SARS-CoV-2 infection of human-induced pluripotent stem cells-derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways.

The COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily affects the lung, particularly the proximal airway and distal alveolar cells. NKX2.1+ primordial lung progenitors of the foregut (anterior) endoderm are the developmental precursors to all adult lung epithelial lineages and are postulated to play an important role in viral tropism. Here, we show that SARS-CoV-2 readily infected and replicated in human-induced pluripotent stem cell-derived proximal airway cells, distal alveolar cells, and lung progenitors. In addition to the upregulation of antiviral defense and immune responses, transcriptomics data uncovered a robust epithelial cell-specific response, including perturbation of metabolic processes and disruption in the alveolar maturation program. We also identified spatiotemporal dysregulation of mitochondrial heme oxygenase 1 (HMOX1), which is associated with defense against antioxidant-induced lung injury. Cytokines, such as TNF-α, INF-γ, IL-6, and IL-13, were upregulated in infected cells sparking mitochondrial ROS production and change in electron transport chain complexes. Increased mitochondrial ROS then activated additional proinflammatory cytokines leading to an aberrant cell cycle resulting in apoptosis. Notably, we are the first to report a chemosensory response resulting from SARS-CoV-2 infection similar to that seen in COVID-19 patients. Some of our key findings were validated using COVID-19-affected postmortem lung tissue sections. These results suggest that our in vitro system could serve as a suitable model to investigate the pathogenetic mechanisms of SARS-CoV-2 infection and to discover and test therapeutic drugs against COVID-19 or its consequences.

Neuronal and cardiac toxicity of pharmacological compounds identified through transcriptomic analysis of human pluripotent stem cell-derived embryoid bodies.

Concurrent with the '3R' principle, the embryonic stem cell test (EST) using mouse embryonic stem cells, developed in 2000, remains the solely accepted in vitro method for embryotoxicity testing. However, the scope and implementation of EST for embryotoxicity screening, compliant with regulatory requirements, are limited. This is due to its technical complexity, long testing period, labor-intensive methodology, and limited endpoint data, leading to misclassification of embryotoxic potential. In this study, we used human induced pluripotent stem cell (hiPSC)-derived embryoid bodies (EB) as an in vitro model to investigate the embryotoxic effects of a carefully selected set of pharmacological compounds. Morphology, viability, and differentiation potential were investigated after exposing EBs to folic acid, all-trans-retinoic acid, dexamethasone, and valproic acid for 15 days. The results showed that the compounds differentially repressed cell growth, compromised morphology, and triggered apoptosis in the EBs. Further, transcriptomics was employed to compare subtle temporal changes between treated and untreated cultures. Gene ontology and pathway analysis revealed that dysregulation of a large number of genes strongly correlated with impaired neuroectoderm and cardiac mesoderm formation. This aberrant gene expression pattern was associated with several disorders of the brain like mental retardation, multiple sclerosis, stroke and of the heart like dilated cardiomyopathy, ventricular tachycardia, and ventricular arrhythmia. Lastly, these in vitro findings were validated using in ovo chick embryo model. Taken together, pharmacological compound or drug-induced defective EB development from hiPSCs could potentially be used as a suitable in vitro platform for embryotoxicity screening.

Long Noncoding RNA RP11-380D23.2 Drives Distal-Proximal Patterning of the Lung by Regulating PITX2 Expression.

Early lung development is a tightly orchestrated process encompassing (a) formation of definitive endoderm, (b) anteriorization of definitive endoderm, followed by (c) specification and maturation of both proximal and distal lung precursors. Several reports detailing the interaction of genes and proteins during lung development are available; however, studies reporting the role(s) of long noncoding RNAs (lncRNA) in lung morphogenesis are limited. To investigate this, we tailored a protocol for differentiation of human-induced pluripotent stem cells into distal and proximal lung progenitors to mimic in vivo lung development. The authenticity of differentiated cells was confirmed by expression of key lung markers such as FoxA2, Sox-17, Nkx2.1, Pitx2, FoxJ1, CC10, SPC, and via scanning as well as transmission electron microscopy. We employed next generation sequencing to identify lncRNAs and categorized them based on their proximity to genes essential for lung morphogenesis. In-depth bioinformatical analysis of the sequencing data enabled identification of a novel lncRNA, RP11-380D23.2, which is located upstream of PITX2 and includes a binding site for PARP1. Chromatin immunoprecipitation and other relevant studies revealed that PARP1 is a repressor for PITX2. Whole genome microarray analysis of RP11-380D23.2/PITX2 knockdown populations of progenitors demonstrated enrichment in proximal progenitors and indicated altered distal-proximal patterning. Dysregulation of WNT effectors in both knockdowns highlighted direct modulation of PITX2 by RP11-380D23.2. Most of these results were validated in four independent hiPSC lines (including a patient-specific CFTR mutant line). Taken together, these findings offer a mechanistic explanation underpinning the role of RP11-380D23.2 during lung morphogenesis via WNT signaling. Stem Cells 2018;36:218-229.

Transcriptional profiling of human neural precursors post alcohol exposure reveals impaired neurogenesis via dysregulation of ERK signaling and miR-145.

Gestational alcohol exposure causes a range of neuropsychological disorders by modulating neurodevelopmental genes and proteins. The extent of damage depends on the stage of the embryo as well as dosage, duration and frequency of exposure. Here, we investigated the neurotoxic effects of alcohol using human embryonic stem cells. Multiple read-outs were engaged to assess the proliferation and differentiation capacity of neural precursor cells upon exposure to 100 mM ethanol for 48 h corresponding to the blood alcohol levels for binge drinkers. Whole-genome analysis revealed a spatiotemporal dysregulation of neuronal- and glial-specific gene expression that play critical roles in central nervous system (CNS) development. Alterations observed in the transcriptome may be attributed to epigenetic constitution witnessed by differential histone H3 Lys-4/Lys-27 modifications and acetylation status. In-depth mRNA and protein expression studies revealed abrogated extracellular signal-regulated kinases signaling in alcohol-treated cells. Consistent with this finding, ingenuity pathway analysis and micro-RNA profiling demonstrated up-regulation of miR-145 by targeting the neural specifier Sox-2. We also show that the neurite branching complexity of tubulin, beta 3 class III+ neurons was greatly reduced in response to alcohol. Finally, in vivo studies using zebrafish embryos reconfirmed the in vitro findings. Employing molecular endpoints in a human model, this report indicates for the first time that acute alcohol exposure could lead to impaired brain development via perturbation of extracellular signal-regulated kinases pathway and miR-145. However, it still needs to be addressed whether these modulations sustain throughout development, compromising the ability of the individual during adulthood and aging.

Dysregulation of Wnt-Signaling and a Candidate Set of miRNAs Underlie the Effect of Metformin on Neural Crest Cell Development.

Neural crest cells (NCC) are a population of epithelial cells that arise from the dorsal tube and undergo epithelial-mesenchymal transition (EMT) eventually generating tissues from peripheral nervous system, melanocytes, craniofacial cartilage, and bone. The antidiabetic drug metformin reportedly inhibits EMT in physiological conditions like cancer and fibrosis. We hypothesize that perturbation of EMT may also contribute to developmental disabilities associated with neural crest (NC) development. To understand the molecular network underlying metformin action during NC formation, we first differentiated murine embryonic stem (ES) cells into NCC and characterized them by demonstrating spatiotemporal regulation of key markers. Metformin treatment prompted a delay in delamination of NCC by inhibiting key markers like Sox-1, Sox-9, HNK-1, and p-75. We then revealed that metformin impedes Wnt axis, a major signaling pathway active during NC formation via DVL-3 inhibition and impairment in nuclear translocation of ß-catenin. Concomitantly we identified and tested a candidate set of miRNAs that play a crucial role in NC cell fate determination. Further studies involving loss and gain of function confirmed that NCC specifiers like Sox-1 and Sox-9 are direct targets of miR-200 and miR-145, respectively and that they are essentially modulated by metformin. Our in vitro findings were strongly supported by in vivo studies in zebrafish. Given that metformin is a widely used drug, for the first time we demonstrate that it can induce a delayed onset of developmental EMT during NC formation by interfering with canonical Wnt signaling and mysregulation of miR-145 and miR-200.

The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration.

The unique properties of mesenchymal stromal/stem cells (MSCs) to self-renew and their multipotentiality have rendered them attractive to researchers and clinicians. In addition to the differentiation potential, the broad repertoire of secreted trophic factors (cytokines) exhibiting diverse functions such as immunomodulation, anti-inflammatory activity, angiogenesis and anti-apoptotic, commonly referred to as the MSC secretome, has gained immense attention in the past few years. There is enough evidence to show that the one important pathway by which MSCs participate in tissue repair and regeneration is through its secretome. Concurrently, a large body of MSC research has focused on characterization of the MSC secretome; this includes both soluble factors and factors released in extracellular vesicles, for example, exosomes and microvesicles. This review provides an overview of our current understanding of the MSC secretome with respect to their potential clinical applications.

Vitiligo patient-derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition.

Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity.

Generation and characterization of retinal pigment epithelium from patient iPSC line to model oculocutaneous albinism (OCA)1A disease.

Oculocutaneous albinism (OCA) is characterized by reduced melanin biosynthesis affecting the retina, thus impairing visual function. The disease pathology of OCA is poorly understood at the cellular level due to unavailability of suitable biological model systems. This study aimed to develop a disease-specific in vitro model for OCA type 1A, the most severe form caused by TYR (tyrosinase) gene mutations, using retinal pigment epithelium (RPE) differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs). A comparative study between healthy and OCA1A RPE cells revealed that while healthy RPE cells exhibited timely onest of pigmentation during differentiation, OCA1A RPE cells failed to pigment even after an extended culture period. This observation was validated by ultrastructural studies using electron microscopy, hinting at melanosome-specific defects. Immunocytochemistry demonstrated abnormal expression patterns of melanogenesis-specific protein markers in OCA1A RPE cells, indicating reduced or absence of melanin synthesis. Next, a quantitative assay was performed to confirm the absence of melanin production in OCA1A RPE cells. Tyrosinase assay showed no activity in OCA1A compared with healthy RPE, suggesting non-functionality of TYR, further corroborated by western blot analysis showing complete absence of the protein. Gene expression by RNA sequencing of healthy and OCA1A RPE cells uncovered differential gene expression associated with lens development, visual perception, transmembrane transporter activity, and key signaling pathways. This disease-in-a-dish model of OCA1A provides an excellent platform to understand disease mechanism, identify potential therapeutic targets, and facilitate gene therapy or gene correction.

Embryonic fibroblasts represent a connecting link between mesenchymal and embryonic stem cells.

It is well established that fibroblasts and mesenchymal stem cells (MSC) share several characteristics with subtle differences. However, no study highlighting the versatility of fibroblasts beyond their multipotentiality has been reported so far. Mouse embryonic fibroblasts (MEFs) are widely used as feeder layers to support the growth of embryonic stem cells (ESC). We hypothesized that MEF may retain ES-like features in concurrence to their developmental hierarchy in addition to their multipotent nature. Hence, we performed a comparative assessment of MEF and ESC to determine their ability to differentiate into cell types other than mesoderm as well as capacity to form teratoma using routine in vitro and in vivo techniques. MEF were derived by trypsin/ EDTA (ethylenediaminetetraacetic acid) digestion from E13.5 embryos after removing heads and viscera following plastic adherence. MEFs robustly proliferated in culture until passage 15 and formed aggregates by hanging drop method. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry revealed the presence of key MSC markers such as CD90, CD73, Sca-1, CD44, CD29, Vimentin and absence of CD45. Additionally, they expressed SSEA-1, Oct-4, Nanog, Sox-2 and ABCG2 as pluripotency markers; Nestin, ß-III tubulin, Otx-2 (ectoderm); MEF-2, Mesp2, GATA-2 (mesoderm) and GATA-4, α-amylase, PDX-1 (endoderm) as tri-lineage markers. Furthermore, MEFs formed representative tissues from all three germ layers upon transplantation into Balb/c mice. These unique abilities of MEF to exhibit pluripotency, in addition to fibroblast characteristics and their ready availability with less ethical concerns and low maintenance requirements make them an attractive model for further exploration as a possible tool for regenerative medicine.

Phenotypic and functional comparison of optimum culture conditions for upscaling of dental pulp stem cells.

Advances in dental pulp stem cell (DPSC) biology and behaviour have promised much in the field of regenerative medicine. Their recent use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients; however they display diverse characteristics under different culture conditions. Since the success of any stem cell culture is regulated by its own micro-environment, it is imperative to optimise the growth conditions and establish a generic protocol for maintenance and scale-up. This study focused on optimisation of long-term culture conditions of human exfoliated deciduous teeth (SHED) in comparison with DPSCs, employing three commonly used basal media - knockout Dulbecco's modified Eagle's medium (KO-DMEM), α-MEM and DMEM/F12. Based on their characterisation with respect to morphology, growth kinetics, cell surface marker expression, differentiation capacity and plating density, our findings suggest that cells can be expanded with the highest efficiency in KO-DMEM medium supplemented with 10% FBS. Additionally, under our standardised xeno-free (10% human plasma) growth conditions, DPSCs displayed and retained their multipotent attributes until late passages. The differences in the growth and differentiation characteristics between SHED and DPSCs are shown, and certify SHED can be a key element in tissue engineering.

A simple and economical route to generate functional hepatocyte-like cells from hESCs and their application in evaluating alcohol induced liver damage.

The in vitro derived hepatocytes from human embryonic stem cells (hESC) is a promising tool to acquire improved knowledge of the cellular and molecular events underlying early human liver development under physiological and pathological conditions. Here we report a simple two-step protocol employing conditioned medium (CM) from human hepatocellular carcinoma cell line, HepG2 to generate functional hepatocyte-like cells from hESC. Immunocytochemistry, flow cytometry, quantitative RT-PCR, and biochemical analyses revealed that the endodermal progenitors appeared as pockets in culture, and the cascade of genes associated with the formation of definitive endoderm (HNF-3ß, SOX-17, DLX-5, CXCR4) was consistent and in concurrence with the up-regulation of the markers for hepatic progenitors [alpha-feto protein (AFP), HNF-4α, CK-19, albumin, alpha-1-antitrypsin (AAT)], followed by maturation into functional hepatocytes [tyrosine transferase (TAT), tryptophan-2, 3-dioxygenase (TDO), glucose 6-phosphate (G6P), CYP3A4, CYP7A1]. We witnessed that the gene expression profile during this differentiation process recapitulated in vivo liver development demonstrating a gradual down-regulation of extra embryonic endodermal markers (SOX-7, HNF-1ß, SNAIL-1, LAMININ-1, CDX2), and the generated hepatic cells performed multiple liver functions. Since prenatal alcohol exposure is known to provoke irreversible abnormalities in the fetal cells and developing tissues, we exposed in vitro generated hepatocytes to ethanol (EtOH) and found that EtOH treatment not only impairs the survival and proliferation, but also induces apoptosis and perturbs differentiation of progenitor cells into hepatocytes. This disruption was accompanied by alterations in the expression of genes and proteins involved in hepatogenesis. Our results provide new insights into the wider range of destruction caused by alcohol on the dynamic process of liver organogenesis.

Cell therapy in critical limb ischemia: current developments and future progress.

Critical limb ischemia (CLI) is a syndrome manifested by ischemic rest pain, non-healing ulcers and tissue loss. CLI patients are at very high risk of amputation and experience poor physical function, leading to severe morbidity and mortality. The fundamental goal for CLI treatment is to relieve ischemic rest pain, heal ulcers, prevent limb loss and improve the quality of life, thereby extending the survival of the patient. Surgical or endovascular revascularization aimed at increasing blood flow is currently available for limb salvage in CLI. However, up to 30% of CLI patients are not suitable for such interventions because of high operative risk or unfavorable vascular anatomy. Therefore exploring new and more effective strategies for revascularization of ischemic limbs is imperative for the establishment of a viable therapeutic alternative. With the emergence of new approaches, this review describes up-to-date progress and developments in cell-based therapy as a novel and promising alternative for CLI treatment. Preliminary clinical data have established the safety, feasibility and efficacy of stem cells, and numerous studies are underway to consolidate this evidence further. However, significant hurdles remain to be addressed before this research can be responsibly translated to the bedside. In particular, we need better understanding of the behavior of cells post-transplantation and to learn how to control their survival and migration proliferation/differentiation in the hostile pathologic environment. Future research should focus on methods of isolation, optimal dosage, appropriate cell type, route of administration, role of tissue-derived factors and supportive endogenous stimulation.

Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells.

In vitro disease modeling using pluripotent stem cells can be a fast track screening tool for toxicological testing of candidate drug molecules. Dimethyl sulfoxide (DMSO) is one of the most commonly used solvents in drug screening. In the present investigation, we exposed 14- to 21-day-old embryoid bodies (EBs) to three different concentrations of DMSO [0.01% (low dose), 0.1% (medium dose) and 1.0% (high dose)] to identify the safest dose that could effectively be used as solvent. We found that DMSO treatment substantially altered the morphology and attachment of cells in concurrence with a significant reduction in cell viability in a dose-dependent manner. Gene expression studies revealed a selective downregulation of key markers associated with stemness (Oct-4, Sox-2, Nanog and Rex-1); ectoderm (Nestin, TuJ1, NEFH and Keratin-15); mesoderm (HAND-1, MEF-2C, GATA-4 and cardiac-actin); and endoderm (SOX-17, HNF-3ß, GATA-6 and albumin), indicating an aberrant and untimely differentiation trajectory. Furthermore, immunocytochemistry, flow cytometry and histological analyses demonstrated substantial decrease in the levels of albumin and CK-18 proteins coupled with a massive reduction in the number of cells positive for PAS staining, implicating reduced deposits of glycogen. Our study advocates for the first time that DMSO exposure not only affects the phenotypic characteristics but also induces significant alteration in gene expression, protein content and functionality of the differentiated hepatic cells. Overall, our experiments warrant that hESC-based assays can provide timely alerts about the outcome of widespread applications of DMSO as drug solvent, cryoprotectant and differentiating agent.

Derivation of Induced Pluripotent Stem Cell (iPSC) Lines from Patient-Specific Peripheral Blood Mononuclear Cells (PBMC) Using Episomal Vectors.

Inherited retinal diseases (IRDs) are a diverse group of rare eye disorders, resulting in vision loss or blindness. The underlying reason is mutation in one or more than 250 different genes associated with the development and normal physiology of retina largely comprising of rod/cone photoreceptors and retinal pigment epithelium. Interestingly, the sub retinal region of an eye has been shown to be immune privileged, broadening the scope of cell-replacement therapies for patients suffering from retinal degeneration. Several groups around the globe, including ours, have demonstrated safety and efficacy in preclinical studies by employing various approaches of retinal cell therapy. This had largely been possible with the advent of induced pluripotent stem cells (iPSC)-reprogrammed from adult somatic cells, that serves as a starting material for generating retinal cells de novo. Here, we describe a detailed procedure for reprogramming peripheral blood mononuclear cells (PBMC) into iPSC using episomal vectors without any physical disruption in the host genome. The lines thus created were tested for sterility, cytogenetic stability, identity, absence of episomal plasmids and further authenticated for pluripotency and tri-lineage differentiation capacity by embryoid body formation and immunocytochemistry. We believe that this feeder-cell free, animal-product free and gene-insertion free protocol would help people to develop and bank patient-specific cell lines for autologous cell therapies for incurable rare diseases.

An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells.

We present an optimized protocol for guided differentiation of retinal pigment epithelium (RPE) cells from human-induced pluripotent stem cells (iPSC). De novo-generated RPE cells are mature, polarized, and mimic the cellular and molecular profile of primary RPE; they are also suitable for in vivo cell transplantation studies. The protocol includes an enrichment step, making it useful for large-scale GMP manufacturing. RPE cells produced following this protocol are appropriate for cell replacement therapy for macular degeneration and disease modeling. For complete details on the use and execution of this protocol, please refer to Surendran et al. (2021).

Implementation of an adapted Sepsis Risk Calculator algorithm to reduce antibiotic usage in the management of early onset neonatal sepsis: a multicentre initiative in Wales, UK.

OBJECTIVE: Assess the impact of introducing a consensus guideline incorporating an adapted Sepsis Risk Calculator (SRC) algorithm, in the management of early onset neonatal sepsis (EONS), on antibiotic usage and patient safety. DESIGN: Multicentre prospective study SETTING: Ten perinatal hospitals in Wales, UK. PATIENTS: All live births ≥34 weeks' gestation over a 12-month period (April 2019-March 2020) compared with infants in the preceding 15-month period (January 2018-March 2019) as a baseline. METHODS: The consensus guideline was introduced in clinical practice on 1 April 2019. It incorporated a modified SRC algorithm, enhanced in-hospital surveillance, ongoing quality assurance, standardised staff training and parent education. The main outcome measure was antibiotic usage/1000 live births, balancing this with analysis of harm from delayed diagnosis and treatment, disease severity and readmissions from true sepsis. Outcome measures were analysed using statistical process control charts. MAIN OUTCOME MEASURES: Proportion of antibiotic use in infants ≥34 weeks' gestation. RESULTS: 4304 (14.3%) of the 30 105 live-born infants received antibiotics in the baseline period compared with 1917 (7.7%) of 24 749 infants in the intervention period (45.5% mean reduction). All 19 infants with culture-positive sepsis in the postimplementation phase were identified and treated appropriately. There were no increases in sepsis-related neonatal unit admissions, disease morbidity and late readmissions. CONCLUSIONS: This multicentre study provides evidence that a judicious adaptation of the SRC incorporating enhanced surveillance can be safely introduced in the National Health Service and is effective in reducing antibiotic use for EONS without increasing morbidity and mortality.

Human embryonic stem cell proliferation and differentiation as parameters to evaluate developmental toxicity.

In vitro models based on embryonic stem cells (ESC) are highly promising for improvement of predictive toxicology screening in humans. After the successful validation of embryonic stem cell test (EST) in 2001; concerns have been raised on the usage of mouse ESC and also the morphological evaluation of beating cell clusters. This requires specialized skill-sets and is highly prone to misjudgement and false positive results. To overcome these limitations, we undertook the present study incorporating improvisations over the conventional EST. Here, we explored the potential of a human ESC (hESC)-based assay to evaluate the potential toxicity of penicillin-G, caffeine, and hydroxyurea. Drug treatment inhibited hESC adhesion and substantially altered the morphology and viability (∼ 50%) of embryoid bodies (EBs). Flow cytometry analysis not only showed a significant increase of apoptotic cells in the highest doses but also induced a diverse pattern in DNA content and cell cycle distribution relative to control. Both semi-quantitative and quantitative RT-PCR studies revealed a selective down regulation of markers associated with stemness (Nanog, Rex1, SOX-2, and hTERT); cardiac mesoderm (Cripto1, MEF-2C, and Brachyury); hepatic endoderm (AFP, HNF-3ß, HNF-4α, GATA-4, and SOX-17); and neuroectoderm (Nestin, SOX-1, NURR1, NEFH, Synaptophysin, TH, and Olig2) in a drug as well as dose dependent manner indicating abnormal differentiation. Furthermore, a decrease in the expression of AFP and GFAP proteins followed by a dose-dependent reduction in the levels of hCG-ß, progesterone-II, and estradiol hormones was demonstrated by immunocytochemistry and ECLIA, respectively. This new and unique approach comprising of DNA cell cycle analysis, germ layer-specific marker expression and hormone levels as endpoints might offer a clinically relevant and commercially viable alternative for predicting in vivo developmental toxicity.

Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells.

Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.

Interventional Strategies for Parkinson Disease: Can Neural Precursor Cells Forge a Path Ahead?

Neural precursor cells (NPCs), derived from pluripotent stem cells (PSCs), with their unique ability to generate multiple neuronal and glial cell types are extremely useful for understanding biological mechanisms in normal and diseased states. However, generation of specific neuronal subtypes (mature) from NPCs in large numbers adequate for cell therapy is challenging due to lack of a thorough understanding of the cues that govern their differentiation. Interestingly, neural stem cells (NSCs) themselves are in consideration for therapy given their potency to form different neural cell types, release of trophic factors, and immunomodulatory effects that confer neuroprotection. With the recent COVID-19 outbreak and its accompanying neurological indications, the immunomodulatory role of NSCs may gain additional significance in the prevention of disease progression in vulnerable populations. In this regard, small-molecule mediated NPC generation from PSCs via NSC formation has become an important strategy that ensures consistency and robustness of the process. The development of the mammalian brain occurs along the rostro-caudal axis, and the establishment of anterior identity is an early event. Wnt signaling, along with fibroblast growth factor and retinoic acid, acts as a caudalization signal. Further, the increasing amount of epigenetic data available from human fetal brain development has enhanced both our understanding of and ability to experimentally manipulate these developmental regulatory programs in vitro. However, the impact on homing and engraftment after transplantation and subsequently on therapeutic efficacy of NPCs based on their derivation strategy is not yet clear. Another formidable challenge in cell replacement therapy for neurodegenerative disorders is the mode of delivery. In this Perspective, we discuss these core ideas with insights from our preliminary studies exploring the role of PSC-derived NPCs in rat models of MPTP-induced Parkinson's disease following intranasal injections.