Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.

BackgroundMesenchymal stem cells have immense potential in stem cell-based therapies, however there is a pre-requisite to develop a curative cell dose. Adipose tissue-derived mesenchymal stem cells are promising mainly due to their potential abundance, immunomodulatory effect and remarkable differentiation potential. Nevertheless, senescence may develop during their in vitro expansion due to the incidence of genetic instability. Hence, it is important to attain an ideal balance between mesenchymal stem cell growth, quality and genetic integrity before their clinical use.MethodsStromal vascular fraction was obtained from omentum tissue of patients undergoing liposuction procedures for morbid obesity. This study standardized a closed system protocol which can be utilized for clinical grade stem cell derivation. Stages of cell growth and characterization of human adipose tissue-derived mesenchymal stem cells were also assessed along with the chromosomal stability in these in vitro cultures.ResultsHuman adipose tissue-derived mesenchymal stem cells maintained their spindle-shaped morphology and were able to proliferate and renew, confirming their suitability for in vitro cultivation and generate clinical grade mesenchymal stem cells. Immunophenotyping indicates that the cells expressed cluster of differentiation (CD)73/CD90/CD105, mesenchymal stem-cell markers, while lacked CD34/CD45/ Human Leukocyte antigen-antigen D related (HLA-DR) expression (hematopoietic cell markers). A cell cycle study demonstrated growth kinetics under in vitro culture conditions. Human adipose tissue derived mesenchymal stem cells expressed normal cell karyotype by chromosomal G-banding indicating their genetic stability at Passage 5. Mesenchymal stem cells also demonstrated trilineage differentiation.ConclusionsAvailability of adipose tissue in abundance is a major advantage for clinical applications. Furthermore, detailed characterization of human adipose tissue-derived mesenchymal stem cells, their genomic stability and differentiation potential from stromal vascular fraction of human adipose tissue would help assist in tissue regeneration and repair.

Directional transdifferentiation of bone marrow precursor cells assumes beta cell like properties in modified tissue microenvironment. The factors that modify the roles of precursor cells to functional beta cells enabling precise, defined and efficient in vitro differentiation protocols are yet to be conclusive. The study aims at the determination of appropriate induction factors that may aid the robust, reproducible transdifferentiation of rat bone marrow derived mesenchymal stem cells (MSCs) to islet-like cells and enhance their transdifferentiation efficiency. High glucose concentration including nicotinamide, β-mercaptoethanol along with β-cellulin, IGF-1 were able to induce bone marrow precursor cells to islet like clusters ex vivo consistently. The four step induction protocol has enhanced the expression of pancreatic islet cell specific transcription and translational factors detectable by immunocytochemistry viz., pro-insulin, glucagon, somatostatin and polypeptide. The functionality was assessed by the glucose challenge assay followed by animal experiment. The streptozotocin (STZ) induced rats demonstrated significant reduction in glucose levels post islet like cell transplantation (P&lt;0.05). The tropic and the growth factors thus used have a profound impact on the induction of the bone marrow precursors to functional islet like cells

The increasing demand for organs for transplantation necessitates the development of substitutes to meet the structural and physiological functions. Tissue decellularization and recellularization aids in retaining the three-dimensional integrity, biochemical composition, tissue ultra-structure, and mechanical behavior, which makes them functionally suitable for organ transplantation. Herein, we attempted to rebuild functional liver grafts in small animal model (Wistar rat) with a potential of translation. A soft approach was adopted using 0.1% SDS (Sodium Dodecyl Sulfate) for decellularization and primary hepatocytes were used as a potential cell source for recellularization. The decellularization process was evaluated and confirmed using histology, DNA content, ultra-structure analysis. The resultant scaffold was re-seeded with the rat hepatocytes and their biocompatibility was assessed by its metabolic functions and gene expression. The structural components of the Extracellular matrix (ECM) (Laminins, Collagen type I, Reticulins) were conserved and the liver cellspecific proteins like CK-18, alpha-fetoprotein, albumin were expressed in the recellularized scaffold. The functionality and metabolic activity of the repopulated scaffold were evident from the albumin and urea production. Expression of , Glucose 6-Phosphatase (G6P), Albumin, Gamma Glutamyl Transferase (GGT) genes has distinctly confirmed the translational signals after the repopulation process. Our study clearly elucidates that the native extracellular matrix of rat liver can be utilized as a scaffold for effective recellularization for whole organ regeneration.

A 62-year-old male patient with previous history of myocardial infarction, akinetic myocardial segments, and an ejection fraction of 31% with the NYHA class III category was selected for the autologous bone marrow (ABM)-derived mononuclear cell fraction injection during CABG surgery. Nitrate augmented myocardial tracer uptake was imaged by ECG gated SPECT pre- and 1 year post-ABM therapy, using radiotracer Tc99m Sestamibi. The baseline gated SPECT demonstrated full thickness infarct in 40% area of LAD territory. Bone marrow aspirate of 20.0 ml from sternum yielding a mono nuclear cell fraction of 4.5 × 107 cells/ml was suspended in 2.0 ml of sterile normal saline to be injected at eight sites of the injured myocardium. There were no apparent side effects due to the procedure, i.e., life threatening events, major bleeds, reaction, or shock. The case was followed at the end of 1, 3, 6 months by ECG and Holter monitor and ECG gated SPECT at the end of 12 months. The gated SPECT images demonstrated mild but definitely improved tracer uptake within part of the infarcted segments along with improvement in ejection fraction to 45%, and a clinical change in the NYHA Class to II. Cell-based therapy may offer benefits of induction of normal tissue microenvironment.