Commitment of human mesenchymal stromal cells to skeletal lineages is independent of their morphogenetic capacity.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent cell populations obtained from fetal and adult tissues. They share some characteristics with limb bud mesodermal cells such as differentiation potential into osteogenic, chondrogenic, and tenogenic lineages and an embryonic mesodermal origin. Although MSCs differentiate into skeletal-related lineages in vitro, they have not been shown to self-organize into complex skeletal structures or connective tissues, as in the limb. In this work, we demonstrate that the expression of molecular markers to commit MSCs to skeletal lineages is not sufficient to generate skeletal elements in vivo. AIM: To evaluate the potential of MSCs to differentiate into skeletal lineages and generate complex skeletal structures using the recombinant limb (RL) system. METHODS: We used the experimental system of RLs from dissociated-reaggregated human placenta (PL) and umbilical cord blood (UCB) MSCs. After being harvested and reaggregated in a pellet, cultured cells were introduced into an ectodermal cover obtained from an early chicken limb bud. Next, this filled ectoderm was grafted into the back of a donor chick embryo. Under these conditions, the cells received and responded to the ectoderm's embryonic signals in a spatiotemporal manner to differentiate and pattern into skeletal elements. Their response to differentiation and morphogenetic signals was evaluated by quantitative polymerase chain reaction, histology, immunofluorescence, scanning electron microscopy, and in situ hybridization. RESULTS: We found that human PL-MSCs and UCB-MSCs constituting the RLs expressed chondrogenic, osteogenic, and tenogenic molecular markers while differentially committing into limb lineages but could not generate complex structures in vivo. MSCs-RL from PL or UCB were committed early to chondrogenic lineage. Nevertheless, the UCB-RL osteogenic commitment was favored, although preferentially to a tenogenic cell fate. These findings suggest that the commitment of MSCs to differentiate into skeletal lineages differs according to the source and is independent of their capacity to generate skeletal elements or connective tissue in vivo. Our results suggest that the failure to form skeletal structures may be due to the intrinsic characteristics of MSCs. Thus, it is necessary to thoroughly evaluate the biological aspects of MSCs and how they respond to morphogenetic signals in an in vivo context. CONCLUSION: PL-MSCs and UCB-MSCs express molecular markers of differentiation into skeletal lineages, but they are not sufficient to generate complex skeletal structures in vivo.

Adenosine metabolism by mesenchymal stromal cells isolated from different human tissues.

Mesenchymal stromal cells (MSCs) have unique biological properties and play important functions, which make them attractive tools for cell-based therapies. The basic mechanisms of these cells are not fully understood. However, the adenosinergic pathway contributes to the main effects attributed to MSCs. Adenosine is a highly immunosuppressive molecule and exerts a central role in inflammation by neutralizing the proinflammatory ATP influence. This nucleoside is produced by purinergic signaling, an important physiological pathway for MSCs, which involves proliferation, migration, differentiation, and apoptosis. Therefore, in this study, we analyzed the extracellular AMP hydrolysis and consequent adenosine production, as well as the expression of CD73 and adenosine receptors on the cell surface of MSCs isolated from different human tissues: dermis (D-MSCs), adipose tissue (AD-MSCs), and umbilical cord (UC-MSCs). All cells confirmed their multipotent capacity by adipogenic, osteogenic, and chondrogenic differentiation, as well as the expression of cell surface markers including CD44 + , CD105 + , and CD90 + . All MSCs expressed similar levels of CD73 and CD26 without a statistical difference among the different tissues, whereas ADA expression was lower in AD-MSCs. In addition, A1R and A3R mRNA levels were higher in D-MSCs and AD-MSCs, respectively. Enzymatic assay showed that AD-MSCs have the highest hydrolysis rate of AMP, leading to increased amount of adenosine production. Moreover, despite all MSCs completely hydrolyze extracellular AMP generating adenosine, the pattern of nucleosides metabolism was different. Therefore, although MSCs share certain characteristics as the multilineage potential and immunophenotype, they show different adenosinergic profiles according to tissue origin.

Femur bone marrow from brain death deceased donors as source of human mesenchymal stromal cells for cell therapy.

Background: The use of a deceased donor (DD) as an alternative source of human mesenchymal stromal cells (hMSC) is promising, but has been little explored. This study evaluated the potential of femur bone marrow (FBM) from brain-death donors as a source of hMSC and compared this with hMSC from matched iliac crest bone marrow (ICBM). Methods: Sixteen donor-matched FBM and ICBM samples were processed from brain-death donors. We analyzed the starting material and compared cell yield, phenotypic profile and differentiation capacity of hMSC. Results: Neither the amount of nucleated cells per gram (14.6×106±10.3×106 from FBM vs. 38.8×106±34.6×106 from ICBM, P≥0.09) nor the frequency of CFU-F (0.0042%±0.0036% in FBM vs. 0.0057%±0.0042% in ICBM, P≥0.73) differ significantly from FBM or ICBM. Cell cultures from both sources were obtained and hMSC yields showed that there were no significant differences in hMSC obtained per gram of bone marrow (BM) when comparing femur with iliac crest samples. At passage 2, 12.5×106±12.9×106 and 5.0×106±4.4×106 hMSC per gram of BM were obtained from FBM and ICBM, respectively. FBM and ICBM hMSC express CD73, CD90, CD105, but not hematopoietic lineage markers [CD45, CD34, CD11, CD19 and isotype of HLA clase II (HLA-DR)]. HLA-A expression from both sources was clearly detected, while HLA-B was weakly expressed or undetectable and HLA-DR was undetectable. Cells from both sources were differentiated in vitro into osteoblasts, adipocytes and chondroblasts. Conclusions: To our knowledge, there are no previous studies evaluating BM from femur dead donors as a source of hMSC. Our findings confirm that it is feasible to expand cells from FBM from brain-death donors meeting in vitro characteristics of hMSC, making them a promising source for clinical translation.

Human Adipose-Derived CD146+ Stem Cells Increase Life Span of a Muscular Dystrophy Mouse Model More Efficiently than Mesenchymal Stromal Cells.

Duchenne muscular dystrophy is the most common and severe form of progressive muscular dystrophy. Previous results showed an increased survival in double knockout mice (dko) when treated with adipose-derived CD146+ cells. In this study, we analyzed the effect of CD146+ cells compared to mesenchymal stem/stromal cells (MSCs) derived from the same human adipose sample when injected in the dko mouse model without immunosuppression. Both CD146+ cells and MSCs increased the survival of treated mice when compared to vehicle-injected mice, with a more prominent effect of CD146+ cells than MSCs. Both CD146+ cells and MSCs suppressed peripheral blood mononuclear cell proliferation, indicating immunomodulatory properties. Co-culture experiments showed that MSCs have a more inflammatory profile expression, and angiogenesis assay showed that CD146+ cells can improve blood vessel formation. CD146+ cells can extend survival of muscular dystrophy mice more efficiently than MSCs, possibly due to immunomodulatory and angiogenic properties. Further investigations focusing on exogenous CD146+ cell role in vivo will improve cell therapy understanding and effectiveness.

IL-8, GRO and MCP-1 produced by hepatocellular carcinoma microenvironment determine the migratory capacity of human bone marrow-derived mesenchymal stromal cells without affecting tumor aggressiveness.

New therapies are needed for advanced hepatocellular carcinoma (HCC) and the use of mesenchymal stromal cells (MSCs) carrying therapeutic genes is a promising strategy. HCC produce cytokines recruiting MSCs to the tumor milieu and modifying its biological properties. Our aim was to study changes generated on human MSCs exposed to conditioned media (CM) derived from human HCC fresh samples and xenografts. All CM shared similar cytokines expression pattern including CXCL1-2-3/GRO, CCL2/MCP-1 and CXCL8/IL-8 being the latter with the highest concentration. Neutralizing and knockdown experiments of CCL2/MCP-1, CXCL8/IL-8, CXCR1 and CXCR2 reduced in vitro MSC migration of ≥20%. Simultaneous CXCR1 and CXCR2 neutralization resulted in 50% of MSC migration inhibition. MSC stimulated with CM (sMSC) from HuH7 or HC-PT-5 showed a 2-fold increase of migration towards the CM compared with unstimulated MSC (usMSC). Gene expression profile of sMSC showed ~500 genes differentially expressed compared with usMSC, being 46 genes related with cell migration and invasion. sMSC increased fibroblasts and endothelial cells chemotaxis. Finally, sMSC with HuH7 CM and then inoculated in HCC tumor bearing-mice did not modify tumor growth. In this work we characterized factors produced by HCC responsible for the changes in MSC chemotactic capacity with would have an impact on therapeutic use of MSCs for human HCC.

HTLV-1 infects human mesenchymal stromal cell in vitro and modifies their phenotypic characteristics.

The typical characteristics of mesenchymal stem cells (MSCs) can be affected by inflammatory microenvironment; however, the exact contribution of HTLV-1 to MSC dysfunction remains to be elucidated. In this study, we demonstrated that MSC cell surface molecules VCAM-1 and ICAM-1 are upregulated by contact with HTLV-1, and HLA-DR was most highly expressed in MSCs co-cultured with MT2 cells. The expression levels of VCAM-1 and HLA-DR were increased in MSCs cultured in the presence of PBMCs isolated from HTLV-1-infected symptomatic individuals compared with those cultured with cells from asymptomatic infected individuals or healthy subjects. HTLV-1 does not impair the MSC differentiation process into osteocytes and adipocytes. In addition, MSCs were efficiently infected with HTLV-1 in vitro through direct contact with HTLV-1-infected cells; however, cell-free virus particles were not capable of causing infection. In summary, HTLV-1 can alter MSC function, and this mechanism may contribute to the pathogenesis of this viral infection.