Murine Mesenchymal Stromal Cells Research Articles

Novel Stem/Progenitor Populations: Implications for Cell Therapy

Mesenchymal stromal cells (MSCs) are increasingly used in human cell therapy protocols. Two major issues, however, have yet to be addressed with this intriguing cell population: the likelihood of true transdifferentiation and isolation of MSC stem cells. To date, neither has been convincingly demonstrated. We have attempted to address both issues here. The demonstration of self‐renewal and differentiation ‐ key criteria for stem cell characteristics ‐ have yet to be accomplished. To address this issue, we rigorously generated true clones from the highly proliferative population of MSCs derived from non‐transformed human umbilical cord perivascular cells (HUCPVCs) that share the properties and immunophenotype of bone marrow‐derived MSCs. We demonstrated a hierarchy of differentiation potential along adipocytic, osteogenic, chondrogenic, myogenic and fibroblastic lineages. Future studies will be aimed at characterizing clones for the regeneration of specific tissues.To address the issue of transdifferentiation, we employed a novel in vitro system in which embryonic rat cardiomyocytes are co‐cultured with adult murine MSCs derived from transgenic animals carrying the cardiac‐specific myosin heavy chain promoter driving green fluorescence protein (GFP). We found that some cells expressed GFP and, additionally, had acquired several cardiac‐specific markers but nevertheless had retained all previously assayed MSC determinants, suggesting the generation of a unique cell population that we also showed had not arisen from cell fusion. Furthermore, electrophysiological studies revealed that these unique GFP+ cells do not generate action potentials or ionic currents typical of cardiomyocytes, indicating that the cells are functionally, stromal and not cardiomyocytes. The presence of this population in vivo was confirmed in studies with a murine experimental acute myocardial infarction model. The implications of these findings on the nature of tissue regeneration with exogenous MSCs will be discussed in detail.

Oct4 Expression and Mesenchymal Stromal Cell Plasticity

Mesenchymal stromal cells (MSCs) have the capacity to differentiate along multiple lineages and are now in cell therapy clinical trials, especially for injured myocardium. Mechanisms mediating tissue regeneration remain unclear and are likely multifactorial. We recently showed that bone marrow-derived murine MSCs can acquire cardiac markers but retain MSCs properties when co-cultured with rat embryonic cardiomyocytes (RECs) (Rose et. al., Stem Cells. Aug7, 2008). The aim of our study was to determine whether expression of the embryonic transcription factor, Oct4 was modulated in this model of MSC plasticity. Wild-type Fvb mouse MSCs (passage 4) were co-cultured with RECs for 5 days and expression of Oct4, Nanog and Sox2, was analyzed by qRT-PCR with mouse-specific primers. Oct4 protein was assessed by immunocytochemistry (ICC) and flow cytometry (FC). The MSCs expressed Oct4, Nanog, and Sox2 transcripts. After co-culture, mRNA levels for Oct4 and Sox2 were upregulated 2.6±1.2-fold (p<0.05) and 2.4±0.4-fold (p<0.05), respectively, compared with MSCs controls, in contrast to Nanog gene expression which remained unchanged (1.2±0.3-fold; p=ns). 83±9% of MSCs nuclei were positive for Oct4 by ICC. To distinguish mouse from rat cells in co-culture, cells were stained with an anti-mouse CD44 antibody which does not cross react with rat CD44. CD44 is expressed on all MSCs and absent on RECs. Flow cytometry showed that Oct4 was over-expressed in CD44+ cells after co-culture. Our data demonstrate that these embryonic transcription factors are constitutively expressed in murine MSCs and that Oct4 and Sox2 transcript levels are increased after co-culture with RECs. Oct4, Nanog and Sox2 are known to maintain pluripotency of embryonic stem cells. Moreover, induced pluripotent stem cells can be generated from mouse fibroblasts by the introduction of Oct4 and Sox2 without the need for Nanog (Cell 126:663, 2006). Our data infer that embryonic transcription factors are involved in MSCs progression to different cell lineages. Our findings suggest new mechanisms that may mediate MSC plasticity.

Mesenchymal Stromal Cells (MSC) Isolated from Human Osteosarcomas Show a High Progenitor Cell Frequency, Typical MSC Morphology, Surface Marker Profile, and Differentiation Capacity, and They Are Considerably Affected by Tyrosine Kinase Inhibitors in Vitro.

Bone-marrow derived mesenchymal stromal cells (MSC, also referred to as mesenchymal stem cells) are multipotent cells with intriguing properties (proliferation and differentiation capacity, stroma function, immune modulation) making them promising candidates for clinical use. Recently, it has been reported that culture-derived MSC can acquire chromosomal abnormalities, and abnormal culture-derived murine MSC have been found to give rise to osteosarcomas in transplantation experiments. Furthermore, MSC have been implicated in osteogenic sarcoma, and we therefore aimed to isolate and characterize MSC from primary human osteosarcomas (OS). Single cell suspensions were prepared from OS specimens and cells could be clearly identified morphologically as tumor cells. Stromal progenitor content was assessed using the CFU-F assay. CFU-F frequencies in the OS specimens were 980 ± 450 colonies per 1 × 105 cells (n=6), which is considerably higher than in normal bone marrow (1.3 ± 0.2 colonies per 1 × 105 cells, n=8, bone marrow from healthy volunteer donors). Culture-derived MSC could be generated from every OS sample tested using standard tissue culture flasks and serum-containing MSC medium. OS-derived MSC were similar to bone marrow (BM) derived MSC showing typical MSC morphology and typical MSC surface marker profile, i.e. cells were positive for CD105, CD73, CD90, CD44, HLA-class I, CD166, and negative for CD45, CD34, CD14, CD19, HLA-DR, and CD31. Furthermore, three of three tested OS-derived MSC samples could be differentiated into the osteoblastic and chondroblastic lineages, and all but one showed adipocytic differentiation capacity. Karyotyping of OS-derived MSC showed that the majority of MSC samples were normal, as were the results of FISH analyses for chromosomes 7, 10, 13, and 17. MSC derived from one OS specimen contained cells that showed a complex abnormal karyotype, which, however, was different from the karyotype of the primary tumor. BM- and OS-derived MSC cultures exposed to the first and second generation tyrosine-kinase inhibitors (TKI) imatinib (IM) and nilotinib (NI) (0 – 10 μM) showed a significant inhibition of MSC growth at every time point studied (0 – 4 weeks). After 4 weeks, BM-derived MSC were reduced by IM to 31.4 ± 12.0% (0.625 μM), 24.6 ± 9.1% (1.25 μM), 23.8 ± 12.3% (2.5 μM), 10.6 ± 0.9% (5.0 μM), and 0.9 ± 0.1% (10.0 μM) compared to controls. The lowest numbers of OS-MSC were observed after 2 weeks exposure to IM (0.625 μM, 6.9 ± 4.6%; 1.25 μM, 8.5 ± 4.5%;2.5 μM, 10.3 ± 6.4%; 5.0 μM, 9.2 ± 4.9%; 10 μM, 0.6 ± 0.1%). BM-MSC and OS-MSC growth was also considerably inhibited by NI, but the effects were less pronounced for most doses and time points studied; minimum numbers of BM-MSC (0.8 ± 0.2% of control) and OS-MSC (0.6 ± 0.1% of control) were observed after 3 weeks culture with 10 μM NI. Taken together, our results show that osteosarcoma samples contain high numbers of mesenchymal progenitor cells and that MSC can be successfully generated from OS. OS-MSC are likely to represent stromal cells, i.e. cancer-associated fibroblasts, rather than the tumor cell population. Interestingly, strong similarities were observed between OSMSC and BM-MSC, implicating that these two cell types are closely related.

Neo-Organoid of Marrow Mesenchymal Stromal Cells Secreting Interleukin-12 for Breast Cancer Therapy

Bone marrow-derived mesenchymal stromal cells (MSCs), beneficial for regenerative medicine applications due to their wide differentiation capabilities, also hold promise as cellular vehicles for the delivery of therapeutic plasma-soluble gene products due to their ease of handling, expansion, and genetic engineering. We hypothesized that MSCs, gene enhanced to express interleukin-12 (IL-12) and then embedded in a matrix, may act as an anticancer neo-organoid when delivered s.c. in autologous/syngeneic hosts. We performed such experiments in mice and noted that primary murine MSCs retrovirally engineered to secrete murine IL-12 can significantly interfere with growth of 4T1 breast cancer cells in vivo, with a more substantial anticancer action achieved when these cells are embedded in a matrix. Plasma of mice that received the IL-12 MSC-containing neo-organoids showed increased levels of IL-12 and IFN-gamma. Histopathologic analysis revealed less tumor cells in implants of 4T1 cells with IL-12 MSCs, and the presence of necrotic tumor islets and necrotic capillaries, suggesting antiangiogenesis. We also showed that the anticancer effect exerted by the IL-12 MSCs is immune mediated because it is absent in immunodeficient mice, is not due to systemic IL-12 delivery, and also occurs in a B16 melanoma model. This study therefore establishes the feasibility of using gene-enhanced MSCs in a cell-based neo-organoid approach for cancer treatment.

Mesenchymal stromal cells genetically engineered to overexpress IGF-I enhance cell-based gene therapy of renal failure-induced anemia

We previously demonstrated that erythropoietin (EPO)-secreting mesenchymal stromal cells (MSC) can be used for the long-term correction of renal failure-induced anemia. The present study provides evidence that coimplantation of insulin-like growth factor I (IGF-I)-overexpressing MSC (MSC-IGF) improves MSC-based gene therapy of anemia by providing paracrine support to EPO-secreting MSC (MSC-EPO) within a subcutaneous implant. IGF-I receptor RNA expression in murine MSC was demonstrated by RT-PCR. Functional protein expression was confirmed by immunoblots and MSC responsiveness to IGF-I stimulation in vitro. IGF-I was also shown to improve MSC survival following staurosporin-induced apoptosis in vitro. A cohort of C57Bl/6 mice was rendered anemic by right kidney electrocoagulation and left nephrectomy. MSC-EPO were subsequently admixed in a bovine collagen matrix and implanted, in combination with MSC-IGF or MSC null, by subcutaneous injection in renal failure mice. In mice receiving MSC-EPO coimplanted with MSC-IGF, hematocrit elevation was greater and enhanced compared with control mice; heart function was also improved. MSC-IGF coimplantation, therefore, represents a promising new strategy for enhancing MSC survival within implanted matrices and for improving cell-based gene therapy of renal anemia.

Design and Fabrication of 3D Porous Scaffolds to Facilitate Cell-Based Gene Therapy

Biomaterials capable of efficient gene delivery by embedded cells provide a fundamental tool for the treatment of acquired or hereditary diseases. A major obstacle is maintaining adequate nutrient and oxygen diffusion to cells within the biomaterial. In this study, we combined the solid free-form fabrication and porogen leaching techniques to fabricate three-dimensional scaffolds, with bimodal pore size distribution, for cell-based gene delivery. The objective of this study was to design micro-/macroporous scaffolds to improve cell viability and drug delivery. Murine bone marrow-derived mesenchymal stromal cells (MSCs) genetically engineered to secrete erythropoietin (EPO) were seeded onto poly-l-lactide (PLLA) scaffolds with different microporosities. Over a period of 2 weeks in culture, an increase in cell proliferation and metabolic activity was observed with increasing scaffold microporosity. The concentration of EPO detected in supernatants also increased with increasing microporosity level. Our study shows that these constructs can promote cell viability and release of therapeutic proteins, and clearly demonstrates their capacity for a dual role as scaffolds for tissue regeneration and as delivery systems for soluble gene products.

Design and Fabrication of 3D Porous Scaffolds to Facilitate Cell-Based Gene Therapy

Biomaterials capable of efficient gene delivery by embedded cells provide a fundamental tool for the treatment of acquired or hereditary diseases. A major obstacle is maintaining adequate nutrient and oxygen diffusion to cells within the biomaterial. In this study, we combined the solid free-form fabrication and porogen leaching techniques to fabricate three-dimensional scaffolds, with bimodal pore size distribution, for cell-based gene delivery. The objective of this study was to design micro-/macroporous scaffolds to improve cell viability and drug delivery. Murine bone marrow-derived mesenchymal stromal cells (MSCs) genetically engineered to secrete erythropoietin (EPO) were seeded onto poly-L-lactide (PLLA) scaffolds with different microporosities. Over a period of 2 weeks in culture, an increase in cell proliferation and metabolic activity was observed with increasing scaffold microporosity. The concentration of EPO detected in supernatants also increased with increasing microporosity level. Our study shows that these constructs can promote cell viability and release of therapeutic proteins, and clearly demonstrates their capacity for a dual role as scaffolds for tissue regeneration and as delivery systems for soluble gene products.

PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts

Conditioned medium (secretome) derived from an enriched stem cell culture stimulates chemotaxis of human fibroblasts. These cells are classified as multipotent murine mesenchymal stromal cells (mMSC) by immunochemical analysis of marker proteins. Proteomic analysis of mMSC secretome identifies nineteen secreted proteins, including extracellular matrix structural proteins, collagen processing enzymes, pigment epithelium-derived factor (PEDF) and cystatin C. Immunodepletion and reconstitution experiments show that PEDF is the predominant fibroblast chemoattractant in the conditioned medium, and immunofluorescence microscopy shows strong staining for PEDF in the cytoplasm, at the cell surface, and in intercellular space between mMSCs. This stimulatory effect of PEDF on fibroblast chemotaxis is in contrast to the PEDF-mediated inhibition of endothelial cell migration, reported previously. These differential functional effects of PEDF toward fibroblasts and endothelial cells may serve to program an ordered temporal sequence of scaffold building followed by angiogenesis during wound healing.

CD34 expression on murine marrow-derived mesenchymal stromal cells: impact on neovascularization

CD34 is a sialomucin often expressed by cells with hemangiopoietic potential and widely serves as a surrogate marker of stem cell potential. Mesenchymal stromal cells (MSCs) also express CD34, although the functional significance of its expression remains undefined. In this study, we determined whether CD34(pos) MSCs are functionally distinct from CD34(null) MSCs. MSCs derived from C57Bl/6 mice were transduced to express the green fluorescent protein (GFP) from which pure CD34(pos) MSC and CD34(null) MSC clones were selected. In vitro, clones were examined by microarray analysis, while in vivo subcutaneous implantation of matrix-embedded MSCs was used to assess cell survival, differentiation, and neovascularization. The flow cytometric phenotype of CD34(pos) and CD34(null) MSCs were similar, as was gene expression of vascular endothelial growth factors (VEGFs) A and B. However, CD34(pos) MSCs upregulated a number of supplementary angiogenesis-associated genes and showed a greater expression of gene associated with vascular differentiation. At 15 days postimplantation, cell survival between CD34(pos) and CD34(null) MSCs was similar, however, CD34(pos) MSCs evoked a significantly greater host-derived response (4.2 +/- 0.7 vs 1.9 +/- 0.5 x 10(6) cells; p < 0.05). GFP-expressing CD34(pos) MSC implants acquired significantly more CD31 expression compared to CD34(null) MSC cells (10.7% +/- 8.4% vs 3.1% +/- 0.6%; p < 0.05), as well as a significantly greater host-derived endothelial cell influx (CD31(+)/CD45(-)). CD34 expression by MSCs correlates with enhanced vasculogenic and angiogenic potential in vivo.

Mesenchymal Stromal Cells Genetically Engineered To Overexpress Insulin-Like Growth Factor-1 Provide Paracrine Support for Cell-Based Erythropoietin Therapy of Chronic Renal Failure-Induced Anemia.

Mesenchymal stromal cells (MSC) are a population of non-hematopoietic progenitors native to the bone marrow that are amenable to genetic engineering, making them attractive delivery vehicles for the in vivo production of therapeutic proteins, such as erythropoietin (Epo). We have previously demonstrated that MSC engineered to secrete Epo can be used for the long-term correction of renal failure-induced anemia [Eliopoulos et al., J Am Soc Nephrol . June 2006]. However, limited long-term transplanted cell survival compromises the efficacy of MSC-based gene therapy approaches. The current study provides evidence that co-implantation of MSC overexpressing Insulin-like growth factor-1 (IGF-I) improves MSC-based gene therapy of anemia by providing paracrine support to Epo-secreting MSC within a synthetic subcutaneous organoid. The IGF-I receptor was found to be expressed in murine MSC by RT-PCR, and protein expression was confirmed by immunoblot. We also demonstrated MSC MAPK pathway responsiveness to IGF-I stimulation in vitro and subsequent improvement of MSC survival following staurosporin-induced apoptosis. Murine MSC were transduced to overexpress either Epo or IGF-I (hereafter MSC-Epo and MSC-IGF) using retroviral vectors. MSC-Epo were subsequently admixed in a collagen matrix and implanted by subcutaneous injection in both naïve mice and a murine model of chronic renal failure, in combination with either MSC-IGF or null MSC. Mice receiving MSC-Epo in conjunction with MSC-IGF experienced a greater and significantly sustained elevation in hematocrit compared to control mice. In addition, mice co-implanted with MSC-IGF and MSC-Epo demonstrated a significant improvement in cardiac function compared to controls. In conclusion, cell-based gene therapy using co-implanted MSC-IGF represents a promising new strategy for the treatment of renal failure-induced anemia, as well as for the improvement of gene-enhanced MSC survival within implanted matrices.

Regulation of MHC Class II Expression and Antigen Processing in Murine and Human Mesenchymal Stromal Cells by IFN-γ, TGF-β, and Cell Density

Mesenchymal stromal cells (MSC) possess immunosuppressive properties, yet when treated with IFN-gamma they acquire APC functions. To gain insight into MSC immune plasticity, we explored signaling pathways induced by IFN-gamma required for MHC class II (MHC II)-dependent Ag presentation. IFN-gamma-induced MHC II expression in mouse MSC was enhanced by high cell density or serum deprivation and suppressed by TGF-beta. This process was regulated by the activity of the type IV CIITA promoter independently of STAT1 activation and the induction of the IFN regulatory factor 1-dependent B7H1/PD-L1 encoding gene. The absence of direct correlation with the cell cycle suggested that cellular connectivity modulates IFN-gamma responsiveness for MHC II expression in mouse MSC. TGF-beta signaling in mouse MSC involved ALK5 and ALK1 TGF-beta RI, leading to the phosphorylation of Smad2/Smad3 and Smad1/Smad5/Smad8. An opposite effect was observed in human MSC where IFN-gamma-induced MHC II expression occurred at the highest levels in low-density cultures; however, TGF-beta reduced IFN-gamma-induced MHC II expression and its signaling was similar as in mouse MSC. This suggests that the IFN-gamma-induced APC features of MSC can be modulated by TGF-beta, serum factors, and cell density in vitro, although not in the same way in mouse and human MSC, via their convergent effects on CIITA expression.

Treatment of diabetic wounds with fetal murine mesenchymal stromal cells enhances wound closure

Diabetes impairs multiple aspects of the wound-healing response. Delayed wound healing continues to be a significant healthcare problem for which effective therapies are lacking. We have hypothesized that local delivery of mesenchymal stromal cells (MSC) at a wound might correct many of the wound-healing impairments seen in diabetic lesions. We treated excisional wounds of genetically diabetic (Db-/Db-) mice and heterozygous controls with either MSC, CD45(+) cells, or vehicle. At 7 days, treatment with MSC resulted in a decrease in the epithelial gap from 3.2 +/- 0.5 mm in vehicle-treated wounds to 1.3 +/- 0.4 mm in MSC-treated wounds and an increase in granulation tissue from 0.8 +/- 0.3 mm(2) to 2.4 +/- 0.6 mm(2), respectively (mean +/- SD, P < 0.04). MSC-treated wounds also displayed a higher density of CD31(+) vessels and exhibited increases in the production of mRNA for epidermal growth factor, transforming growth factor beta 1, vascular endothelial growth factor, and stromal-derived growth factor 1-alpha. MSC also demonstrated greater contractile ability than fibroblast controls in a collagen gel contraction assay. The effects of locally applied MSC are thus sufficient to improve healing in diabetic mice. Possible mechanisms of this effect include augmented local growth-factor production, improved neovascularization, enhanced cellular recruitment to wounds, and improved wound contraction.

Comparative analysis of mesenchymal stromal cells from murine bone marrow and amniotic fluid

Murine mesenchymal stromal cells (mMSC) are a good model for pre-clinical investigations, and alternative sources of mMSC are subject to intensive experiments. In the present study, we obtained mMSCs from amniotic fluid (AF) and compared their characteristics with mMSCs from bone marrow (BM). NMRI mice, 4-6 weeks old, were killed and AF and BM cells collected from those in the second week of pregnancy. MSC were achieved by adhesion to cell culture plastic. Isolated cells were assessed for clonogenic capacity, some surface markers (epitopes) and differentiation potential. We achieved AF mMSC more readily than BM mMSC. Differences concerning colony assay and some surface markers of mMSC derived from these two source cells were observed. Most strikingly, AF mMSC showed no adipogenic differentiation capacity, in contrast with BM mMSC. Our results show that mMSC from AF are an appropriate source for pre-clinical investigation. Furthermore, mMSC from different sources of mice vary in clonogenic capacity, surface markers and differentiation potential.

Chondrogenic differentiation of murine and human mesenchymal stromal cells in a hyaluronic acid scaffold: Differences in gene expression and cell morphology

Chondrogenesis is a complex process characterized by a sequence of different steps that start with the condensation of the cells, followed by the expression of specific components, such as collagens and proteoglycans. We evaluated in vitro chondrogenic differentiation of C3H10T1/2 murine mesenchymal cells and compared them with human mesenchymal stromal cells (h-MSCs) in a hyaluronic acid scaffold. We analyzed (from day 0 to day 28) cellular morphology, proliferation, and chondrogenic/osteogenic gene expression at different time points. Our data demonstrate that, during chondrogenic differentiation, murine cells proliferate both in the absence and presence of TGFbeta, while h-MSCs require the presence of this activating factor. Murine cells, even if viable, differentiate on hyaluronan scaffold, maintain a fibroblastic morphology, and form a capsule outside the scaffold. At the mRNA level, murine cells showed a decrease in collagen type I combined with a significant increase in collagen type II (from day 0), and aggrecan (on day 28), as found for h-MSCs. Immunohistochemical data confirmed that chondrogenic differentiation of murine cells, induced by TGFbeta, occurred only in some restricted areas inside the scaffold that were positive to collagen type II, but did not show a cartilage-like tissue structure, as we had found using h-MSCs. These data demonstrate that C3H10T1/2 murine cells, widely used as a chondrogenic model, show a different sequence of chondrogenic events in hyaluronic acid scaffold, compared with primary h-MSCs.