Towards Early Prediction of Human iPSC Reprogramming Success

This paper presents advancements in automated early-stage prediction of the success of reprogramming human induced pluripotent stem cells (iPSCs) as a potential source for regenerative cell therapies.The minuscule success rate of iPSC-reprogramming of around $0.01$ to $0.1$ makes it labor-intensive, time-consuming, and exorbitantly expensive to generate a stable iPSC line. Since that requires culturing of millions of cells and intense biological scrutiny of multiple clones to identify a single optimal clone. The ability to reliably predict which cells are likely to establish as an optimal iPSC line at an early stage of pluripotency would therefore be ground-breaking in rendering this a practical and cost-effective approach to personalized medicine. Temporal information about changes in cellular appearance over time is crucial for predicting its future growth outcomes. In order to generate this data, we first performed continuous time-lapse imaging of iPSCs in culture using an ultra-high resolution microscope. We then annotated the locations and identities of cells in late-stage images where reliable manual identification is possible. Next, we propagated these labels backwards in time using a semi-automated tracking system to obtain labels for early stages of growth. Finally, we used this data to train deep neural networks to perform automatic cell segmentation and classification. Our code and data are available at https://github.com/abhineet123/ipsc_prediction.Comment: Accepted for publication at the Journal of Machine Learning for Biomedical Imaging (MELBA) https://melba-journal.org/2023:01

A Small Molecule Swertisin from Enicostemma littorale

Aim. Stem cell therapy is one of the upcoming therapies for the treatment of diabetes. Discovery of potent differentiating agents is a prerequisite for increasing islet mass. The present study is an attempt to screen the potential of novel small biomolecules for their differentiating property into pancreatic islet cells using NIH3T3, as representative of extra pancreatic stem cells/progenitors. Methods. To identify new agents that stimulate islet differentiation, we screened various compounds isolated from Enicostemma littorale using NIH3T3 cells and morphological changes were observed. Characterization was performed by semiquantitative RT-PCR, Q-PCR, immunocytochemistry, immunoblotting, and insulin secretion assay for functional response in newly generated islet-like cell clusters (ILCC). Reversal of hyperglycemia was monitored after transplanting ILCC in STZ-induced diabetic mice. Results. Among various compounds tested, swertisin, an isolated flavonoid, was the most effective in differentiating NIH3T3 into endocrine cells. Swertisin efficiently changed the morphology of NIH3T3 cells from fibroblastic to round aggregate cell cluster in huge numbers. Dithizone (DTZ) stain primarily confirmed differentiation and gene expression studies signified rapid onset of differentiation signaling cascade in swertisin-induced ILCC. Molecular imaging and immunoblotting further confirmed presence of islet specific proteins. Moreover, glucose induced insulin release (in vitro) and decreased fasting blood glucose (FBG) (in vivo) in transplanted diabetic BALB/c mice depicted functional maturity of ILCC. Insulin and glucagon expression in excised islet grafts illustrated survival and functional integrity. Conclusions. Rapid induction for islet differentiation by swertisin, a novel herbal biomolecule, provides low cost and readily available differentiating agent that can be translated as a therapeutic tool for effective treatment in diabetes

Inducible Pluripotent Stem Cells as a Potential Cure for Diabetes

Over the last century, diabetes has been treated with subcutaneous insulin, a discovery that enabled patients to forego death from hyperglycemia. Despite novel insulin formulations, patients with diabetes continue to suffer morbidity and mortality with unsustainable costs to the health care system. Continuous glucose monitoring, wearable insulin pumps, and closed-loop artificial pancreas systems represent an advance, but still fail to recreate physiologic euglycemia and are not universally available. Islet cell transplantation has evolved into a successful modality for treating a subset of patients with ‘brittle’ diabetes but is limited by organ donor supply and immunosuppression requirements. A novel approach involves generating autologous or immune-protected islet cells for transplant from inducible pluripotent stem cells to eliminate detrimental immune responses and organ supply limitations. In this review, we briefly discuss novel mechanisms for subcutaneous insulin delivery and define their shortfalls. We describe embryological development and physiology of islets to better understand their role in glycemic control and, finally, discuss cell-based therapies for diabetes and barriers to widespread use. In response to these barriers, we present the promise of stem cell therapy, and review the current gaps requiring solutions to enable widespread use of stem cells as a potential cure for diabetes

Inducible Pluripotent Stem Cells as a Potential Cure for Diabetes

Over the last century, diabetes has been treated with subcutaneous insulin, a discovery that enabled patients to forego death from hyperglycemia. Despite novel insulin formulations, patients with diabetes continue to suffer morbidity and mortality with unsustainable costs to the health care system. Continuous glucose monitoring, wearable insulin pumps, and closed-loop artificial pancreas systems represent an advance, but still fail to recreate physiologic euglycemia and are not universally available. Islet cell transplantation has evolved into a successful modality for treating a subset of patients with ‘brittle’ diabetes but is limited by organ donor supply and immunosuppression requirements. A novel approach involves generating autologous or immune-protected islet cells for transplant from inducible pluripotent stem cells to eliminate detrimental immune responses and organ supply limitations. In this review, we briefly discuss novel mechanisms for subcutaneous insulin delivery and define their shortfalls. We describe embryological development and physiology of islets to better understand their role in glycemic control and, finally, discuss cell-based therapies for diabetes and barriers to widespread use. In response to these barriers, we present the promise of stem cell therapy, and review the current gaps requiring solutions to enable widespread use of stem cells as a potential cure for diabetes

Pan-caspase inhibitor F573 mitigates liver ischemia reperfusion injury in a murine model.

BACKGROUND:Liver ischemia reperfusion injury (IRI) remains a challenge in liver transplantation. A number of compounds have previously demonstrated efficacy in mitigating IRI. Herein, we applied three specific additive strategies to a mouse IRI screening model to determine their relative potencies in reducing such injury, with a view to future testing in a large animal and clinical ex situ normothermic perfusion setting: 1) F573, a pan-caspase inhibitor, 2) anti-inflammatory anakinra and etanrecept and 3) BMX-001, a mimetic of superoxide dismutase. METHODS:A non-lethal liver ischemia model in mice was used. Additives in the treatment groups were given at fixed time points before induction of injury, compared to a vehicle group that received no therapeutic treatment. Mice were recovered for 6 hours following the ischemic insult, at which point blood and tissue samples were obtained. Plasma was processed for transaminase levels. Whole liver tissue samples were processed for histology, markers of apoptosis, oxidative stress, and cytokine levels. RESULTS:In an in vivo murine IRI model, the F573 treatment group demonstrated statistically lower alanine aminotransferase (ALT) levels (p = 0.01), less evidence of apoptosis (p = 0.03), and lower cytokine levels compared to vehicle. The etanercept with anakinra treatment group demonstrated significantly lower cytokine levels. The BMX-001 group demonstrated significantly decreased apoptosis (p = 0.01) evident on TUNEL staining. CONCLUSIONS:The administration of pan-caspase inhibitor F573 in a murine in vivo model likely mitigates liver IRI based on decreased markers of cellular injury, decreased evidence of apoptosis, and improved cytokine profiles. Anakinra with etanercept, and BMX-001 did not demonstrate convincing efficacy at reducing IRI in this model, and likely need further optimization. The positive findings set rational groundwork for future translational studies of applying F573 during normothermic ex situ liver perfusion, with the aim of improving the quality of marginal grafts

Insulin fluorescence and Insulin content per unit cytoplasm quantification in Swertisin induced islet-like clusters derived from PANC-1 cells with activin-A and MAPKinase Inhibitor.

<p>(A) shows fluorescent images for insulin expression in islet differentiation pathway inhibited using p38 MAP kinase inhibitor SB-203580 added in conjunction with Swertisin throughout ten days. The islet-like clusters were immunostained for insulin (green) on 10^th day. Nuclear DNA was stained with DAPI (blue). (B) represents insulin content in islet like clusters after p-30 MAPK pathway inhibition. Graph represents insulin fluorescence quantification per unit cytoplasm in differentiated cells with and without inhibition of MAPK pathway using p38 MAP kinase inhibitor SB-203580 added in conjunction with Swertisin throughout ten days. Data is represented as Mean±SEM. *** and ** shows p value <0.001 and 0.01 Vs Activin-A and Swertisin alone groups respectively.</p

Differentiation of mouse intra-islet progenitor cells and immunoblotting of key transcription factors and islet markers under differentiation with activin, swertisin and presence of p-38 MAPK inhibitor.

<p>(A) depicts mIP cells cultured in complete media at day 0 which were then subjected to differentiation using activin-A and swertisin for 10 days. Bright field image shows, cells under differentiation on 8^th day at 20X magnification, and dithizone stained clusters on day 10^th. A fluorescent image represents immunostaining for insulin (green) and glucagon (red) in clusters from SFM/ITS, activin-a and swertisin groups. DAPI was used as nuclear stain. (B) shows immunoblotting of E-cadherin, N-cadherin, Ngn-3, P-p38, Native p-38 MAP kinase pathway proteins in presence and absence of MAP kinase pathway inhibitor SB203580. Ponceau S stain blot was shown as loading control. (C) shows immunoblotting of key differentiation pathway parameters that indicate the conversion of Mouse intra-islet progenitor cells into islet like clusters. Key transcription factors and MAP Kinase pathway proteins like P-p38, Erk1/2 Ngn-3, Pax-4, and Smad proteins under differentiation were monitored. Beta actin was used as loading control.</p

In-vivo analysis of molecular mechanism by Swertisin differentiation in Ppx mice model.

<p>(A) confocal images from regenerating pancreas for assessment of various islet transcription factors and signaling proteins of TGF-beta pathway from tissue of Ppx sham and Swertisin treated animals. Various markers like Ki67, Nestin, Ngn-3, CK19, p-smad-2, Smad-7, Insulin and Ck-19 were probed and analyzed. (B) showed western blot profile of stem cell markers, key transcription factors in islet differentiation pathway and cell death markers in PPx Sham and Swertisin treated animal pancreatic tissues.</p

Swertisin an Anti-Diabetic Compound Facilitate Islet Neogenesis from Pancreatic Stem/Progenitor Cells <i>via</i> p-38 MAP Kinase-SMAD Pathway: An <i>In-Vitro</i> and <i>In-Vivo</i> Study

<div><p>Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported “Swertisin” as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes.</p></div