Overview of the National Ethics Committee Registry for Biomedical and Health Research in India: Stepping up to safeguard the ethical aspect of research involving human participants.

As notified in the New Drugs and Clinical Trials Rules 2019, ethics committees (ECs), intending to review and oversee the conduct of Biomedical and Health Research (BHR) shall be required to register with the authority designated by the Central Government in the Ministry of Health and Family Welfare, Department of Health Research (DHR). The entire process of receiving and processing such applications is done online through the Naitik portal. Vide these rules, it has become mandatory for all institutions/entities whether publicly or privately conducting or intending to conduct BHR involving the human participants, to get their EC, registered with the DHR. A status report of the functioning of the National Ethics Registry and an analysis of ECs registered with the DHR are presented in this manuscript. A workflow of the processing involved in EC registration is given with sectorial segregation, and analysis of data on ECs across the country is made for the dissemination and information. This article elaborates on the registration requirements and process of the EC registry with the necessity of being registered with the DHR. 2100 login requests and more than 1560 applications for registration have been received; private hospitals and medical colleges have been the front-runner in getting their organization registered, and organizations in the commercial sector are faring better in terms of EC registration. Further dissemination and outreach efforts have to be made to draw the attention of various stakeholders regarding this requirement and thereby ensuring that all ECs in the country are registered with the DHR.

Peripheral Blood As a Source of Stem Cells for Regenerative Medicine: Emphasis Towards Corneal Epithelial Reconstruction-An In Vitro Study.

BACKGROUND: Mesenchymal stem cell-based treatments are now emerging as a therapy for corneal epithelial damage. Although bone marrow, adipose tissue and umbilical cord blood are the main sources of mesenchymal stem cells (MSCs), other tissues like the peripheral blood also harbor mesenchymal-like stem cells called peripheral blood-derived mononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasive collection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneal limbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymal cell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explant conditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiated PBMNCs were engineered into a cell sheet using an in-house developed thermo-responsive polymer. RESULTS: These blood derived cells were demonstrated to have similar properties to mesenchymal stem cells. Corneal epithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker thereby demonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitro cell sheet that was then demonstrated for cell sheet transfer on an ex vivo excised rabbit eye. CONCLUSION: PBMNCs as an alternative autologous cell source for limbal stem cells is envisaged as an effective therapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency.

Bioengineered corneal epithelial cell sheet from mesenchymal stem cells-A functional alternative to limbal stem cells for ocular surface reconstruction.

Limbal stem cell deficiency (LSCD) is the loss of limbal stem cells that reside in the corneoscleral junction resulting in vision loss or blindness. Bilateral LSCD is usually treated by allogeneic corneal transplantation, with instances of tissue rejection or failure in long-term follow-up. This study aims to use adipose mesenchymal stem cells (ASC) as an alternative autologous cell source for treating bilateral limbal deficiency conditions. ASCs derived from rabbit fat tissue were differentiated into corneal epithelial lineage using limbal explant condition media. Apart from transdifferentiation, ASC sheets were developed to facilitate effective delivery of these cells to the damage site. A thermoresponsive polymer N-isopropylacrylamide-co-glycidylmethacrylate (NGMA) was synthesized and characterized to demonstrate ASC sheet formation. Transdifferentiated ASCs showed positive expression of corneal epithelial marker CK3/12 on immunostaining, supported by gene expression studies. in vivo studies by transplanting cell sheet in rabbit models of corneal injury showed clear and smooth cornea in comparison to the sham models. Histology revealed a sheet of cells aligned and integrated on to the injured corneal surface, 1 month posttransplantation. Identifying ASCs as an alternative cell source along with cell sheet technology will be a novel step in the field of corneal surface therapies.

Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics.

Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared to asynchronous proliferating (pre-G0) BMSC. Global gene expression profiling revealed reprogramming of the transcriptome during G0 state including significant alterations in relevant pathways and expression of secreted factors, suggesting altered autocrine and paracrine signaling by G0 state-BMSC and a possible mechanism for enhanced bone formation. G0 state-BMSC might provide a clinically relevant model for understanding the in vivo biology of BMSC.

Cell density overrides the effect of substrate stiffness on human mesenchymal stem cells' morphology and proliferation.

The effect of substrate stiffness on the cellular morphology, proliferation, and differentiation of human mesenchymal stem cells (hMSCs) has been extensively researched and well established. However, the majority of these studies are done with a low seeding density where cell to cell interactions do not play a significant role. While these conditions permit an analysis of cell-substratum interactions at the single cell level, such a model system fails to capture a critical aspect of the cellular micro-environment in vivo, i.e. the cell-cell interaction via matrix deformation (i.e., strain). To address this question, we seeded hMSCs on soft poly-acrylamide (PAA) gels, at a seeding density that permits cells to be mechanically interacting via the underlying substrate. We found that as the intercellular distance decreases with the increasing seeding density, cellular sensitivity towards the substrate rigidity becomes significantly diminished. With the increasing seeding density, the cell spread area increased on a soft substrate (500 Pa) but reduced on an even slightly stiffer substrate (2 kPa) as well as on glass making them indistinguishable at a high seeding density. Not only in terms of cell spread area but also at a high seeding density, cells formed mature focal adhesions and prominent stress fibres on a soft substrate similar to that of the cells being cultured on a stiff substrate. The decreased intercellular distance also influenced the proliferation rate of the cells: higher seeding density on the soft substrate showed cell cycle progression similar to that of the cells on glass substrates. In summary, this paper demonstrates how the effect of substrate rigidity on the cell morphology and fate is a function of inter-cellular distance when seeded on a soft substrate. Our AFM data suggest that such changes happen due to local strain stiffening of the soft PAA gel, an effect that has been rarely reported in the literature so far.

Post implantation fate of adipogenic induced mesenchymal stem cells on Type I collagen scaffold in a rat model.

Regenerative medicine via its application in soft tissue reconstruction through novel methods in adipose tissue engineering (ATE) has gained remarkable attention and investment despite simultaneous reports on clinical incidence of graft resorption and impaired vascularization. The underlying malaise here once identified may play a critical role in optimizing implant function. Our work attempts to determine the fate of donor cells and the implant in recipient micro environment using adipose-derived mesenchymal stem cells (ASCs) on a type I collagen sponge, an established scaffold for ATE. Cell components within the construct were identified 21 days post implantation to delineate cell survival, proliferation & terminal roles in vivo. ASC's are multipotent, while collagen type I is a natural extra cellular matrix component. Commercially available bovine type I collagen was characterized for its physiochemical properties and cyto-compatibility. Nile red staining of induced ASCs identified red globular structures in cell cytoplasm indicating oil droplet accumulation. Similarly, in vivo implantation of the cell seeded collagen construct in rat model for 21 days in the dorsal muscle, showed genesis of chicken wire network of fat-like cells, which was demonstrated histologically using a variety of staining techniques. Furthermore, fluorescent in situ hybridization (FISH) technique established the efficiency of transplantation wherein the male donor cells with labeled Y chromosome was identified 21 days post implantation from female rat model. Retrieved samples at 21 days indicated adipogenesis in situ, with donor cells highlighted via FISH. The study provides an insight to stem cells in ATE from genesis to functionalization.

Adipogenesis on biphasic calcium phosphate using rat adipose-derived mesenchymal stem cells: in vitro and in vivo.

Developing adipose tissue-engineered construct to mend soft tissue defects arising from traumatic injury, tumor resections, and maxillofacial abnormalities is of prime importance in plastic and reconstructive surgical procedures. It is apparent that the clinical outcome of classic techniques like adipose tissue transplantation is unpredictable, with graft resorption, lack of vascularization, and impaired functionality. In this prospective, the concept of tissue engineering was adopted to fabricate a combination product with biphasic calcium phosphate (BCP) and rat adipose-derived mesenchymal stem cells (ASCs) toward the development of an adipose tissue construct. BCP, a combination of hydroxyapatite and α-tricalcium phosphate, was characterized for its physiochemical properties, and ASCs were characterized for their stemness. The cell-ceramic interactions were demonstrated in vitro, whereas adipogenesis was picturesquely depicted by Nile red-stained multilocular adipocyte-like cells. Subsequently, the three-dimensional cell-ceramic-engineered construct was implanted in the rat dorsal muscle for a period of 3 weeks to demonstrate the efficacy of the tissue construct in vivo. Interestingly, the histology of the postimplanted tissue construct revealed the distribution of chicken wire net-like fat cells within the vicinity of the construct. The efficacy of cell transplantation via the scaffold was traced using fluorescent in situ hybridization by labeling the Y chromosome. Thus, the ceramic-based construct may be a good option for reconstruction therapies.