Isolation and primary characterization of WJ-MSCs.

<p>(A) WJ-MSCs Passage 5 in HPL; (B) WJ-MSCs masson’s trichrome staining; (C) Adipocyte differentiation of WJ-MSCs; (D) Oil Red O Staining; Osteocyte differentiation, Von kossa Staining (E) and Alkaline Phosphatase staining (F); (G) Chondrocyte Differentiation, Safranin O Staining; (H) Flow cytometry Analysis of WJ-MSCs; (I) Neural Differentiation, Neuroglia2 immunostaining (J); (K) Smooth muscle actin staining; (L) Photoreceptor cell differentiation, Rhodopsin staining (M); (N) Pancreatic Progenitor differentiation, (O) Insulin, (P) PDX1.</p

Human Wharton’s Jelly Mesenchymal Stem Cells Plasticity Augments Scar-Free Skin Wound Healing with Hair Growth

<div><p>Human mesenchymal stem cells (MSCs) are a promising candidate for cell-based transplantation and regenerative medicine therapies. Thus in the present study Wharton’s Jelly Mesenchymal Stem Cells (WJ-MSCs) have been derived from extra embryonic umbilical cord matrix following removal of both arteries and vein. Also, to overcome the clinical limitations posed by fetal bovine serum (FBS) supplementation because of xenogeneic origin of FBS, usual FBS cell culture supplement has been replaced with human platelet lysate (HPL). Apart from general characteristic features of bone marrow-derived MSCs, wharton jelly-derived MSCs have the ability to maintain phenotypic attributes, cell growth kinetics, cell cycle pattern, <i>in vitro</i> multilineage differentiation plasticity, apoptotic pattern, normal karyotype-like intrinsic mesenchymal stem cell properties in long-term <i>in vitro</i> cultures. Moreover, the WJ-MSCs exhibited the <i>in vitro</i> multilineage differentiation capacity by giving rise to differentiated cells of not only mesodermal lineage but also to the cells of ectodermal and endodermal lineage. Also, WJ-MSC did not present any aberrant cell state upon <i>in vivo</i> transplantation in SCID mice and <i>in vitro</i> soft agar assays. The immunomodulatory potential assessed by gene expression levels of immunomodulatory factors upon exposure to inflammatory cytokines in the fetal WJ-MSCs was relatively higher compared to adult bone marrow-derived MSCs. WJ-MSCs seeded on decellularized amniotic membrane scaffold transplantation on the skin injury of SCID mice model demonstrates that combination of WJ-MSCs and decellularized amniotic membrane scaffold exhibited significantly better wound-healing capabilities, having reduced scar formation with hair growth and improved biomechanical properties of regenerated skin compared to WJ-MSCs alone. Further, our experimental data indicate that indocyanin green (ICG) at optimal concentration can be resourcefully used for labeling of stem cells and <i>in vivo</i> tracking by near infrared fluorescence non-invasive live cell imaging of labelled transplanted cells, thus proving its utility for therapeutic applications.</p></div

Mice skin injury model and <i>in vivo</i> tracking of cells.

<p>(A) Dermal injury; (B) Amniotic membrane containing WJ-MSCs grafting; Area of injury after 14 days, (C) Control, (D) WJ-MSCs injection, (E) Amniotic membrane + WJ-MSCs; H&E staining, (F) Control, (G) WJ-MSCs injection, (H) Amniotic membrane + WJ-MSCs; <i>in vitro</i> analysis of ICG, (I) 2D, (J) 3D; (K) Total flux of ICG in different concentration; (L) GFP labeled WJ-MSCs seeded on amniotic membrane, (i) Phase contrast, (ii) fluorescent image and (iii) merged image. (M) IVIS image of amniotic membrane containing ICG labeled WJ-MSCs grafted mice showing fluorescence for 11 days, (N) decrease in total flux of ICG with passage of time; (O) Singer score of regenerated tissue.</p

Systematic evaluation of markers used for the identification of human induced pluripotent stem cells

Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs)