Ian Michael Rogers

Hair follicles (HFs) are highly accessible mini-organs that house diverse somatic and stem cell populations with broad therapeutic potential. In this study, we investigate the untapped utility of plucked HFs as a non-invasive tissue source for regenerative medicine. We demonstrate the successful isolation and expansion of keratinocytes and mesenchymal stem cells (MSCs) from plucked follicles using an enzyme-free explant culture method. HF-derived keratinocytes retained their epithelial identity and were efficiently reprogrammed into induced pluripotent stem cells (iPSCs). These iPSCs were further directed toward definitive endoderm and pancreatic progenitor fates, confirming their robust autologous regenerative capacity. Flow cytometric analysis of HF-MSCs validated a characteristic mesenchymal profile, and these cells exhibited classical trilineage plasticity alongside the ability to differentiate into dopaminergic neural progenitors. Furthermore, proteomic and vesicular characterization of the autologous HF secretome (aHFS) revealed a rich enrichment of regenerative cytokines and exosomes. The aHFS demonstrated potent wound-healing bioactivity in vitro. Collectively, these findings establish the plucked hair follicle as a highly practical, scalable source for both cell-based and cell-free therapies, highlighting the clinical value of early-stage follicular biobanking for personalized medicine.

Background: Modeling precancerous stages holds the promise to understand early transformation events, thereby offering the potential for personalized, targeted treatment. Because cancer hijacks developmental pathways, precancerous stages could potentially be modeled by reprogramming cancer cells to an induced pluripotent stem cell state and subsequently differentiating them to the target organs using organoid models. Methods: We attempted reprogramming of patient-derived clear cell renal cell carcinoma (ccRCC) cell lines and adjacent normal renal epithelial cell lines using lentivirus or episomal reprogramming vectors. Results: The cancer cells failed to reprogram while the adjacent normal cells reprogrammed with high efficiency. The von Hippel–Lindau factor (VHL) gene was re-expressed in ccRCC cells in an attempt to restore the wild-type phenotype and restore reprogramming. The VHL gene is the major tumor suppressor in ccRCC pathogenesis and a conductor of oxidative-glycolytic glucose metabolism. While its re-expression did restore the epithelial phenotype and oxidative regulation of ccRCC cells, they still failed to stably reprogram. With an optimized reprogramming workflow, VHL-corrected ccRCC cells generate NANOG+ cells; however, they remained dependent on the ectopic expression of the reprogramming factors. Conclusions: We concluded that while VHL expression is necessary for cellular reprogramming of ccRCC cells, other genetic lesions in the ccRCC cells could be preventing the stabilization of the pluripotent state.

Ex vivo organ perfusion (EVOP) is used for whole organ preservation, and the main focus is to improve the outcome of donor organs for transplantation. Recently, EVOP has found application in disease modeling, drug development, and tissue regeneration. We discuss progress in EVOP research involving small animal organs using benchtop and incubator‐based EVOP systems, highlighting innovative designs of EVOP systems, technical specifications of each system, and their versatile applications across a range of research fields.

The advent of induced pluripotent stem cell (iPSC) technology has brought about transformative advancements in regenerative medicine, offering novel avenues for disease modeling, drug testing, and cell-based therapies. Patient-specific iPSC-based treatments hold the promise of mitigating immune rejection risks. However, the intricacies and costs of producing autologous therapies present commercial challenges. The hair follicle is a multi-germ layered versatile cell source that can be harvested at any age. It is a rich source of keratinocytes, fibroblasts, multipotent stromal cells, and the newly defined Hair Follicle-Associated Pluripotent Stem Cells (HAP). It can also be obtained non-invasively and transported via regular mail channels, making it the ideal starting material for an autologous biobank. In this study, cryopreserved hair follicle-derived iPSC lines (HF-iPS) were established through integration-free vectors, encompassing a diverse cohort. These genetically stable lines exhibited robust expression of pluripotency markers, and showcased tri-lineage differentiation potential. The HF-iPSCs effectively differentiated into double-positive cKIT⁺/CXCR4⁺ definitive endoderm cells and NKX6.1⁺/PDX1⁺ pancreatic progenitor cells, affirming their pluripotent attributes. We anticipate that the use of plucked hair follicles as an accessible, non-invasive cell source to obtain patient cells, in conjunction with the use of episomal vectors for reprogramming, will improve the future generation of clinically applicable pancreatic progenitor cells for the treatment of Type I Diabetes.

Respiratory diseases, claim over eight million lives annually. However, the transition from preclinical to clinical phases in research studies is often hindered, partly due to inadequate representation of preclinical models in clinical trials. To address this, we conducted a proof‐of‐concept study using an ex vivo model to identify lung pathologies and to screen therapeutics in a humanized rodent model. We extracted and decellularized mouse heart‐lung tissues using a detergent‐based technique. The lungs were then seeded and cultured with human cell lines (BEAS‐2B, A549, and Calu3) for 6−10 days, representing healthy lungs, cancerous states, and congenital pathologies, respectively. By manipulating cultural conditions and leveraging the unique characteristics of the cell lines, we successfully modeled various pathologies, including advanced‐stage solid tumors and the primary phase of SARS‐CoV‐2 infection. Validation was conducted through histology, immunofluorescence staining, and pathology analysis. Additionally, our study involved pathological screening of the efficacy and impact of key anti‐neoplastic therapeutics (Cisplatin and Wogonin) in cancer models. The results highlight the versatility and strength of the ex vivo model in representing crucial lung pathologies and screening therapeutics during the preclinical phase. This approach holds promise for bridging the gap between preclinical and clinical research, aiding in the development of effective treatments for respiratory diseases, including lung cancer.

The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies. Here we show that the overexpression of eight immunomodulatory transgenes (Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically ‘cloak’ the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses. Moreover, by using the previously reported FailSafe transgene system, which transcriptionally links a gene essential for cell division with an inducible and cell-proliferation-dependent kill switch, we generated cloaked tissues from mESCs that served as immune-privileged subcutaneous sites that protected uncloaked allogeneic and xenogeneic cells from rejection in immune-competent hosts. The combination of cloaking and FailSafe technologies may allow for the generation of safe and allogeneically accepted cell lines and off-the-shelf cell products.

The majority of nucleated somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). The process of reprogramming involves epigenetic remodelling to turn on pluripotency-associated genes and turn off lineage-specific genes. Some evidence shows that iPSCs retain epigenetic marks of their cell of origin and this “epigenetic memory” influences their differentiation potential, with a preference towards their cell of origin. Here, we reprogrammed proximal tubule cells (PTC) and tail tip fibroblasts (TTF), from a reprogrammable mouse to iPSCs and differentiated the iPSCs to renal progenitors to understand if epigenetic memory plays a role in renal differentiation. This model allowed us to eliminate experimental variability due to donor genetic differences and transfection of the reprogramming factors such as copy number and integration site. In this study we demonstrated that early passage PTC iPSCs and TTF iPSCs expressed low levels of renal progenitor genes and high levels of pluripotency-associated genes, and the transcriptional levels of these genes were not significantly different between PTC iPSCs and TTF iPSCs. We used ChIP-seq of H3K4me3, H3K27me3, H3K36me3 and global DNA methylation profiles of PTC iPSCs and TTF iPSCs to demonstrate that global epigenetic marks were not different between the cells from the two different sets of tissue samples. There were also no epigenetic differences observed when kidney developmental genes and pluripotency-associated genes were closely examined. We did observe that during differentiation to renal progenitor cells the PTC iPSC-derived renal cells expressed higher levels of three renal progenitor genes compared to progenitors derived from TTF iPSCs but the underlying DNA methylation and histone methylation patterns did not suggest an epigenetic memory basis for this.

Regenerative medicine requires better pre-clinical tools in order to increase the efficiency of novel therapies transitioning to the clinic. Current monolayer cell culture methods are suboptimal for effectively testing new therapies and live mouse models are expensive, time consuming and require invasive procedures. Fetal organ culture, organoids, microfluidics and culture of thick sections of adult organs all aim to fill the knowledge gap between monolayer culture and live mouse studies. Here we report on an ex vivo organ perfusion system that can support whole adult mouse organs. Ex vivo perfusion of healthy and diseased mouse organs allows for real-time analysis that provides immediate feedback and accurate data collection throughout the experiment. Having a suitable normothermic ex vivo perfusion system for mouse organs provides a tool that will help contribute to our understanding of kidney physiology and disease and can take advantage of the many mouse models of human disease that already exist. Furthermore, an ex vivo kidney perfusion system can be used for testing novel cell therapies, drug screening, drug validation and for the detection of nephrotoxic substances. Critical to the success of mouse ex vivo organ perfusion is having a suitable bioreactor to maintain the organ. Here we have focused on the mouse kidney and mathematically modeled, built and validated a bioreactor that can maintain a kidney for 7 days. The long duration of the ex vivo perfusion will help to advance studies on kidney disease and can rapidly test for new regenerative medicine therapies compared to whole animal studies.