BackgroundA culture system that closely recapitulates marrow physiology is essential to study the nichemediated regulation of hematopoietic stem cell fate at a molecular level. We investigated the key features that play a crucial role in the formation of a functional niche in vitro. Design and MethodsHydrogel-based cultures of human placenta-derived mesenchymal stromal cells were established to recapitulate the fibrous three-dimensional architecture of the marrow. Plastic-adherent mesenchymal stromal cells were used as controls. Human bone marrow-derived CD34 + cells were co-cultured with them. The output hematopoietic cells were characterized by various stem cell-specific phenotypic and functional parameters. ResultsThe hydrogel-cultures harbored a large pool of primitive hematopoietic stem cells with superior phenotypic and functional attributes. Most importantly, like the situation in vivo, a significant fraction of these cells remained quiescent in the face of a robust multi-lineage hematopoiesis. The retention of a high percentage of primitive stem cells by the hydrogel-cultures was attributed to the presence of CXCR4-SDF1α axis and integrin beta1-mediated adhesive interactions. The hydrogel-grown mesenchymal stromal cells expressed high levels of several molecules that are known to support the maintenance of hematopoietic stem cells. Yet another physiologically relevant property exhibited by the hydrogel cultures was the formation of hypoxia-gradient. Destruction of hypoxia-gradient by incubating these cultures in a hypoxia chamber destroyed their specialized niche properties. ConclusionsOur data show that hydrogel-based cultures of mesenchymal stromal cells form a functional in vitro niche by mimicking key features of marrow physiology.

Donor age is one of the major concerns in bone marrow transplantation, as the aged hematopoietic stem cells (HSCs) fail to engraft efficiently. Here, using murine system, we show that a brief interaction of aged HSCs with young mesenchymal stromal cells (MSCs) rejuvenates them and restores their functionality via inter-cellular transfer of microvesicles (MVs) containing autophagy-related mRNAs. Importantly, we show that MSCs gain activated AKT signaling as a function of aging. Activated AKT reduces the levels of autophagy-related mRNAs in their MVs, and partitions miR-17 and miR-34a into their exosomes, which upon transfer into HSCs downregulate their autophagy-inducing mRNAs. Our data identify previously unknown mechanisms operative in the niche-mediated aging of HSCs. Inhibition of AKT in aged MSCs increases the levels of autophagy-related mRNAs in their MVs and reduces the levels of miR-17 and miR-34a in their exosomes. Interestingly, transplantation experiments showed that the rejuvenating power of these "rescued" MVs is even better than that of the young MVs. We demonstrate that such ex vivo rejuvenation of aged HSCs could expand donor cohort and improve transplantation efficacy. Stem Cells 2018;36:420-433.

Mesenchymal stem cells (MSCs) show immunoregulatory properties. Here, we compared MSCs obtained from placenta (P-MSCs) and umbilical cord (C-MSCs) from the same donor, for their immunomodulatory efficacy. P-MSCs and C-MSCs showed similar morphology and phenotypic profile, but different clonogenic ability. Importantly, they showed a significant difference in their immunosuppressive properties as assessed in mixed leukocyte reaction (MLR). The P-MSCs affected the antigen presenting ability of mononuclear cells (MNCs) and dendritic cells (DCs) significantly as compared to C-MSCs resulting in a reduced T-cell proliferation. P-MSC conditioned medium (CM) showed a significant reduction in T cell proliferation as compared to C-MSC CM, thus suggesting that a cell to cell contact is not essential. We found increased levels of IL-10 and TGFβ1 and reduction in levels of IFNγ in P-MSC MLRs as compared to C-MSC MLRs. Furthermore, the CD3+ CD4+ CD25+ T regulatory cells were enriched in case of P-MSCs in both, MSC-MNC and MSC-DC co-cultures. This observation was further supported by increased mRNA expression of FoxP3 in P-MSCs. Presently, cord-derived MSCs are being employed in transplantation therapies parallel to the bone marrow-derived MSCs. Our findings suggest that P-MSCs can be a better alternative to C-MSCs, to provide aid in immunological ailments.

IntroductionEx vivo expansion of umbilical cord blood (UCB) is attempted to increase cell numbers to overcome the limitation of cell dose. Presently, suspension cultures or feeder mediated co-cultures are performed for expansion of hematopoietic stem cells (HSCs). Mesenchymal stem cells (MSCs) have proved to be efficient feeders for the maintenance of HSCs. Here, we have established MSCs-HSCs co-culture system with MSCs isolated from less invasive and ethically acceptable sources like umbilical cord tissue (C-MSCs) and placenta (P-MSCs). MSCs derived from these tissues are often compared with bone marrow derived MSCs (BM-MSCs) which are considered as a gold standard. However, so far none of the studies have directly compared C-MSCs with P-MSCs as feeders for ex vivo expansion of HSCs. Thus, we for the first time performed a systematic comparison of hematopoietic supportive capability of C and P-MSCs using paired samples.MethodsUCB-derived CD34+ cells were isolated and co-cultured on irradiated C and P-MSCs for 10 days. C-MSCs and P-MSCs were isolated from the same donor. The cultures comprised of serum-free medium supplemented with 25 ng/ml each of SCF, TPO, Flt-3 L and IL-6. After 10 days cells were collected and analyzed for phenotype and functionality.ResultsC-MSCs and P-MSCs were found to be morphologically and phenotypically similar but exhibited differential ability to support ex vivo hematopoiesis. Cells expanded on P-MSCs showed higher percentage of primitive cells (CD34+CD38−), CFU (Colony forming unit) content and LTC-IC (Long term culture initiating cells) ability. CD34+ cells expanded on P-MSCs also exhibited better in vitro adhesion to fibronectin and migration towards SDF-1α and enhanced NOD/SCID repopulation ability, as compared to those grown on C-MSCs. P-MSCs were found to be closer to BM-MSCs in their ability to expand HSCs. P-MSCs supported expansion of functionally superior HSCs by virtue of reduction in apoptosis of primitive HSCs, higher Wnt and Notch activity, HGF secretion and cell-cell contact. On the other hand, C-MSCs facilitated expansion of progenitors (CD34+CD38+) and differentiated (CD34−CD38+) cells by secretion of IL1-α, β, MCP-2, 3 and MIP-3α.ConclusionsP-MSCs were found to be better feeders for ex vivo maintenance of primitive HSCs with higher engraftment potential than the cells expanded with C-MSCs as feeders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0194-y) contains supplementary material, which is available to authorized users.