We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF.hsa-mir199a ͉ hsa-mir346 ͉ stem cells ͉ plasticity H uman multipotent stromal cells (hMSCs) from bone marrow, also known as mesenchymal stem cells, are progenitor cells capable of differentiating into a variety of mature tissues (1). hMSCs can be isolated easily as single cell clones and differentiated into osteoblasts and adipocytes as well as other cellular phenotypes in culture. The cells hold great promise for therapy but the molecular mechanisms that govern the plasticity and differentiation remain unclear. Recently, the existence and function of a class of small, noncoding RNA molecules known as microRNAs (miRNAs) have gained attention as regulatory molecules. These genomically encoded RNAs undergo several modifications before being converted into mature 21-23 base pair transcripts capable of gene silencing. Biogenesis of functional miRNAs involves several enzymes, including Dicer and Drosha. Previous studies demonstrated that decreased expression of Dicer in Drosophila, zebrafish, and mice restricts the differentiation potential of stem cells (2-5). Studies have also demonstrated that specific miRNAs regulate gene expression during germ line development and cellular differentiation (2-6), including playing key roles in hematopoiesis as well as myogenic and neurogenic function (7-11). However, much of the work on miRNAs has focused on simpler organisms with very little data on human miRNAs. In this study we investigated the role of miRNAs in hMSCs, focusing first on the need for miRNA processing enzymes in hMSC differentiation. Then, we identified the key miRNAs that may regulate differentiation of hMSCs. Finally, we demonstrated that two of the miRNAs target leukemia inhibitory factor (LIF), the expression of which decreases as hMSCs differentiate. Results Generation of a Stable Knockdown of Dicer and Drosha in hMSCs.As a first step toward identifying the role of miRNAs in the differentiation of hMSCs, we analyzed the effect of decreasing the expression of key proteins...

Recent reports have indicated that mesenchymal stromal cells (MSCs) from bone marrow have a potential in vascular remodeling and angiogenesis. Here, we report a unique phenomenon that under serum-deprived conditions MSCs survive and replicate. Secretome analysis of MSCs grown under serum-deprived conditions (SD-MSCs) identified a significant upregulation of prosurvival and angiogenic factors including VEGF-A, ANGPTs, IGF-1, and HGF. An ex vivo rat aortic assay demonstrated longer neovascular sprouts generated from rat aortic rings cultured in SD-MSC-conditioned media compared to neovascular sprouts from aortas grown in MSC-conditioned media. With prolonged serum deprivation, a subpopulation of SD-MSCs began to exhibit an endothelial phenotype. This population expressed endothelial-specific proteins including VEGFR2, Tie2/TEK, PECAM/CD31, and eNOS and also demonstrated the ability to uptake acetylated LDL. SD-MSCs also exhibited enhanced microtubule formation in an in vitro angiogenesis assay. Modified chick chorioallantoic membrane (CAM) angiogenesis assays showed significantly higher angiogenic potential for SD-MSCs compared to MSCs. Analysis of CAMs grown with SD-MSCs identified human-specific CD31-positive cells in vascular structures. We conclude that under the stress of serum deprivation MSCs are highly angiogenic and a population of these cells has the potential to differentiate into endothelial-like cells.

Recent studies have implicated multipotential mesenchymal stem cells (MSCs) as an aid to breast cancer cell proliferation and metastasis, partly as a result of the MSCs secretome. As the tumor gets beyond 2 mm in diameter, the stromal cells could undergo starvation due to the lack of sufficient nutrients in solid tumor microenvironment. In this study, we investigated the survival mechanisms used by stressed stromal cells in breast cancers. We used serum-deprived mesenchymal stem cells (SD-MSCs) and MCF-7 breast cancer cells as model system with a hypothesis that stromal cells in the nutrient-deprived core utilize survival mechanisms for supporting surrounding cells. We tested this hypothesis using in vivo tumor xenografts in immunodeficient mice, which indicated that SD-MSCs supported MCF-7 tumor growth by protection from apoptosis. Histochemical assays showed that SD-MSCs-injected tumors exhibited higher cellularity, decreased apoptosis and decreased differentiation. Beclin-1 staining indicated autophagic areas surrounded by actively proliferating cells. Furthermore, in vitro studies demonstrate that SD-MSCs survive using autophagy and secrete paracrine factors that support tumor cells following nutrient/serum deprivation. Western blot and immunocytochemistry analysis of SD-MSCs demonstrated upregulation and perinuclear relocation of autophagy key regulators such as beclin-1, ATG10, ATG12, MAP-LC3 and lysosomes. Electron microscopic analysis detected a time-dependent increase in autophagosome formation and HDAC6 activity assays indicated the upregulation of autophagy. Taken together, these data suggest that under nutrient-deprived conditions that can occur in solid tumors, stromal cells utilize autophagy for survival and also secrete anti-apoptotic factors that can facilitate solid tumor survival and growth.

With the aggressive resource conservation necessary to face the COVID-19 pandemic, vascular surgeons face unique challenges to managing the health of their high-risk patients. Early analysis of patient outcomes following pandemic-related practice changes suggest that patients with chronic limb threatening ischemia (CLTI) have been presenting with more severe foot infections and are more likely to require major limb amputation compared to 6 months prior. As our society and health care system adapt to the new changes required in the post-COVID era, it is critical that we pay special attention to the most vulnerable subsets of patients with vascular disease, particularly those with CLTI and limited access to care.

ABSTRACT:In the present study, phosphorylation of cAMP-response element binding protein (pCREB) and expression of c-Fos were measured in the dorsal and ventral hippocampus, as well as in a control region, the retrosplenial cortex, of rats following acquisition and recall of a socially transmitted food preference (STFP). Behavioral analyses revealed that STFP-trained rats showed a stronger preference for the demonstrated food than did rats in social-control or odor-control conditions. Rats in a social ؉ odor control condition displayed an intermediate preference that was not significantly different from either STFP-trained rats or the socialor odor-controls. Immunocytochemical analyses revealed increased pCREB-immunoreactivity (ir) in the ventral hippocampus of STFP-trained rats in comparisons with rats in all three control conditions and increased pCREB-ir in the dorsal hippocampus in comparisons with the social-and odor-control conditions. In contrast, c-Fos-ir was greater in the dorsal hippocampus of STFP-trained rats in comparisons with all three control conditions and greater in the ventral hippocampus than rats in the socialand odor-control conditions. Comparisons of pCREB-ir and c-Fos-ir were made also between STFP-trained rats and social-controls following either acquisition or a 48-h recall test. c-Fos-ir was greater in STFP-trained rats after both acquisition and recall, whereas pCREB was greater after recall only. There were no differences in either c-Fos-ir or pCREB-ir in comparisons between trained and control rats in the retrosplenial cortex. The current results indicate that the activity of transcription factors in the hippocampus is related to both acquisition and retention of a socially transmitted food preference.