isolation-characterization-mesenchymal-stromal-cells-from-human

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Oakland University

OU-WB Institute for Stem Cell and Regenerative Medicine

To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells.

Efficacy and safety of anticancer drugs are traditionally studied using cancer cell lines and animal models. The thienodiazepine class of BET inhibitors, such as JQ1, has been extensively studied for the potential treatment of hematological malignancies and several small molecules belonging to this class are currently under clinical investigation. While these compounds are well known to inhibit cancer cell growth and cause apoptosis, their effects on stem cells, particularly mesenchymal stem cells (MSCs), which are important for regeneration of damaged cells and tissues, are unknown. In this study we employed umbilical cord derived MSCs as a model system to evaluate the safety of JQ1.

BET protein inhibitor JQ1 inhibits growth and modulates WNT signaling in mesenchymal stem cells.

Human umbilical cord (hUC) blood and tissue are non-invasive sources of potential stem/progenitor cells with similar cell surface properties as bone marrow stromal cells (BMSCs). While they are limited in cord blood, they may be more abundant in hUC. However, the hUC is an anatomically complex organ and the potential of cells in various sites of the hUC has not been fully explored. We dissected the hUC into its discrete sites and isolated hUC cells from the cord placenta junction (CPJ), cord tissue (CT), and Wharton's jelly (WJ). Isolated cells displayed fibroblastoid morphology, and expressed CD29, CD44, CD73, CD90, and CD105, and showed evidence of differentiation into multiple lineages in vitro. They also expressed low levels of pluripotency genes, OCT4, NANOG, SOX2 and KLF4. Passaging markedly affected cell proliferation with concomitant decreases in the expression of pluripotency and other markers, and an increase in chondrogenic markers. Microarray analysis further revealed the differences in the gene expression of CPJ-, CT- and WJ-hUC cells. Five coding and five lncRNA genes were differentially expressed in low vs. high passage hUC cells. Only MAEL was expressed at high levels in both low and high passage CPJ-hUC cells. They displayed a greater proliferation limit and a higher degree of multi-lineage differentiation in vitro and warrant further investigation to determine their full differentiation capacity, and therapeutic and regenerative medicine potential.

Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord.

Intervertebral disc (IVD) degeneration is characterized by the loss of nucleus pulposus (NP), which is a common cause for lower back pain. Although, currently, there is no cure for the degenerative disc disease, stem cell therapy is increasingly being considered for its treatment. In this study, we investigated the feasibility and efficacy of human umbilical cord mesenchymal stem cells (MSCs) and chondroprogenitor cells (CPCs) derived from those cells to regenerate damaged IVD in a rabbit model. Transplanted cells survived, engrafted and dispersed into NP in situ. Significant improvement in the histology, cellularity, extracellular matrix proteins, and water and glycosaminoglycan contents in IVD recipients of CPCs was observed compared to MSCs. In addition, IVDs receiving CPCs exhibited higher expression of NP-specific human markers, SOX9, aggrecan, collagen 2, FOXF1 and KRT19. The novelty of the study is that in vitro differentiated CPCs derived from umbilical cord MSCs, demonstrated far greater capacity to regenerate damaged IVDs, which provides basis and impetus for stem cell based clinical studies to treat degenerative disc disease. Copyright © 2016 John Wiley & Sons, Ltd.

Human umbilical cord derivatives regenerate intervertebral disc.

Embryonic stem cells (ESCs) are an ideal source for chondrogenic progenitors for the repair of damaged cartilage tissue. It is currently difficult to induce uniform and scalable ESC differentiation in vitro, a process required for stem cell therapy. This is partly because stem cell fate is determined by complex interactions with the native microenvironment and mechanical properties of the extracellular matrix. Mechanical signaling is considered to be one of the major factors regulating the proliferation and differentiation of chondrogenic cells both in vitro and in vivo. We used biocompatible and elastic polydimethylsiloxane (PDMS) scaffolds, capable of transducing mechanical signals including compressive stress in vitro. ESCs seeded into the PDMS scaffolds and subjected to mechanical loading resulted in induction of differentiation. Differentiated ESC derivatives in three-dimensional (3-D) PDMS scaffolds exhibited elongated single cell rather than round clonal ESC morphology. They expressed chondrogenic marker, Col2 with concomitant reduction in the expression of pluripotent marker, Oct4. Immunocytochemical analysis also showed that the expression of COL2 protein was significantly higher in ESCs in 3-D scaffolds subjected to compressive stress. Further analysis showed that compressive stress also resulted in expression of early chondrogenic makers, Sox9 and Acan but not hypertrophic chondrogenic markers, Runx2, Col10, and Mmp13. Compressive stress induced differentiation caused a reduction in the expression of β-Catenin and an increase in the expression of genes, Rhoa, Yap and Taz, which are known to be affected by mechanosignaling. The chondroinductive role of RhoA was confirmed by its downregulation with simultaneous decrease in the transcriptional and translational expression of early chondrogenic markers, SOX9, COL2, and ACAN, when ESCs in PDMS scaffolds were subjected to compressive stress and treated with RhoA inhibitor, CCG-1432. Based on these observations, a model for compression induced chondrogenic differentiation of ESCs in 3-D scaffolds was proposed.

Christina McKee

Department of Biological Sciences

Oakland University

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta