Extracellular Vesicles and Their Emerging Roles as Cellular Messengers in Endocrinology: An Endocrine Society Scientific Statement.

During the last decade, there has been great interest in elucidating the biological role of extracellular vesicles (EVs), particularly, their hormone-like role in cell-to-cell communication. The field of endocrinology is uniquely placed to provide insight into the functions of EVs, which are secreted from all cells into biological fluids and carry endocrine signals to engage in paracellular and distal interactions. EVs are a heterogeneous population of membrane-bound vesicles of varying size, content, and bioactivity. EVs are specifically packaged with signaling molecules, including lipids, proteins, and nucleic acids, and are released via exocytosis into biofluid compartments. EVs regulate the activity of both proximal and distal target cells, including translational activity, metabolism, growth, and development. As such, EVs signaling represents an integral pathway mediating intercellular communication. Moreover, as the content of EVs is cell-type specific, it is a "fingerprint" of the releasing cell and its metabolic status. Recently, changes in the profile of EV and bioactivity have been described in several endocrine-related conditions including diabetes, obesity, cardiovascular diseases, and cancer. The goal of this statement is to highlight relevant aspects of EV research and their potential role in the field of endocrinology.

Analysis of deep sequencing microRNA expression profile from human embryonic stem cells derived mesenchymal stem cells reveals possible role of let-7 microRNA family in downstream targeting of hepatic nuclear factor 4 alpha.

BACKGROUND: Recent literature has revealed that genetic exchange of microRNA between cells can be essential for cell-cell communication, tissue-specificity and developmental processes. In stem cells, as in other cells, this can be accomplished through microvesicles or exosome mediated transfer. However, molecular profiles and functions of microRNAs within the cells and in their exosomes are poorly studied. Next generation sequencing technologies could provide a broad-spectrum of microRNAs and their expression and identify possible microRNA targets. In this work, we performed deep sequencing of microRNAs to understand the profile and expression of the microRNAs in microvesicles and intracellular environment of human embryonic stem cells derived mesenchymal stem cells (hES-MSC). We outline a workflow pertaining to visualizing, statistical analysis and interpreting deep sequencing data of known intracellular and extracellular microRNAs from hES-MSC). We utilized these results of which directed our attention towards establishing hepatic nuclear factor 4 alpha (HNF4A) as a downstream target of let-7 family of microRNAs. RESULTS: In our study, significant differences in expression profile of microRNAs were found in the intracellular and extracellular environment of hES-MSC. However, a high level of let-7 family of microRNAs is predominant in both intra- and extra- cellular samples of hES-MSC. Further results derived from visualization of our alignment data and network analysis showed that let-7 family microRNAs could affect the downstream target HNF4A, which is a known endodermal differentiation marker. The elevated presence of let-7 microRNA in both intracellular and extra cellular environment further suggests a possible intercellular signalling mechanism through microvesicles transfer. We suggest that let-7 family microRNAs might play a signalling role via such a mechanism amongst populations of stem cells in maintaining self renewal property by suppressing HNF4A expression. This is in line with recent paradigm where microRNAs regulate self-renewal and differentiation pathways of embryonic stem cells by forming an integral biological network with transcription factors. CONCLUSION: In summary, our study using a combination of alignment, statistical and network analysis tools to examine deep sequencing data of microRNAs in hES-MSC has led to a result that (i) identifies intracellular and exosome microRNA expression profiles of hES-MSC with a possible mechanism of miRNA mediated intercellular regulation by these cells and (ii) placed HNF4A within the cross roads of regulation by the let-7 family of microRNAs.

Autogeneic feeders for the culture of undifferentiated human embryonic stem cells in feeder and feeder-free conditions.

Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture while retaining their ability to differentiate to any cell type in the body. Conventionally, hESC are cultured either directly on feeders or on an extracellular matrix supplemented with conditioned medium (CM) from feeders. To minimize the risk of xenozootic infections, several sources of primary human feeders have been identified. However, this does not eliminate the risk of contaminating hESC with infectious agents from the donor human feeders. In this study, we evaluated the use of the CD105+ /CD24 hESC-derived mesenchymal stem cell (MSC) line, HuES9.E1, for its ability to support the growth of undifferentiated hESC in feeder and feeder-free cultures. This line was previously reported to be karyotypically stable and phenotypically displayed MSC-like surface antigens and gene transcription profiles. In addition, like adult MSC, HuES9.E1 can be differentiated to adipocytes, osteocytes, and chondrocytes in vitro. When tested for its ability to support hESC growth, it was found that hESC maintained the undifferentiated morphology for >12 continuous passages in coculture with HuES9.E1 and >8 passages in feeder-free cultures supplemented with CM from HuES9.E1. Furthermore, the hESC cultures continued to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, and retained a normal karyotype. When injected into severe combined immunodeficient (SCID) mice, hESC differentiated to form teratomas comprising of tissues representative of the three embryonic germ layers. Potentially, the ability to derive and use autogeneic feeders may provide a safe and accessible source of feeders for the expansion of hESC required in clinical applications.