progression mechanisms. We report herein the spontaneous in vitro formation of heterotypic hybrids between human bone marrow-derived multipotent stromal cells (MSCs) and two different breast carcinoma cell lines, MDA-MB-231 (MDA) and MA11. Hybrids showed predominantly mesenchymal morphological characteristics, mixed gene expression profiles, and increased DNA ploidy. Both MA11 and MDA hybrids were tumorigenic in immunodeficient mice, and some MDA hybrids had an increased metastatic capacity. Both in culture and as xenografts, hybrids underwent DNA ploidy reduction and morphological reversal to breast carcinoma-like morphological characteristics, while maintaining a mixed breast cancer-mesenchymal expression profile. Analysis of coding single-nucleotide polymorphisms by RNA sequencing revealed genetic contributions from both parental partners to hybrid tumors and metastasis. Because MSCs migrate and localize to breast carcinoma, our findings indicate that formation of MSC-breast cancer cell hybrids is a potential mechanism of the generation of invasive/metastatic breast cancer cells. Our findings reconcile the fusion theory of cancer progression with the common observation that breast cancer metastases are generally aneuploid, but not tetraploid, and are histopathologically similar to the primary neoplasm.

In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

BackgroundMultipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs) possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs) and adult dermal skin (hADSSCs) using explants cultures and were compared with bone marrow (hMSC-TERT) and adipose tissue-derived mesenchymal stem cells (hADMSCs) for their potential differentiation into osteoblasts, adipocytes, and endothelial cells.ResultsConcordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC), with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA) was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues.ConclusionsOur data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs represents a novel source of stem/stromal cells for tissue regeneration and the vascularization of engineered tissues. Moreover, the CD146 investigations suggested that the microenvironmental niche might contribute to direct stromal cells multipotency toward certain lineages, which warrants further investigation.

Neurosteroid Hormones Modulate the Differentiation of Adult Human Multipotent Mesenchymal Stromal Cells

Objective: To examine the effect of neurosteroids on human multipotent mesenchymal stromal cell (hMSCs) neuroglial differentiation. Materials and methods: Human MSCs were isolated and expanded in vitro. The expression of neurosteroid receptors by hMSCs was examined using immunocytochemistry and the effect of neurosteroids on the proliferation and differentiation of hMSCs was investigated using immunocytochemistry and the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) survival assay. Results: Human MSCs express receptors for neurosteroids. Nestin expression is decreased by neurosteroids. Neurosteroids also exert differential effects depending on the gender of the hMSC donor; dihydrotestosterone (DHT) was responsible for maximal A2B5 expression in hMSCs from male donors whereas 17-β estradiol (E2) exerted the greatest effect on differentiation of ‘female’ hMSCs. Maximal effects of E2 and DHT were observed at a concentration of 100nM, progesterone (PROG) at 250nM. The neurosteroid-induced increase in oligodendroglial differentiation was abrogated by specific receptor antagonists for E2, PROG and DHT. High concentrations of neurosteroids were toxic for hMSCs, irrespective of gender. Conclusions: These results suggest a significant role for neurosteroid hormones in the differentiation of hMSCs and may have important implications for the development of MSC transplantation therapy for multiple sclerosis and in autoimmune diseases.

Assessment of DNA Damage in Human Bone Marrow Cells and Multipotent Mesenchymal Stromal Cells

We carried out a comparative analysis of DNA damage (percentage of DNA in comet tail) and frequencies of comets in apoptotic cells in BM samples and cultures of BM multipotent mesenchymal stromal cells at different terms of culturing (passages 3-11). The levels of DNA damage in mesenchymal stromal cells remained unchanged during culturing (3.5 ± 0.9 and 4.4 ± 1.2%) and did not differ from those in BM cells (3.6 ± 0.8%). In BM samples, 10-28% atypical cells with high level of DNA damage were detected. In mesenchymal stromal cells, 2.8 ± 0.9 and 3.6 ± 1.8% apoptotic cells were detected at early and late passages, respectively.