Efficient Generation of Pancreatic Progenitor Cells from Induced Pluripotent Stem Cells Derived from a Non-Invasive and Accessible Tissue Source-The Plucked Hair Follicle.

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.

Using ex vivo bioengineered lungs to model pathologies and screening therapeutics: A proof-of-concept study.

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.

Correction: Won et al. Ex Vivo Perfusion Using a Mathematical Modeled, Controlled Gas Exchange Self-Contained Bioreactor Can Maintain a Mouse Kidney for Seven Days. Cells 2022, 11, 1822.

In the original publication [...].

Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts.

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.

Determining epigenetic memory in kidney proximal tubule cell derived induced pluripotent stem cells using a quadruple transgenic reprogrammable mouse.

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.

Ex Vivo Perfusion Using a Mathematical Modeled, Controlled Gas Exchange Self-Contained Bioreactor Can Maintain a Mouse Kidney for Seven Days.

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.

Decellularization of porcine kidney with submicellar concentrations of SDS results in the retention of ECM proteins required for the adhesion and maintenance of human adult renal epithelial cells.

When decellularizing kidneys, it is important to maintain the integrity of the acellular extracellular matrix (ECM), including associated adhesion proteins and growth factors that allow recellularized cells to adhere and migrate according to ECM specificity. Kidney decellularization requires the ionic detergent sodium dodecyl sulfate (SDS); however, this results in a loss of ECM proteins important for cell adherence, migration, and growth, particularly glycosaminoglycan (GAG)-associated proteins. Here, we demonstrate that using submicellar concentrations of SDS results in a greater retention of structural proteins, GAGs, growth factors, and cytokines. When porcine kidney ECM scaffolds were recellularized using human adult primary renal epithelial cells (RECs), the ECM promoted cell survival and the uniform distribution of cells throughout the ECM. Cells maintained the expression of mature renal epithelial markers but did not organize on the ECM, indicating that mature cells are unable to migrate to specific locations on ECM scaffolds.

Rapid target validation in a Cas9-inducible hiPSC derived kidney model.

Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

Recapitulating kidney development in vitro by priming and differentiating mouse embryonic stem cells in monolayers.

In order to harness the potential of pluripotent stem cells, we need to understand how to differentiate them to our target cell types. Here, we developed a protocol to differentiate mouse embryonic stem cells (ESCs) to renal progenitors in a step-wise manner. Microarrays were used to track the transcriptional changes at each stage of differentiation and we observed that genes associated with metanephros, ureteric bud, and blood vessel development were significantly upregulated as the cells differentiated towards renal progenitors. Priming the ESCs and optimizing seeding cell density and growth factor concentrations helped improve differentiation efficiency. Organoids were used to determine the developmental potential of the renal progenitor cells. Aggregated renal progenitors gave rise to organoids consisting of LTL+/E-cadherin+ proximal tubules, cytokeratin+ ureteric bud-derived tubules, and extracellular matrix proteins secreted by the cells themselves. Over-expression of key kidney developmental genes, Pax2, Six1, Eya1, and Hox11 paralogs, during differentiation did not improve differentiation efficiency. Altogether, we developed a protocol to differentiate mouse ESCs in a manner that recapitulates embryonic kidney development and showed that precise gene regulation is essential for proper differentiation to occur.

Umbilical Cord Tissue as a Source of Young Cells for the Derivation of Induced Pluripotent Stem Cells Using Non-Integrating Episomal Vectors and Feeder-Free Conditions.

The clinical application of induced pluripotent stem cells (iPSC) needs to balance the use of an autologous source that would be a perfect match for the patient against any safety or efficacy issues that might arise with using cells from an older patient or donor. Drs. Takahashi and Yamanaka and the Office of Cellular and Tissue-based Products (PMDA), Japan, have had concerns over the existence of accumulated DNA mutations in the cells of older donors and the possibility of long-term negative effects. To mitigate the risk, they have chosen to partner with the Umbilical Cord (UC) banks in Japan to source allogeneic-matched donor cells. Production of iPSCs from UC blood cells (UCB) has been successful; however, reprogramming blood cells requires cell enrichment with columns or flow cytometry and specialized growth media. These requirements add to the cost of production and increase the manipulation of the cells, which complicates the regulatory approval process. Alternatively, umbilical cord tissue mesenchymal stromal cells (CT-MSCs) have the same advantage as UCB cells of being a source of young donor cells. Crucially, CT-MSCs are easier and less expensive to harvest and grow compared to UCB cells. Here, we demonstrate that CT-MSCs can be easily isolated without expensive enzymatic treatment or columns and reprogramed well using episomal vectors, which allow for the removal of the reprogramming factors after a few passages. Together the data indicates that CT-MSCs are a viable source of donor cells for the production of clinical-grade, patient matched iPSCs.

The cause of pyloric stenosis of infancy: A hyperacidity pathogenesis.

The cause of pyloric stenosis of infancy (PS) is at present unknown. A theory of causation is proposed which is consistent with all the known clinical features of this condition. It is based on the knowledge that PS babies are hypersecretors of acid which pre-dates the development of PS and is an inherited constitutional feature. This acidity will become temporarily and dangerously high due to an insensitivity of the negative feed-back between gastrin and gastric acidy within the first few weeks of life. Normal babies who have inherited normal acidity will also experience peak acid secretions at that time but will be much less acid than babies destined to develop PS. Acid entering the duodenum causes contraction of the pyloric sphincter. Hyperacidity will naturally lead to repeated pyloric sphincter contractions and sphincter hypertrophy. Inappropriate repeated feeding of the vomiting PS baby by a first-time overanxious mother to her ever hungry baby, by provoking feed related sphincter contraction is considered to play a significant part in pathogenesis. Should the baby with PS survive beyond the age of around 6weeks, the matured negative feed-back between gastrin and acid will ensure that dangerous hyperacidity is kept in check. This coupled with the natural pyloric canal widening with age, will lead then to an long lasting cure. This theory explains satisfactorily all the known and hitherto unexplained features of this condition.

Limitations of recellularized biological scaffolds for human transplantation.

A shortage of donor organs for transplantation and the dependence of the recipients on immunosuppressive therapy have motivated researchers to consider alternative regenerative approaches. The answer may reside in acellular scaffolds generated from cadaveric human and animal tissues. Acellular scaffolds are expected to preserve the architectural and mechanical properties of the original organ, permitting cell attachment, growth, and differentiation. Although theoretically, the use of acellular scaffolds for transplantation should pose no threat to the recipient's immune system, experimental data have revealed significant immune responses to allogeneic and xenogeneic transplanted scaffolds. Herein, we review the various factors of the scaffold that could trigger an inflammatory and/or immune response, thereby compromising its use for human transplant therapy. In addition, we provide an overview of the major cell types that have been considered for recellularization of the scaffold and their potential contribution to triggering an immune response.

Milk vomiting or regurgitation in the first 3 months of life: Something old-something new.

The age of presentation of reflux symptoms and their self-cure in babies without a sliding hernia parallel those of mild pyloric stenosis of infancy (PS). It is proposed that this is because PS and, at least some cases of reflux, share the same cause-a temporary hold-up at the pyloric sphincter owing to acid provoked hypertrophy of the pyloric sphincter. In support of this theory, the written observations of John Thomson, Pediatrician from Edinburgh, in 1921 and Isabella Forshall, Pediatric Surgeon from Alder Hey Hospital, Liverpool, in 1958 are revisited. An analysis of both papers provides supportive evidence that, in at least some cases diagnosed as simple reflux, an underlying temporary hold up is present owing to early hypertrophy of the sphincter. It is recommended that sphincter thickness measurements should be made by ultrasonic assessment whenever uncomplicated reflux is diagnosed within the first 3 months of life.

Banking Mesenchymal Stromal Cells from Umbilical Cord Tissue: Large Sample Size Analysis Reveals Consistency Between Donors.

Mesenchymal stromal cells (MSCs) have emerged as candidate cells with therapeutic potential to treat different pathologies. The underlying mechanism is paracrine signaling. The cells secrete proteins that can impact inflammation, apoptosis, angiogenesis, and cell proliferation. All are important in wound healing and tissue regeneration. Although the bone marrow has been the most widely used source of MSCs, umbilical cord tissue (CT) presents a source that is just starting to be used in the clinic, yet can be obtained with more ease and easily stored. Here, we characterize CT-MSCs obtained from multiple donors by analyzing cell surface proteins, differentiation capacity, and proteome profile. Analysis of low, medium, and high passage cells indicates that the morphology and proliferation rate stay constant and with the exception of cluster of differentiation (CD) 105 at late passage, there are no changes in the cell surface protein characteristics, indicating the population does not change with passage. TNF-stimulated gene 6 protein was measured in a subset of samples and variable expression was observed, but this did not impact the ability of the cells to enhance skin regeneration. In conclusion, CT-MSC represents a consistent, easily accessible source of cells for cell therapy. Stem Cells Translational Medicine 2019;8:1041-1054.

Decellularizing and Recellularizing Adult Mouse Kidneys.

Decellularization is the process by which resident cells are lysed and cellular debris is removed from the tissue, leaving behind the extracellular matrix scaffold. The extracellular matrix scaffold can be used for three-dimensional culturing of cells. Here we describe methods of decellularizing whole and thick sections of mouse kidneys using a 0.1% sodium dodecyl sulfate (SDS) detergent solution and strategies to repopulate whole and thick sections of decellularized mouse kidneys with cells.

Topical Application of Culture-Expanded CD34+ Umbilical Cord Blood Cells from Frozen Units Accelerates Healing of Diabetic Skin Wounds in Mice.

Chronic and nonhealing wounds are constant health issues facing patients with type 2 diabetes. As the incidence of type 2 diabetes mellitus (T2DM) increases, the incidence of chronic wounds and amputations will rise. T2DM is associated with peripheral arterial occlusive disease, which leads to the development of nonhealing skin ulcers after minor trauma. Patients develop severe pain limiting their mobility and ability to work and take care of themselves, thus putting a significant burden on the family and society. CD34+ cells from umbilical cord blood (UCB) grown in fibroblast growth factor-4 (FGF-4), stem cell factor, and Flt3-ligand produced a population of cells that have the ability to proliferate and develop properties enabling them to enhance tissue regeneration. The goal of this study was to assess in vitro cultured CD34+ cells in a setting where they would eventually be rejected so we could isolate paracrine signaling mediated therapeutic effect from the therapeutic effect due to engraftment and differentiation. To achieve this, we used db/db mice as a model for diabetic skin ulcers. Here, we report that in vitro cultured UCB CD34+ cells from frozen units can accelerate wound healing and resulted in the regeneration of full thickness skin. This study demonstrates a new indication for banked UCB units in the area of tissue regeneration. Stem Cells Translational Medicine 2018;7:591-601.

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

Periostin is critical for improving the therapeutic properties of adipocyte-derived stem cells.

Periostin is a matricellular protein that is reactivated during tissue damage and repair and has been shown to be a critical regulator of multiple biological pathways involved in the repair of tissue after myocardial infarction, peripheral vascular disease, and skin wounds. The tissue repair properties attributed to periostin make it an ideal candidate to enhance the therapeutic properties of donor cells such as mesenchymal stem cells from adipocyte tissue. In a recent article in Stem Cell Research & Therapy, Qin et al. demonstrated enhanced therapeutic properties of adipocyte-derived stem cells by genetically engineering them to express periostin.