Measurement of the Bio-Mechanical Properties of Two Different Feeder Layer Cells

Introduction: We here present our findings on 2 types of feeder layers, one composed of mouse embryonic fibroblasts (MEF) and the second one of mouse skeletal myoblasts (C2Cl2) feeder cells. Methods: The 2 feeder layers present a dramatic variance of intrinsic stiffness (142.68 ± 17.21 KPa and 45.78 ± 9.81 KPa, respectively). Results and Conclusion: This information could be used for a better understanding of cells and cell microenvironment mechano-physical characteristics that are influencing stem cell commitment, in order to develop a suitable engineered tissue for cardiac and skeletal muscle repair and a bio-actuator.

Direct differentiation of cord blood derived mesenchymal stem cells into keratinocytes without feeder layers and cAMP inducers

Objectives: The purpose of our study was isolation of umbilical cord blood derived mesenchymal stem (UCB-MSCs), their direct differentiation towards keratinocytes without using feeder layers, cAMP inducers and hormones known for morphological maintenance and proliferation of keratinocytes and characterization of UCB-MSCs through flowcytometry and keratinocytes through immunofluorescence. Methods: We have isolated and cultured UCB-MSCs (n=4) following critical parameters for successful isolation like sample processing within an hour of collection, gestational age not more than 38 weeks, no co-morbid and blood volume at least 80 ml. Cord blood mononuclear cells were isolated through ficoll based density-gradient centrifugation then cultured to isolate MSCs, defined by minimum criteria of International Society for Cellular Therapy. UCB-MSCs were then differentiated directly into keratinocytes. Differentiation was confirmed by morphology and characterized through immunofluorescence staining. UCB samples were collected from gynae/obstetric ward of OJHA campus under sterile conditions and processed at Stem cells and Regenerative medicine Lab, Dow Research Institute of Biotechnology and Biomedical Sciences, Ojha campus. The total duration of study was approximately 12 months. Results: We have successfully isolated UCB-MSCs that were plastic adherent, spindle shaped, showed trilineage mesodermal differentiation potential and were positive for CD90, CD73 and CD105 and negative for CD34 markers. UCB-MSCs were directly differentiated towards keratinocytes without using cAMP inducers, hormones or feeder layers. Differentiated keratinocytes attained typical honeycomb morphology and were stained positive on immunofluorescence for anti-pan cytokeratin antibody. Conclusion: Our study concludes possibility of direct differentiation of isolated and cultured UCB-MSCs into keratinocytes without using feeder layers and conventional keratinocyte culture media. doi: https://doi.org/10.12669/pjms.36.5.1566 How to cite this:Ghauri AK, Wahid M, Mirza T, Uddin JAA. Direct differentiation of cord blood derived mesenchymal stem cells into keratinocytes without feeder layers and cAMP inducers. Pak J Med Sci. 2020;36(5):---------. doi: https://doi.org/10.12669/pjms.36.5.1566 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Xenofree generation of limbal stem cells for ocular surface advanced cell therapy

Background Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives. Methods We compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA. Results XSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells. Conclusions The combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy. Graphical abstract

IVM of mouse fully grown germinal vesicle oocytes upon a feeder layer of selected cumulus cells enhances their developmental competence

In the ovary, acquisition of oocyte developmental competence depends on a bidirectional exchange between the gamete and its companion cumulus cells (CCs). In this study we investigated the contribution of CCs surrounding oocytes of known developmental competence or incompetence to the acquisition of oocyte developmental competence. To this end, feeder layers of CCs (FL-CCs) were prepared using CCs isolated either from: (1) developmentally competent mouse oocytes whose nucleolus was surrounded by a chromatin ring (FL-SN-CCs); or (2) developmentally incompetent mouse oocytes whose nucleolus was not surrounded by a chromatin ring (FL-NSN-CCs). Denuded, fully grown oocytes (DOs) were matured to the MII stage on either FL-SN-CCs or FL-NSN-CCs, inseminated with spermatozoa and cultured throughout preimplantation development. FL-SN-CCs significantly improved the acquisition of oocyte developmental competence, with a blastocyst development rate equal to that for maturation of intact cumulus–oocyte–complexes. In contrast, DOs matured on FL-NSN-CCs or in the absence of CCs exhibited developmental failure, with embryos arresting at either the 4-cell or morula stage. These results set a culture platform to further improve the protocols for the maturation of DOs and to unravel the molecules involved in the cross-talk between the gamete and its companion CCs during the germinal vesicle to MII transition.