Development of Bioengineered Organ Using Biological Acellular Rat Liver Scaffold and Hepatocytes.

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 cell-specific 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 Cytokeratin-19 (CK-19), 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.

Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue

Abstract Background Mesenchymal 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. Methods Stromal 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. Results Human 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. Conclusions Availability 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.

Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue.

BACKGROUND: Mesenchymal 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. METHODS: Stromal 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. RESULTS: Human 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. CONCLUSIONS: Availability 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.

Proliferation and differentiation potential of human adipose-derived stem cells grown on chitosan hydrogel.

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.

Fluorescent magnetic iron oxide nanoparticles for cardiac precursor cell selection from stromal vascular fraction and optimization for magnetic resonance imaging.

Fluorescent magnetic iron oxide nanoparticles have been used to label cells for imaging as well as for therapeutic purposes. The purpose of this study was to modify the approach to develop a nanoprobe for cell selection and imaging with a direct therapeutic translational focus. The approach involves physical coincubation and adsorption of superparamagnetic iron oxide nanoparticle-polyethylene glycol (SPION-PEG) complexes with a monoclonal antibody (mAb) or a set of antibodies. Flow cytometry, confocal laser scanning microscopy, transmission electron microscopy, iron staining, and magnetic resonance imaging were used to assess cell viability, function, and labeling efficiency. This process has been validated by selecting adipose tissue-derived cardiac progenitor cells from the stromal vascular fraction using signal regulatory protein alpha (SIRPA)/kinase domain receptor (KDR) mAbs. These markers were chosen because of their sustained expression during cardiomyocyte differentiation. Sorting of cells positive for SIRPA and KDR allowed the enrichment of cardiac progenitors with 90% troponin-I positivity in differentiation cultures. SPION labeled cardiac progenitor cells (1×10(5) cells) was mixed with gel and used for 3T magnetic resonance imaging at a concentration, as low as 12.5 µg of iron. The toxicity assays, at cellular and molecular levels, did not show any detrimental effects of SPION. Our study has the potential to achieve moderate to high specific cell selection for the dual purpose of imaging and therapy.

A study on FoxP3 and Tregs in paired samples of peripheral blood and synovium in rheumatoid arthritis.

There is an increasing evidence suggesting the role of fork head boxP3 (FoxP3) in the development and the regulation of CD4(+)CD25(+) Treg cells. T-cell regulatory mechanisms in rheumatoid arthritis patients were evaluated by the contributing factors such as pro-inflammatory cytokines, circulating immune complexes, HLA DR expression, ligand binding biomarkers, FoxP3 expression in paired samples of peripheral blood (PB) and synovial fluid (SF). These cellular responses were further correlated with the humoral immune responses such as anti-cyclic citrullinated peptides IgG (CCP), circulating immune complex-c1q IgG (CIC), immunoglobulin G (IgG) and immunoglobulin M (IgM) of the rheumatoid arthritis factor (RAF). The results suggest a definitive role of Tregs in the homeostatic control because there is an increase in FoxP3 (37%) and HLA-DR (45%) expression in the synovial fluid as compared to PB. Furthermore, humoral responses as a downstream effector mechanism are positively correlated with the pathogenesis of rheumatoid arthritis (RA). A positive relationship exists between quantitative anti-CCP production and the expression of HLA-DR. The study relates an increased and pivotal role of B cell activation in the synovial fluid thereby permitting the need to ablate the targeted B cell immune responses.

Evaluation of the autologous bone marrow mononuclear therapy and functional restoration in the scarred myocardium by imaging analysis.

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 × 10(7) 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.