Engraftment and Proliferation of Thermoreversible-Gelation-Polymer-Encapsulated Human Corneal Limbal-Stem-Cells on Ocular Surface of a Cadaver Cornea.

PURPOSE: Corneal limbal stem cell (LSC) transplantation has been reported as a potential approach to treat the damaged corneal epithelium. Scaffolds such as human amniotic membrane (hAM) are commonly employed for the in vitro culture and as a carrier during in vivo transplantation. However, they carry the risk of biological contamination and donor to donor variability. To overcome these disadvantages, we herein report the capabilities of a synthetic thermoreversible gelation polymer (TGP) scaffold to serve as an encapsulation support during LSC transplantation and to enable engraftment for corneal regeneration. METHODS: Sixteen discarded human corneas were used to isolate the corneal epithelium which was cultured in TGP and hAM. The cell proliferation and characteristics between TGP and hAM culture methods were evaluated by microscopic observation, 3H Thymidine incorporation assay, immunoperoxidase and immunofluorescence staining. RESULTS: The 3H Thymidine assay's results showed that TGP allowed human-donor cornea-derived LSCs to proliferate well in vitro, compared to hAM and the cells encapsulated in TGP and transplanted ex vivo onto a human cadaver donor cornea denuded of its epithelium, migrated on the ocular surface, and proliferated to form a continuous layer in 25 days. Immunoperoxidase and Immunofluorescence staining of TGP-cultured cells were positive for LSC markers (p63, ABCG2, Connexin 43 and Integrin ß), proving that the TGP helps to preserve the limbal cells' stemness. CONCLUSION: TGP is found to be a multipurpose scaffold for (i) in vitro culture, (ii) ex vivo encapsulation, and in vivo transplantation (iii), enabling engraftment of LSCs in this study, with potentials to extend its application in cell-based therapies in several regenerative medicine approaches.

In Vitro Culture Expansion and Characterization of Buccal Mucosal Epithelial Cells for Tissue Engineering Applications in Urethral Stricture After Transportation Using a Thermoreversible Gelation Polymer.

Introduction: The transportation of tissues from hospitals to clinical laboratories for cell therapy is an essential component of regenerative medicine. Previously, we used laboratory-cultured mucosal cells from buccal epithelium expanded and encapsulated using a scaffold-hybrid approach to the urethral stricture (BEES-HAUS) procedure. In this study, to improve the outcomes, we compared the thermoreversible gelation polymer (TGP) transportation procedure with conventional culture methods, and reported its advantages. Methods: Human buccal mucosal tissues in Phase I of the study were transported in Euro-Collins solution (ECS) and the cells obtained were cultured in two-dimensional (2D) Dulbecco's modified Eagle's medium (DMEM), CnT-Prime epithelial 2D differentiation medium (CnT-PR), and a three-dimensional (3D)-TGP scaffold. In Phase II, tissues were transported in a TGP cocktail and the ECS. The cells were cultured in 2D-DMEM and 3D-TGP, quantified, and characterized by immunohistochemistry. Results: The cells in 3D-TGP culture maintained epithelial morphology in a better manner compared with 2D-DMEM, in which they developed fibroblast-like morphology. The TGP-transported cells grew rapidly. Immunohistochemical analysis results for AE1/AE3, EGFR, integrin-ß1, p63, and p75 were intensely positive in 3D-TGP. Conclusion: The TGP-based cocktail used in human buccal tissue transportation yielded cells with better morphology maintenance. The TGP scaffold provides an optimal in vitro environment wherein epithelial cells better maintain their native phenotype compared to those cultured through conventional methods. These results suggest using TGP for the transportation and culture of human buccal tissues for clinical applications. In addition, the use of a TGP-based cocktail for the transport of other tissues for regenerative medicine applications is worth further analysis.

Successful engraftment of epithelial cells derived from autologous rabbit buccal mucosal tissue, encapsulated in a polymer scaffold in a rabbit model of a urethral stricture, transplanted using the transurethral approach.

BACKGROUND: A pilot study reported an autologous buccal mucosal cell transplant in humans through the trans-urethral route using the buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS), a minimally invasive approach to treat urethral stricture. Although successful outcomes were achieved in that study, for further validation, it is essential to prove that the transplanted buccal epithelium was engrafted over the urothelium through histological examination of the urethra, harvested post-transplant, which is infeasible in humans. Herein, we report the successful creation of an animal model of urethral stricture and the engraftment of epithelial cells derived from autologous buccal mucosal tissue, encapsulated in a thermo-reversible gelation polymer (TGP) scaffold, transplanted by trans-urethral route. METHODS: An animal model of urethral stricture was created in Japanese white male rabbits using electro-coagulation. Buccal tissue was harvested from the rabbits and subjected to enzyme digestion, followed by 5-7 days of in vitro culture in conventional two-dimensional (2D) culture and in a 3D platform of thermo-reversible gelation polymer (3D-TGP) culture. The cells harvested from the groups were mixed and encapsulated and transplanted with TGP, by transurethral catheterization. Fourteen days later, the urethra was harvested and subjected to histological examination. The buccal biopsy tissue, cells after digestion and cells post-culture were also subjected to histological examination. Urethrogram and endoscopy images were recorded at different time points. RESULTS: The stricture was successfully created, with the coagulated area markedly stenosed. Histological staining of the cells after in vitro processing showed that the cells grew with native epithelial and rounded cell morphology in 3D-TGP while they differentiated into fibroblast like-cells in 2D culture. Histological staining of the urethral tissue after transplantation revealed the engraftment of the transplanted buccal mucosal cells, with stratified squamous epithelium over the specialized stratified urothelium in the urethrotomy site. CONCLUSION: We used histology to prove the successful engraftment of TGP-encapsulated buccal mucosal epithelial cells in an animal model of urethral injury with healing of the injured tissue. The model of urethral stricture and cell therapy, using a transurethral approach, recapitulates the previously reported BEES-HAUS approach and lays the foundation for larger multi-centric translational clinical studies.

Cell-based immunotherapy in stage IIIA inflammatory breast cancer with declining innate immunity following successive chemotherapies: A case report.

Cancer stem cells in breast cancer migrating to the bone marrow may cause future metastasis, particularly during periods of decreased immunity. Natural killer (NK) cells have a role in immune surveillance and are able to target cancer stem cells. The present study reported a case in which NK cell-based autologous immune enhancement therapy was used combined with conventional treatments in a patient with stage IIIA breast cancer, yielding >28 months of disease-free survival. However, there was a gradual decline in the in vitro expansion of NK cells with subsequent chemotherapeutic treatments. As this NK cell decline following chemotherapy may contribute to cancer cell immune evasion and future metastasis; modifying current cancer therapies in order to avoid potentially compromising the immune system may lead to improved treatment outcomes.

The known-unknowns in spinal cord injury, with emphasis on cell-based therapies - a review with suggestive arenas for research.

INTRODUCTION: In spite of extensive research, the progress toward a cure in spinal cord injury (SCI) is still elusive, which holds good for the cell- and stem cell-based therapies. We have critically analyzed seven known gray areas in SCI, indicating the specific arenas for research to improvise the outcome of cell-based therapies in SCI. AREAS COVERED: The seven, specific known gray areas in SCI analyzed are: i) the gap between animal models and human victims; ii) uncertainty about the time, route and dosage of cells applied; iii) source of the most efficacious cells for therapy; iv) inability to address the vascular compromise during SCI; v) lack of non-invasive methodologies to track the transplanted cells; vi) need for scaffolds to retain the cells at the site of injury; and vii) physical and chemical stimuli that might be required for synapses formation yielding functional neurons. EXPERT OPINION: Further research on scaffolds for retaining the transplanted cells at the lesion, chemical and physical stimuli that may help neurons become functional, a meta-analysis of timing of the cell therapy, mode of application and larger clinical studies are essential to improve the outcome.

Smallpox still haunts scientists: results of a questionnaire-based inquiry on the views of health care and life science experts and students on preserving the remaining variola virus stocks.

The World Health Organization (WHO) declared eradication of the dreadful disease "smallpox" in 1980. Though the disease has died down, the causative virus "variola" has not, as it has been well preserved in two high security laboratories-one in USA and another in Russia. The debate on whether the remaining stocks of the smallpox virus should be destroyed or not is ongoing, and the World Health Assembly (WHA) in 2011 has decided to postpone the review on this debate to the 67th WHA in 2014. A short questionnaire-based inquiry was organized during a one-day stem cell meeting to explore the views of various health care and life science specialists especially students on this aspect. Among the 200 participants of the meeting, only 66 had answered the questionnaire. 60.6% of participants who responded to the questionnaire were for preserving the virus for future reference, while 36.4% of the participants were for destroying the virus considering the magnitude with which it killed millions. However, 3% of the respondents were not able to decide on any verdict. Therefore, this inquiry expresses the view that "what we cannot create, we do not have the right to destroy."

In vitro culture and characterization of enteric neural precursor cells from human gut biopsy specimens using polymer scaffold.

In vitro expansion and characterization of neural precursor cells from human gut biopsy specimens with or without Hirschsprung's disease using a novel thermoreversible gelation polymer (TGP) is reported aiming at a possible future treatment. Gut biopsy samples were obtained from five patients undergoing gut resection for Hirschsprung's disease (n = 1) or gastrointestinal disorders (n = 4). Cells isolated from the smooth muscle layer and the myenteric plexus were cultured in two groups for 18 to 28 days; Group I: conventional culture as earlier reported and Group II: using TGP scaffold. Neurosphere like bodies (NLBs) were observed in the cultures between 8th to 12th day and H & E staining was positive for neural cells in both groups including aganglionic gut portion from the Hirschsprung's disease patient. Immunohistochemistry using S-100 and neuron specific enolase (NSE) was positive in both groups but the TGP group (Group II) showed more number of cells with intense cytoplasmic granular positivity for both NSE and S-100 compared to Group I. TGP supports the in vitro expansion of human gut derived neuronal cells with seemingly better quality NLBs. Animal Studies can be tried to validate their functional outcome by transplanting the NLBs with TGP scaffolds to see whether this can enhance the outcome of cell based therapies for Hirschsprung's disease.