@article {4451, title = {SARS-CoV-2 infection of human-induced pluripotent stem cells-derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {J Cell Physiol}, year = {2022}, month = {2022 Apr 23}, abstract = {

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.

}, issn = {1097-4652}, doi = {10.1002/jcp.30755}, author = {Surendran, Harshini and Kumar, Saurabh and Narasimhaiah, Swathi and Ananthamurthy, Anuradha and Varghese, P S and D{\textquoteright}Souza, George A and Medigeshi, Guruprasad and Pal, Rajarshi} } @article {1699, title = {Derivation of Induced Pluripotent Stem Cell (iPSC) Lines from Patient-Specific Peripheral Blood Mononuclear Cells (PBMC) Using Episomal Vectors [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Methods Mol Biol}, year = {2021}, month = {2021 Mar 27}, abstract = {

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.

}, issn = {1940-6029}, doi = {10.1007/7651_2021_385}, author = {Konala, Vijay Bhaskar Reddy and Nandakumar, Swapna and Surendran, Harshini and Pal, Rajarshi} } @article {1854, title = {Interventional Strategies for Parkinson Disease: Can Neural Precursor Cells Forge a Path Ahead? [Eyestem Research, a C-CAMP startup]}, journal = {ACS Chemical Neuroscience}, year = {2021}, month = {10/2021}, type = {Journal Article}, abstract = {

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{\textquoteright}s disease following intranasal injections.

}, keywords = {cell transplantation, induced pluripotent stem cells, neural precursor cell, neural stem cell., Parkinson disease}, doi = {https://doi.org/10.1021/acschemneuro.1c00525}, url = {https://pubs.acs.org/doi/abs/10.1021/acschemneuro.1c00525}, author = {Nandakumar, Swapna and Shahani, Pradnya and Datta, Indrani and Pal, Rajarshi} } @article {1639, title = {Transplantation of retinal pigment epithelium and photoreceptors generated concomitantly via small molecule-mediated differentiation rescues visual function in rodent models of retinal degeneration [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Stem Cell Res Ther}, volume = {12}, year = {2021}, month = {2021 Jan 19}, pages = {70}, abstract = {

BACKGROUND: Age-related macular degeneration (AMD) is a result of degeneration/damage of the retinal pigment epithelium (RPE) while retinitis pigmentosa (RP), an inherited early-onset disease, results from premature loss of photoreceptors. A promising therapeutic approach for both is the replacement of lost/damaged cells with human induced pluripotent stem cell (hiPSC)-derived retinal cells.

METHODS: The aim of this study was to investigate the in vivo functionality of RPE and photoreceptor progenitor (PRP) cells derived from a clinical-grade hiPSC line through a unified protocol. De novo-generated RPE and PRP were characterized extensively to validate their identity, purity, and potency.

RESULTS: RPE expressed tight junction proteins, showed pigmentation and ciliation, and secreted polarization-related factors vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PRP expressed neural retina proteins and cone and rod markers, and responded to KCl-induced polarization. Transcriptomic analysis demonstrated an increase in the expression of mature retinal tissue-specific genes coupled with concomitant downregulation of genes from undesired lineages. RPE transplantation rescued visual function in RCS rats shown via optokinetic tracking and photoreceptor rescue. PRP transplantation improved light perception in NOD.SCID-rd1 mice, and positive electroretinography signals indicated functional photoreceptor activity in the host{\textquoteright}s outer nuclear layer. Graft survival and integration were confirmed using immunohistochemistry, and no animals showed teratoma formation or any kind of ectopic growth in the eye.

CONCLUSIONS: To our knowledge, this is the first demonstration of a unified, scalable, and GMP-adaptable protocol indicating strong animal efficacy and safety data with hiPSC-derived RPE and PRP cells. These findings provide robust proof-of-principle results for IND-enabling studies to test these potential regenerative cell therapies in patients.

}, issn = {1757-6512}, doi = {10.1186/s13287-021-02134-x}, author = {Surendran, Harshini and Nandakumar, Swapna and Reddy K, Vijay Bhaskar and Stoddard, Jonathan and Mohan K, Varsha and Upadhyay, Pramod K and McGill, Trevor J and Pal, Rajarshi} } @article {1464, title = {Human induced pluripotent stem cell-derived lung epithelial system for SARS-CoV-2 infection modeling and its potential in drug repurposing [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Stem Cells Dev}, year = {2020}, month = {2020 Aug 31}, abstract = {

The lung is the most vulnerable target for the SARS-CoV-2 infection and respiratory failure causing Acute Respiratory Distress Syndrome (ARDS) is its foremost outcome. However, the current primary in vitro models in use for SARS-CoV-2 display apparent limitations for modeling such complex human respiratory disease. While patient cells can directly model the effects of a drug, their availability and capacity for expansion are limited compared to the transformed/immortalized cells or the tumor-derived cell lines. What{\textquoteright}s more, the latter may harbor genetic and metabolic abnormalities in the course of their derivation, making them unsuitable for drug screening. Therefore, it is important to create physiologically relevant human-cell models that can replicate the pathophysiology of SARS-CoV-2, thus, facilitating drug testing. Here, we show preliminary data on how human induced pluripotent stem cells (hiPSC)-based lung epithelial cell system could emerge as a relevant and sensitive platform for modelling SARS-CoV-2 infection and drug screening.

}, issn = {1557-8534}, doi = {10.1089/scd.2020.0152}, author = {Surendran, Harshini and Nandakumar, Swapna and Pal, Rajarshi} } @article {1061, title = {Differentiating Human Induced Pluripotent Stem Cells (iPSCs) Into Lung Epithelial Cells. [Eyestem, a C-CAMP Startup]}, journal = {Curr Protoc Stem Cell Biol}, year = {2019}, month = {2019 Apr 18}, pages = {e86}, abstract = {

Human induced pluripotent\ stem cells\ (hiPSCs) not only offer great opportunities for the study of human development but also have tremendous potential for future clinical\ cell-based therapies.\ The protocol outlined here is used to differentiate hiPSCs into lung epithelial cell types through a process that faithfully recapitulates the stepwise events observed in vivo. From pluripotency, cells are differentiated to a definitive endoderm fate, followed by progression into anteriorized foregut endoderm that has the ability to give rise to both proximal and distal epithelial cells. Furthermore, this methodology allows for the study of lung dysfunction and disease modeling using patient-derived cells, as well as high-throughput pharmacological screening and eventually personalized therapies. Recently we were able to reproduce this protocol using the working cell bank of an hiPSC line made under current Good Manufacturing Practice (cGMP) conditions, a necessary step for the future clinical application of these\ cells. {\textcopyright} 2019 by John Wiley \& Sons, Inc.

}, issn = {1938-8969}, doi = {10.1002/cpsc.86}, author = {Surendran, Harshini and Rajamoorthy, Mohanapriya and Pal, Rajarshi} } @article {1062, title = {Generation of Transplantable Retinal Pigmented Epithelial (RPE) Cells for Treatment of Age-Related Macular Degeneration (AMD). [Eyestem, a C-CAMP Startup]}, journal = {Methods Mol Biol}, year = {2018}, month = {2018 Jun 13}, abstract = {

Age-related macular degeneration (AMD) is the foremost cause of blindness in people over the age of 60 worldwide. Clinically, this disease starts with distortion in central vision eventually leading to legal blindness. Vision loss has a significant impact on quality of life and incurs a substantial cost to the economy. Furthermore, AMD is a complex and progressive neurodegenerative disorder that triggers visual impairment due to the loss of retinal pigmented epithelium (RPE) and the light-sensitive photoreceptors that they support, protect and provide nutrition. Currently, there is no curative treatment for the most common form of this disease, i.e., dry AMD. A novel approach to treat AMD involves the transplantation of RPE cells derived from human induced pluripotent stem cells (iPSCs) in the outer retina. These iPSC-derived RPE cells not only show characteristics similar to native RPE but also could replace as well as regenerate damaged pathologic RPE and produce supportive growth factors and cytokines. Several clinical trials are being conducted taking advantage of a variety of cell- and tissue engineering-based approaches. Here, we present a simple, cost effective, and scalable cell-culture model for generation of purified RPE thus providing the foundation for developing an allogeneic cell therapy for AMD.

}, issn = {1940-6029}, doi = {10.1007/7651_2018_140}, author = {Surendran, Harshini and Rathod, Reena J and Pal, Rajarshi} }