Radhakrishnan Vishnubalaji

Human Stromal (Mesenchymal) Stem Cells from Bone Marrow, Adipose Tissue and Skin Exhibit Differences in Molecular Phenotype and Differentiation Potential

Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow, but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin (human adult skin stromal cells, (hASSCs) and human new-born skin stromal cells (hNSSCs)) grew readily in culture and the growth rate was highest in hNSSCs and lowest in hATSCs. Compared with phenotype of hBM-MSC, all cell populations were CD34−, CD45−, CD14−, CD31−, HLA-DR−, CD13+, CD29+, CD44+, CD73+, CD90+,and CD105+. When exposed to in vitro differentiation, hATSCs, hASSCs and hNSSCs exhibited quantitative differences in their ability to differentiate into adipocytes and to osteoblastic cells. Using a microarray-based approach we have unveiled a common MSC molecular signature composed of 33 CD markers including known MSC markers and several novel markers e.g. CD165, CD276, and CD82. However, significant differences in the molecular phenotype between these different stromal cell populations were observed suggesting ontological and functional differences. In conclusion, MSC populations obtained from different tissues exhibit significant differences in their proliferation, differentiation and molecular phenotype, which should be taken into consideration when planning their use in clinical protocols.

Comparative investigation of the differentiation capability of bone-marrow- and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis

Mesenchymal stem cells (MSCs) hold promise for cell-based therapy in regenerative medicine. To date, MSCs have been obtained from conventional bone marrow via a highly invasive procedure. Therefore, MSCs are now also isolated from sources such as adipose tissue, cord blood and cord stroma, a subject of growing interest. As the characterization and differentiation potential of adipose-derived MSCs (AD-MSCs) and bone-marrow-derived MSCs (BM-MSCs) have not been documented, we have evaluated and compared the characteristics of both MSC types by qualitative and quantitative analyses. Both cell types show similar morphology and surface protein expression, being positive for stromal-associated markers and negative for hematopoietic and endothelial markers. The colony-forming potential of AD-MSCs is distinctly higher than that of BM-MSCs. Nonetheless, similar adipogenic and osteogenic differentiation is observed in both groups of MSCs. Cytochemical qualitative analysis and calcium mineralization demonstrate higher levels toward osteogenic differentiation in BM-MSCs than in AD-MSCs. On the contrary, the percentage of Nile red oil staining for differentiated adipocytes is higher in AD-MSCs than in BM-MSCs. Quantitative real-time polymerase chain reaction shows similar patterns of osteogenic- and adipogenic-associated gene expression in both cell types. Each of the MSCs respond in functional analysis by exhibiting unique properties at the differentiation level according to their micro-environmental niche. Thus, quantitative analysis might be a valuable means of describing stem cell multipotency, in addition to qualitative investigation.

microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells

The molecular mechanisms promoting lineage-specific commitment of human mesenchymal (skeletal or stromal) stem cells (hMSCs) into adipocytes (ADs) are not fully understood. Thus, we performed global microRNA (miRNA) and gene expression profiling during adipocytic differentiation of hMSC, and utilized bioinformatics as well as functional and biochemical assays, and identified several novel miRNAs differentially expressed during adipogenesis. Among these, miR-320 family (miR-320a, 320b, 320c, 320d and 320e) were ~2.2–3.0-fold upregulated. Overexpression of miR-320c in hMSC enhanced adipocytic differentiation and accelerated formation of mature ADs in ex vivo cultures. Integrated analysis of bioinformatics and global gene expression profiling in miR-320c overexpressing cells and during adipocytic differentiation of hMSC identified several biologically relevant gene targets for miR-320c including RUNX2, MIB1 (mindbomb E3 ubiquitin protein ligase 1), PAX6 (paired box 6), YWHAH and ZWILCH. siRNA-mediated silencing of those genes enhanced adipocytic differentiation of hMSC, thus corroborating an important role for those genes in miR-320c-mediated adipogenesis. Concordant with that, lentiviral-mediated stable expression of miR-320c at physiological levels (~1.5-fold) promoted adipocytic and suppressed osteogenic differentiation of hMSC. Luciferase assay validated RUNX2 (Runt-related transcription factor 2) as a bona fide target for miR-320 family. Therefore, our data suggest miR-320 family as possible molecular switch promoting adipocytic differentiation of hMSC. Targeting miR-320 may have therapeutic potential in vivo through regulation of bone marrow adipogenesis.

In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

BackgroundMultipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs) possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs) and adult dermal skin (hADSSCs) using explants cultures and were compared with bone marrow (hMSC-TERT) and adipose tissue-derived mesenchymal stem cells (hADMSCs) for their potential differentiation into osteoblasts, adipocytes, and endothelial cells.ResultsConcordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC), with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA) was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues.ConclusionsOur data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs represents a novel source of stem/stromal cells for tissue regeneration and the vascularization of engineered tissues. Moreover, the CD146 investigations suggested that the microenvironmental niche might contribute to direct stromal cells multipotency toward certain lineages, which warrants further investigation.

Genome-wide mRNA and miRNA expression profiling reveal multiple regulatory networks in colorectal cancer

Despite recent advances in cancer management, colorectal cancer (CRC) remains the third most common cancer and a major health-care problem worldwide. MicroRNAs have recently emerged as key regulators of cancer development and progression by targeting multiple cancer-related genes; however, such regulatory networks are not well characterized in CRC. Thus, the aim of this study was to perform global messenger RNA (mRNA) and microRNA expression profiling in the same CRC samples and adjacent normal tissues and to identify potential miRNA-mRNA regulatory networks. Our data revealed 1273 significantly upregulated and 1902 downregulated genes in CRC. Pathway analysis revealed significant enrichment in cell cycle, integrated cancer, Wnt (wingless-type MMTV integration site family member), matrix metalloproteinase, and TGF-β pathways in CRC. Pharmacological inhibition of Wnt (using XAV939 or IWP-2) or TGF-β (using SB-431542) pathways led to dose- and time-dependent inhibition of CRC cell growth. Similarly, our data revealed up- (42) and downregulated (61) microRNAs in the same matched samples. Using target prediction and bioinformatics, ~77% of the upregulated genes were predicted to be targeted by microRNAs found to be downregulated in CRC. We subsequently focused on EZH2 (enhancer of zeste homolog 2 ), which was found to be regulated by hsa-miR-26a-5p and several members of the let-7 (lethal-7) family in CRC. Significant inverse correlation between EZH2 and hsa-miR-26a-5p (R2=0.56, P=0.0001) and hsa-let-7b-5p (R2=0.19, P=0.02) expression was observed in the same samples, corroborating the belief of EZH2 being a bona fide target for these two miRNAs in CRC. Pharmacological inhibition of EZH2 led to significant reduction in trimethylated histone H3 on lysine 27 (H3K27) methylation, marked reduction in cell proliferation, and migration in vitro. Concordantly, small interfering RNA-mediated knockdown of EZH2 led to similar effects on CRC cell growth in vitro. Therefore, our data have revealed several hundred potential miRNA-mRNA regulatory networks in CRC and suggest targeting relevant networks as potential therapeutic strategy for CRC.

Skin-derived multipotent stromal cells – an archrival for mesenchymal stem cells

Progenitor stem cells have been identified, isolated and characterized in numerous tissues and organs. However, their therapeutic potential and the use of these stem cells remain elusive except for a few progenitor cells from bone marrow, umbilical cord blood, eyes and dental pulp. The use of bone marrow-derived hematopoietic stem cells (HSC) or mesenchymal stem cells (MSCs) is restricted due to their extreme invasive procedures, low differentiation potential with age and rejection. Thus, we need a clinical grade alternative to progenitor stem cells with a high potential to differentiate, naïve and is relatively easy in in vitro propagation. In this review, we summarize cell populations of adherent and floating spheres derived from different origins of skin, or correctly foreskin, by enzymatic digestion compared with established MSCs. The morphology, phenotype, differentiation capability and immunosuppressive property of the adherent cell populations are comparable with MSCs. Serum-free cultured floating spheres have limited mesodermal but higher neurogenic differentation potential, analogous to neural crest stem cells. Both the populations confirmed their plethora potential in in vitro. Together, it may be noted that the skin-derived adherent cell populations and floating cells can be good alternative sources of progenitor cells especially in cosmetic, plastic and sports regenerative medicine.

Green synthesis of silver nanoparticles using Pimpinella anisum seeds: antimicrobial activity and cytotoxicity on human neonatal skin stromal cells and colon cancer cells

Background The present study focused on a simple and eco-friendly method for the synthesis of silver nanoparticles (AgNPs) with multipurpose anticancer and antimicrobial activities. Materials and methods We studied a green synthesis route to produce AgNPs by using an aqueous extract of Pimpinella anisum seeds (3 mM). Their antimicrobial activity and cytotoxicity on human neonatal skin stromal cells (hSSCs) and colon cancer cells (HT115) were assessed. Results A biophysical characterization of the synthesized AgNPs was realized: the morphology of AgNPs was determined by transmission electron microscopy, energy dispersive spectroscopy, X-ray powder diffraction, and ultraviolet-vis absorption spectroscopy. Transmission electron microscopy showed spherical shapes of AgNPs of P. anisum seed extracts with a 3.2 nm minimum diameter and average diameter ranging from 3.2 to 16 nm. X-ray powder diffraction highlighted the crystalline nature of the nanoparticles, ultraviolet-vis absorption spectroscopy was used to monitor their synthesis, and Fourier transform infrared spectroscopy showed the main reducing groups from the seed extract. Energy dispersive spectroscopy was used to confirm the presence of elemental silver. We evaluated the antimicrobial potential of green-synthesized AgNPs against five infectious bacteria: Staphylococcus pyogenes (29213), Acinetobacter baumannii (4436), Klebsiella pneumoniae (G455), Salmonella typhi, and Pseudomonas aeruginosa. In addition, we focused on the toxicological effects of AgNPs against hSSC cells and HT115 cells by using in vitro proliferation tests and cell viability assays. Among the different tested concentrations of nanoparticles, doses < 10 µg showed few adverse effects on cell proliferation without variations in viability, whereas doses >10 µg led to increased cytotoxicity. Conclusion Overall, our results highlighted the capacity of P. anisum-synthesized AgNPs as novel and cheap bioreducing agents for eco-friendly nanosynthetical routes. The data confirm the multipurpose potential of plant-borne reducing and stabilizing agents in nanotechnology.

CDH1 and IL1-beta expression dictates FAK and MAPKK-dependent cross-talk between cancer cells and human mesenchymal stem cells

IntroductionTumor microenvironment conferred by stromal (mesenchymal) stem cells (MSCs) plays a key role in tumor development, progression, and response to therapy. Defining the role of MSCs in tumorigenesis is crucial for their safe utilization in regenerative medicine. Herein, we conducted comprehensive investigation of the cross-talk between human MSCs (hMSCs) and 12 cancer cell lines derived from breast, prostate, colon, head/neck and skin.MethodsHuman bone marrow-derived MSC line expressing green fluorescence protein (GFP) (hMSC-GFP) were co-cultured with the following cancer cell lines: (MCF7, BT-20, BT-474, MDA-MB-468, T-47D, SK-BR-3, MDA-MB-231, PC-3, HT-29, MDA-MB-435s, and FaDu) and changes in their morphology were assessed using fluorescent microscopy. For cellular tracking, cells were labeled with Vybrant DiO, DiL, and DiD lipophilic dyes. Time-lapse microscopy was conducted using Nikon BioStation IM-Q. Stable expression of mCherry, and luciferase genes was achieved using lentiviral technology. IL1-Beta neutralizing experiments were conducted using soluble recombinant IL-1R (srIL-1R). Changes in gene expression in sorted hMSCs were assessed using Agilent microarray platform while data normalization and bioinformatics were conducted using GeneSpring software.ResultsWe observed a dynamic interaction between cancer cells and hMSCs. High CDH1 (E-cadherin) and low IL1-Beta expression by cancer cells promoted reorganization of hMSCs into a niche-like formation, which was dependent on direct cell-cell contact. Our data also revealed transfer of cellular components between cancer cells and hMSCs as one possible mechanism for intercellular communication. Global gene expression analysis of sorted hMSCs following co-culturing with MCF7 and BT-20 cells revealed enrichment in signaling pathways related to bone formation, FAK and MAPKK signaling. Co-culturing hMSCs with MCF7 cells increased their growth evidenced by increase in Ki67 and PCNA staining in tumor cells in direct contact with hMSCs niche. On the other hand, co-culturing hMSCs with FaDu, HT-29 or MDA-MB-231 cells led remarkable decline in their cell growth.ConclusionsDynamic interaction exists between hMSCs and cancer cells. CDH1 and IL1-Beta expression by cancer cells mediates the crosstalk between hMSCs and cancer cells. We propose a model where hMSCs act as the first line of defense against cancer cell growth and spread.

Teratoma formation in immunocompetent mice after syngeneic and allogeneic implantation of germline capable mouse embryonic stem cells.

BACKGROUND Embryonic stem cells (ESCs) have the potential to form teratomas when implanted into immunodeficient mice, but data in immunocompetent mice are limited. We therefore investigated teratoma formation after implantation of three different mouse ESC (mESC) lines into immunocompetent mice. MATERIALS AND METHODS BALB/c mice were injected with three highly germline competent mESCs (129Sv, BALB/c and C57BL/6) subcutaneously or under the kidney capsule. After 4 weeks, mice were euthanized and examined histologically for teratoma development. The incidence, size and composition of teratomas were compared using Pearson Chi-square, t-test for dependent variables, one-way analysis of variance and the nonparametric Kruskal- Wallis analysis of variance and median test. RESULTS Teratomas developed from all three cell lines. The incidence of formation was significantly higher under the kidney capsule compared to subcutaneous site and occurred in both allogeneic and syngeneic mice. Overall, the size of teratoma was largest with the 129Sv cell line and under the kidney capsule. Diverse embryonic stem cell-derived tissues, belonging to the three embryonic germ layers, were encountered, reflecting the pluripotency of embryonic stem cells. Most commonly represented tissues were nervous tissue, keratinizing stratified squamous epithelium (ectoderm), smooth muscle, striated muscle, cartilage, bone (mesoderm), and glandular tissue in the form of gut- and respiratory-like epithelia (endoderm). CONCLUSIONS ESCs can form teratomas in immunocompetent mice and, therefore, removal of undifferentiated ESC is a pre-requisite for a safe use of ESC in cell-based therapies. In addition the genetic relationship of the origin of the cell lines to the ability to transplant plays a major role.

CUDC-907 Promotes Bone Marrow Adipocytic Differentiation Through Inhibition of Histone Deacetylase and Regulation of Cell Cycle

The role of bone marrow adipocytes (BMAs) in overall energy metabolism and their effects on bone mass are currently areas of intensive investigation. BMAs differentiate from bone marrow stromal cells (BMSCs); however, the molecular mechanisms regulating BMA differentiation are not fully understood. In this study, we investigated the effect of CUDC-907, identified by screening an epigenetic small-molecule library, on adipocytic differentiation of human BMSCs (hBMSCs) and determined its molecular mechanism of action. Human bone marrow stromal cells exposed to CUDC-907 (500 nM) exhibited enhanced adipocytic differentiation (∼2.9-fold increase, P < 0.005) compared with that of control cells. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis, cell cycle, and DNA replication. Chromatin immune precipitation combined with quantitative polymerase chain reaction showed significant increase in H3K9ac epigenetic marker in the promoter regions of AdipoQ, FABP4, PPARγ, KLF15, and CEBPA in CUDC-907-treated hBMSCs. Follow-up experiments corroborated that the inhibition of histone deacetylase (HDAC) activity enhanced adipocytic differentiation, while the inhibition of PI3K decreased adipocytic differentiation. In addition, CUDC-907 arrested hBMSCs in the G0-G1 phase of the cell cycle and reduced the number of S-phase cells. Our data reveal that HDAC, PI3K, and cell cycle genes are important regulators of BMA formation and demonstrate that adipocyte differentiation of hBMSCs is associated with complex changes in a number of epigenetic and genetic pathways, which can be targeted to regulate BMA formation.