CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer and various pathological conditions. However, the phenotypic and functional heterogeneity of human MDSCs represents a major hurdle for the development of therapeutic strategies targeting or regulating MDSCs in tumor progression, inflammation, and graft-versus-host disease (GVHD). We previously shown that circulating HLA-DR-CD14+ monocytic MDSCs are a major contributor to clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we identified, using high-throughput screening, a set of surface markers that are strongly expressed in HLA-DR-CD14+ monocytic MDSCs isolated from the peripheral blood (PB) of patients receiving allo-HSCT. Subsequent experiments showed the consistent dominant expression of CD1d in monocytic MDSCs of allo-HSCT PB in comparison with granulocytic MDSCs. In addition, CD1d-expressing cells isolated from PB of allo-HSCT patients showed the suppressive activity of T cell proliferation and higher expression of MyD88 and IDO compared with CD1d- cells. Our results suggest that CD1d could be a valuable marker for further therapeutic evaluation of human monocytic MDSCs for immune-related diseases, including GVHD.

Paracrine influence of human perivascular cells on the proliferation of adenocarcinoma alveolar epithelial cells.

Understanding the crosstalk mechanisms between perivascular cells (PVCs) and cancer cells might be beneficial in preventing cancer development and metastasis. In this study, we investigated the paracrine influence of PVCs derived from human umbilical cords on the proliferation of lung adenocarcinoma epithelial cells (A549) and erythroleukemia cells (TF-1α and K562) in vitro using Transwell® co-culture systems. PVCs promoted the proliferation of A549 cells without inducing morphological changes, but had no effect on the proliferation of TF-1α and K562 cells. To identify the factors secreted from PVCs, conditioned media harvested from PVC cultures were analyzed by antibody arrays. We identified a set of cytokines, including persephin (PSPN), a neurotrophic factor, and a key regulator of oral squamous cell carcinoma progression. Supplementation with PSPN significantly increased the proliferation of A549 cells. These results suggested that PVCs produced a differential effect on the proliferation of cancer cells in a cell-type dependent manner. Further, secretome analyses of PVCs and the elucidation of the molecular mechanisms could facilitate the discovery of therapeutic target(s) for lung cancer.

Non-enzymatic isolation followed by supplementation of basic fibroblast growth factor improves proliferation, clonogenic capacity and SSEA-4 expression of perivascular cells from human umbilical cord.

Multipotent perivascular cells (PVCs) have recently gained attention as an alternative source for cell-based regenerative medicine. Because of their rarity in human tissues, the development of efficient methods to isolate and expand PVCs from various fetal and adult tissues is necessary to obtain a clinically relevant number of cells that maintain progenitor potency. We report a simple non-enzymatic isolation (NE) method of PVCs from human umbilical cord (HUC) and compare its efficiency with the conventional collagenase treatment method (CT) in terms of proliferation, immunophenotype, clonogenic capacity, and differentiation potential. Cells isolated by NE expressed the accepted surface marker profile of PVCs and possessed multilineage differentiation potential. Whereas both methods provided similar patterns or levels of immunophenotypes and proliferation, PVCs obtained by NE maintained a higher level of CD146(+) frequency compared with that of CT over passages and displayed greater in vitro osteogenic differentiation potential and clonogenic capacity than CT-PVCs. We assess the potential of various exogenous factors to boost the proliferation of NE- and CT-PVCs in vitro. Supplementation of basic fibroblast growth factor (bFGF) provided optimal conditions that significantly enhanced their proliferation rate. This treatment drove the cells into S phase and increased the proportion of stage-specific antigen-4-positive population without altering other immunophenotypes. Thus, the NE method with bFGF supplementation offers an alternative way for obtaining sufficient numbers of HUCPVCs that have good clonogenic and differentiation potential and that are applicable at therapeutic doses for regenerative medicine.

BMP4 and perivascular cells promote hematopoietic differentiation of human pluripotent stem cells in a differentiation stage-specific manner.

The efficient and reproducible derivation and maturation of multipotent hematopoietic progenitors from human pluripotent stem cells (hPSCs) requires the recapitulation of appropriate developmental stages and the microenvironment. Here, using serum-, xeno-, and feeder-free stepwise hematopoietic induction protocols, we showed that short-term and high-concentration treatment of hPSCs with bone morphogenetic protein 4 (BMP4) strongly promoted early mesoderm induction followed by increased hematopoietic commitment. This method reduced variations in hematopoietic differentiation among hPSC lines maintained under chemically defined Essential 8 medium compared to those maintained under less-defined mTeSR medium. We also found that perivascular niche cells (PVCs) significantly augmented the production of hematopoietic cells via paracrine signaling mechanisms only when they were present during the hematopoietic commitment phase. A protein array revealed 86 differentially expressed (>1.5-fold) secretion factors in PVC-conditioned medium compared with serum-free control medium, of which the transforming growth factor-ß inducible gene H3 significantly increased the number of hematopoietic colony-forming colonies. Our data suggest that BMP4 and PVCs promote the hematopoietic differentiation of hPSCs in a differentiation stage-specific manner. This will increase our understanding of hematopoietic development and expedite the development of hPSC-derived blood products for therapeutic use.

Effects of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system-Based Deletion of miR-451 in Mouse Embryonic Stem Cells on Their Self-Renewal and Hematopoietic Differentiation.

Pluripotent stem cells (PSCs) are a useful source of cells for exploring the role of genes related with early developmental processes and specific diseases due to their ability to differentiate into all somatic cell types. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system has proven to be a robust tool for targeted genetic modification. Here, we generated miR-451-deficient PSCs using the CRISPR/Cas9 system with PCR-based homologous recombination donor and investigated the impact of its deletion on self-renewal and hematopoietic development. CRISPR/Cas9-mediated miR-451 knockout did not alter the gene expressions of pluripotency, cellular morphology, and cell cycle, but led to impaired erythrocyte development. These findings propose that a combination of PSCs and CRISPR/Cas9 system could be useful to promote biomedical applications of PSCs by elucidating the function and manipulating of specific miRNAs during lineage specification and commitment.

Gestational Diabetes Affects the Growth and Functions of Perivascular Stem Cells.

Gestational diabetes mellitus (GDM), one of the common metabolic disorders of pregnancy, leads to functional alterations in various cells including stem cells as well as some abnormalities in fetal development. Perivascular stem cells (PVCs) have gained more attention in recent years, for the treatment of various diseases. However, the effect of GDM on PVC function has not been investigated. In our study, we isolated PVCs from umbilical cord of normal pregnant women and GDM patients and compared their phenotypes and function. There is no significant difference in phenotypic expression, response to bFGF exposure and adipogenic differentiation capacity between normal (N)-PVCs and GDM-PVCs. However, when compared with N-PVCs, early passage GDM-PVCs displayed decreased initial rates of cell yield and proliferation as well as a reduced ability to promote wound closure. These results suggest that maternal metabolic dysregulation during gestation can alter the function of endogenous multipotent stem cells, which may impact their therapeutic effectiveness.

Hormonal regulation of hematopoietic stem cells and their niche: a focus on estrogen.

Self-renewal and differentiation are hallmarks of stem cells and controlled by various intrinsic and extrinsic factors. Increasing evidence indicates that estrogen (E2), the primary female sex hormone, is involved in regulating the proliferation and lineage commitment of adult and pluripotent stem cells as well as modulating the stem cell niche. Thus, a detailed understanding of the role of E2 in behavior of stem cells may help to improve their therapeutic potential. Recently, it has been reported that E2 promotes cell cycle activity of hematopoietic stem and progenitor cells and induces them to megakaryocyte-erythroid progenitors during pregnancy. This study paves the way towards a previously unexplored endocrine mechanism that controls stem cell behavior. In this review, we will focus on the scientific findings regarding the regulatory effects of E2 on the hematopoietic system including its microenvironment.