From Rejection to Regeneration: Developing Hypoimmune Human Pluripotent Stem Cells for Vascular Disease Therapies

Authors:

Ziyan Liu, Umji Lee, Juan Melero-Martin

Date Created:

2025-01-01

Course Title:

Professor:

Not specified

About Paper:

Human Induced Pluripotent Stem (hIPS) cell therapies hold those ligands on hIPSCs, we hypothesize that we will be able significant potential for the treatment of a broad range of to induce hypoimmunogenicity. Using the piggyBac transposon conditions, including neurodegenerative, cardiovascular, and system, we will introduce 4 inhibitory surface ligands—HLA- autoimmune disorders. However, current hIPSC-based therapies G, CD47, PD-L1, and CD300a TASR— onto dKO-hIPSCs to for vascular diseases are limited by their immunogenicity, which generatehypoimmunogenicIPSCs(HIPs). ThethreehIPSCs(WT, requires donor cells to be closely matched to the genetic makeup dKO,HIP)willthenbedifferentiatedintoinducedendothelialcells of the recipient. Existing strategies to reduce immunogenicity (iECs) and induced mural progenitor cells (iMPCs), before being through the deletion of human leukocyte antigen (HLA) class I cocultured with NK cells. The degranulation of NK cells and and II molecules by B2M and CIITA deletions (dKO-hIPSCs) the survival rate of the iECs and iMPCs obtained from the WT-, have proven successful in evading T-cell activation. However, dKO-, and HIP-IPSCs will be measured via flow cytometry and dKO leaves cells vulnerable to attacks by the natural killer (NK)fluorescent imaging. If our hypothesis is supported, we would cells in the innate immune system. Our research this summer expect our iECs and iMPCs derived from HIPs to have a higher aims to develop a strategy for genetically engineering hIPS cellssurvival rate compared to those derived from dKO cells, and a to induce protection against both the adaptive and innate immune similarsurvivalratecomparedtothosederivedfromWTcells. The systems. Prior research suggests that a few inhibitory ligands creation of hypoimmune IPS cells will allow “off-the-shelf” stem were effective in reducing NK cell activation; thus, by inducing cell therapies, thus enhancing their applicability. 54 Harvard Stem Cell Institute Internship Program Lineage Tracing and Characterization of Pi16+ Cells in Development and Repair Keely Romine-West, Yizhong Hu, Yingzi Yang Wesleyan University | Molecular and Cellular Biology | 2027 Stem cells are fundamental to development, tissue maintenance, line Pi16CreER, which were crossed with tdTomato to perform and regeneration, playing a central role in both normal standard lineage tracing of healthy mice to assess the distribution physiological processes and responses to disease or injury. In of Pi16 cells over time (two days, one month, and three months a previous study investigating +eriodontal tissue regeneration in post tamoxifen injection). Preliminary analysis using fluorescent mice, our lab found that Gli1 cells expand in the periodontal dark-field microscopy images revealed Pi16 expression across ligament (PDL) and surrounding periodontal tissues following multiple tissues including the lung, heart, intestine, liver, spleen, injury. However, due to the broad nature of Gli1 expression, kidney, femur, stomach, molar, and periodontal tissue. Temporal single-cell RNA sequencing was employed to further resolve the comparisons of the first two time points demonstrated a significant cellular heterogeneity. This analysis identified Pi16 (peptidase increase in Pi16 signal intensity in several tissues, including the inhibitor 16) as a marker of a distinct population that may give riselung,heart,intestine,femur,spleen,kidney,molar,andperiodontal to various progenitor populations within the PDL and periodontal regions. Current work aims to identify the specific cell populations tissues. Although Pi16 has been described in scientific literature labeled by Pi16 to clarify its functional roles in these contexts. Our as marking a distinct population of fibroblasts involved in tissue goal of assessing whether Pi16 may serve as a marker of a novel remodeling, immune regulation, and barrier function, its potential stem cell population in both healthy and damaged tissues may role as a stem cell remains to be explored. To identify the provide new insights into tissue regeneration and open avenues for expression pattern of Pi16, our lab generated knock-in mouse targeted therapeutic strategies in regenerative medicine. The Investigation of Estrogen Treatment on the Liver: Developmental or Functional Ahmad Shamsi, Patrice Delaney, Wolfram Goessling Pomona College | Human Developmental and Regenerative Biology | 2027 Hepatocytes and biliary epithelial cells (BECs) are the principal 3-6 dpf, when liver architecture matures and functional activity cell types of the liver, responsible for coordinating key liver begins. Our preliminary data show that E2 increases hepatocyte functions, such as metabolism, detoxification, and bile secretion. populations, regardless of developmental timing, suggesting a role Estrogen is a critical hormonal regulator of liver development, inpromotinghepatocytegrowthindependentofliverdevelopment. function, and regeneration yet its role remains complex and In contrast, bile duct network formation is selectively impaired context-dependent in liver pathologies. In this study, I investigatedwhen E2 is administered during early liver, implicating E2 in the whether estrogen-induced liver abnormalities are caused by disrupt of hepatoblast to BEC differentiation. On the other hand, developmental disruption or functional interference. Using when we assessed liver functionality, the data indicated that while zebrafish with fluorescently labeled hepatocytes and BECs, we E2 exposure during the 3-6 dpf window led to complete bile flow exposed embryos and larvae to 17▯-estradiol (E2) during two loss, the liver was able to recover bile flow following E2 exposure distinct developmental windows: (1) 0-3 days post-fertilization during the 0-3 dpf window. Together, these findings suggest that (dpf), when the common BEC and hepatocyte progenitor E2 differentially impacts liver functionality and cell populations hepatoblasts differentiate and liver morphogenesis begins, and (2) depending on timing of exposure. Ideal Mechanical Loading Regimens for Culturing Osteochondral Tissues In Vitro Lucy Shen, Nereida Ramirez, April Craft Columbia University | Biomedical Engineering | 2028 Osteoarthritis (OA) is an incurable cartilage disease that causes the unloaded, free-swelling control tissues, we found that loaded pain and immobility. Studying the pathology of OA in tissues had higher viability at day 1 and day 4 as assessed by a patients is challenging due to the limited availability of primary LIVE/DEAD™ stain and Quant-iT™ PicoGreen™ dsDNA assay. tissues. Moreover, current models to study OA in vitro fail to Moreover, we found that the loaded tissues also produced more incorporate mechanical loading. Consequently, using CellScale’s glycosaminoglycans (a unique component of the articular cartilage MechanoTX™ device, we developed an in vitro loading regime extracellular matrix) via a DMMB (1,9-dimethylmethylene blue) that promotes cartilage tissue viability and matrix deposition. Theassay and Safarin O staining. However, we also observed that study was performed in vitro with 6 millimeter by 2 millimeter comparedtothefree-swellingcontrols,theloadedtissuesexhibited osteochondral cores (cartilage thickness 1 ± 0.5 millimeters) morecelldeathinthemostsuperficialregion. Overall,theseresults removed from the stifle joints of 7- to 9-month-old wild-type suggest that loading the tissues under 10% strain for 1 cycle leads sheep. Tissues were either treated with cyclic compressive strain to more viability in the tissues, with similar but less pronounced or maintained under free-swelling conditions and then collected results for 3 cycles. Our cartilage loading regimen can be utilized after 1 and 4 days. In a single loading cycle, loaded tissues were in the future to load tissues in vitro to study the efficacy of treated with 10% compressive strain at a frequency of 0.5 hertz forosteoarthritis treatments, thus providing a physiologically relevant 2 hours, followed by a rest period of 16 ± 1 hours. Compared to model with which to study the disease. Differentiation and Characterization of hiPSC-derived Corneal Endothelial Cells Aditya Shivaswamy, Vinay K. Pulimamidi, Sunil K. Chauhan Johns Hopkins University | Neuroscience | 2028 Corneal endothelial cells (CEnCs), which line the innermost trilineage differentiation potential. iPSCs were differentiated into corneal layer, do not proliferate in vivo following disease or neural crest cells (NCCs) and further differentiated into CEnCs injury, leading to reduced cell density and subsequent loss of using defined culture conditions. The iPSC-derived NCCs and their pump and barrier functions. CEnC loss is a leading cause CEnCs were characterized based on the expression of NCC and of vision impairment and the primary indication for corneal CEnC-specific markers by qRT-PCR and immunofluorescence. transplantation worldwide. However, the global shortage of donor corneas, corneal graft rejection, and use of immunosuppression in The iPSC line expressed the pluripotency markers Oct4, Nanog, SSEA4 and TRA-1-60. The iPSCs were differentiated into the keratoplasty highlight the urgent need for alternative therapeutic three germ layers and expressed ectoderm (OTX2), endoderm strategies. Deriving functional CEnCs from human induced (SOX2), and mesoderm (Brachyury) specific markers. In pluripotent stem cells (hiPSCs) offers a promising solution to address this limitation and reduce dependence on donor tissue. In differentiation cultures at D10, the pluripotency markers were thisstudy,weareoptimizingtheprotocoltoderivetheCEnCsfrom downregulated and NCC-specific markers were upregulated. On human iPSCs in a xeno-free and temporally efficient manner. D20, the iPSC-derived CEnCs showed increased expression of CEnC-specific markers. Human fibroblast-derived iPSC line was purchased from Wi Cell (Madison, WI). iPSCs were cultured on Matrigel-coated Our approach for differentiation of hiPSCs into CEnC-like cells seems optimal. Future research will focus on functional validation tissue culture dishes in mTeSR1 medium. The iPSC line was of the hiPSC-derived CEnCs in vitro and in vivo. characterized by its expression of pluripotency markers and its

Abstract:

Human Induced Pluripotent Stem (hIPS) cell therapies hold those ligands on hIPSCs, we hypothesize that we will be able significant potential for the treatment of a broad range of to induce hypoimmunogenicity. Using the piggyBac transposon conditions, including neurodegenerative, cardiovascular, and system, we will introduce 4 inhibitory surface ligands—HLA- autoimmune disorders. However, current hIPSC-based therapies G, CD47, PD-L1, and CD300a TASR— onto dKO-hIPSCs to for vascular diseases are limited by their immunogenicity, which generatehypoimmunogenicIPSCs(HIPs). ThethreehIPSCs(WT, requires donor cells to be closely matched to the genetic makeup dKO,HIP)willthenbedifferentiatedintoinducedendothelialcells of the recipient. Existing strategies to reduce immunogenicity (iECs) and induced mural progenitor cells (iMPCs), before being through the deletion of human leukocyte antigen (HLA) class I cocultured with NK cells. The degranulation of NK cells and and II molecules by B2M and CIITA deletions (dKO-hIPSCs) the survival rate of the iECs and iMPCs obtained from the WT-, have proven successful in evading T-cell activation. However, dKO-, and HIP-IPSCs will be measured via flow cytometry and dKO leaves cells vulnerable to attacks by the natural killer (NK)fluorescent imaging. If our hypothesis is supported, we would cells in the innate immune system. Our research this summer expect our iECs and iMPCs derived from HIPs to have a higher aims to develop a strategy for genetically engineering hIPS cellssurvival rate compared to those derived from dKO cells, and a to induce protection against both the adaptive and innate immune similarsurvivalratecomparedtothosederivedfromWTcells. The systems. Prior research suggests that a few inhibitory ligands creation of hypoimmune IPS cells will allow “off-the-shelf” stem were effective in reducing NK cell activation; thus, by inducing cell therapies, thus enhancing their applicability. 54 Harvard Stem Cell Institute Internship Program Lineage Tracing and Characterization of Pi16+ Cells in Development and Repair Keely Romine-West, Yizhong Hu, Yingzi Yang Wesleyan University | Molecular and Cellular Biology | 2027 Stem cells are fundamental to development, tissue maintenance, line Pi16CreER, which were crossed with tdTomato to perform and regeneration, playing a central role in both normal standard lineage tracing of healthy mice to assess the distribution physiological processes and responses to disease or injury. In of Pi16 cells over time (two days, one month, and three months a previous study investigating +eriodontal tissue regeneration in post tamoxifen injection). Preliminary analysis using fluorescent mice, our lab found that Gli1 cells expand in the periodontal dark-field microscopy images revealed Pi16 expression across ligament (PDL) and surrounding periodontal tissues following multiple tissues including the lung, heart, intestine, liver, spleen, injury. However, due to the broad nature of Gli1 expression, kidney, femur, stomach, molar, and periodontal tissue. Temporal single-cell RNA sequencing was employed to further resolve the comparisons of the first two time points demonstrated a significant cellular heterogeneity. This analysis identified Pi16 (peptidase increase in Pi16 signal intensity in several tissues, including the inhibitor 16) as a marker of a distinct population that may give riselung,heart,intestine,femur,spleen,kidney,molar,andperiodontal to various progenitor populations within the PDL and periodontal regions. Current work aims to identify the specific cell populations tissues. Although Pi16 has been described in scientific literature labeled by Pi16 to clarify its functional roles in these contexts. Our as marking a distinct population of fibroblasts involved in tissue goal of assessing whether Pi16 may serve as a marker of a novel remodeling, immune regulation, and barrier function, its potential stem cell population in both healthy and damaged tissues may role as a stem cell remains to be explored. To identify the provide new insights into tissue regeneration and open avenues for expression pattern of Pi16, our lab generated knock-in mouse targeted therapeutic strategies in regenerative medicine. The Investigation of Estrogen Treatment on the Liver: Developmental or Functional Ahmad Shamsi, Patrice Delaney, Wolfram Goessling Pomona College | Human Developmental and Regenerative Biology | 2027 Hepatocytes and biliary epithelial cells (BECs) are the principal 3-6 dpf, when liver architecture matures and functional activity cell types of the liver, responsible for coordinating key liver begins. Our preliminary data show that E2 increases hepatocyte functions, such as metabolism, detoxification, and bile secretion. populations, regardless of developmental timing, suggesting a role Estrogen is a critical hormonal regulator of liver development, inpromotinghepatocytegrowthindependentofliverdevelopment. function, and regeneration yet its role remains complex and In contrast, bile duct network formation is selectively impaired context-dependent in liver pathologies. In this study, I investigatedwhen E2 is administered during early liver, implicating E2 in the whether estrogen-induced liver abnormalities are caused by disrupt of hepatoblast to BEC differentiation. On the other hand, developmental disruption or functional interference. Using when we assessed liver functionality, the data indicated that while zebrafish with fluorescently labeled hepatocytes and BECs, we E2 exposure during the 3-6 dpf window led to complete bile flow exposed embryos and larvae to 17▯-estradiol (E2) during two loss, the liver was able to recover bile flow following E2 exposure distinct developmental windows: (1) 0-3 days post-fertilization during the 0-3 dpf window. Together, these findings suggest that (dpf), when the common BEC and hepatocyte progenitor E2 differentially impacts liver functionality and cell populations hepatoblasts differentiate and liver morphogenesis begins, and (2) depending on timing of exposure. Ideal Mechanical Loading Regimens for Culturing Osteochondral Tissues In Vitro Lucy Shen, Nereida Ramirez, April Craft Columbia University | Biomedical Engineering | 2028 Osteoarthritis (OA) is an incurable cartilage disease that causes the unloaded, free-swelling control tissues, we found that loaded pain and immobility. Studying the pathology of OA in tissues had higher viability at day 1 and day 4 as assessed by a patients is challenging due to the limited availability of primary LIVE/DEAD™ stain and Quant-iT™ PicoGreen™ dsDNA assay. tissues. Moreover, current models to study OA in vitro fail to Moreover, we found that the loaded tissues also produced more incorporate mechanical loading. Consequently, using CellScale’s glycosaminoglycans (a unique component of the articular cartilage MechanoTX™ device, we developed an in vitro loading regime extracellular matrix) via a DMMB (1,9-dimethylmethylene blue) that promotes cartilage tissue viability and matrix deposition. Theassay and Safarin O staining. However, we also observed that study was performed in vitro with 6 millimeter by 2 millimeter comparedtothefree-swellingcontrols,theloadedtissuesexhibited osteochondral cores (cartilage thickness 1 ± 0.5 millimeters) morecelldeathinthemostsuperficialregion. Overall,theseresults removed from the stifle joints of 7- to 9-month-old wild-type suggest that loading the tissues under 10% strain for 1 cycle leads sheep. Tissues were either treated with cyclic compressive strain to more viability in the tissues, with similar but less pronounced or maintained under free-swelling conditions and then collected results for 3 cycles. Our cartilage loading regimen can be utilized after 1 and 4 days. In a single loading cycle, loaded tissues were in the future to load tissues in vitro to study the efficacy of treated with 10% compressive strain at a frequency of 0.5 hertz forosteoarthritis treatments, thus providing a physiologically relevant 2 hours, followed by a rest period of 16 ± 1 hours. Compared to model with which to study the disease. Differentiation and Characterization of hiPSC-derived Corneal Endothelial Cells Aditya Shivaswamy, Vinay K. Pulimamidi, Sunil K. Chauhan Johns Hopkins University | Neuroscience | 2028 Corneal endothelial cells (CEnCs), which line the innermost trilineage differentiation potential. iPSCs were differentiated into corneal layer, do not proliferate in vivo following disease or neural crest cells (NCCs) and further differentiated into CEnCs injury, leading to reduced cell density and subsequent loss of using defined culture conditions. The iPSC-derived NCCs and their pump and barrier functions. CEnC loss is a leading cause CEnCs were characterized based on the expression of NCC and of vision impairment and the primary indication for corneal CEnC-specific markers by qRT-PCR and immunofluorescence. transplantation worldwide. However, the global shortage of donor corneas, corneal graft rejection, and use of immunosuppression in The iPSC line expressed the pluripotency markers Oct4, Nanog, SSEA4 and TRA-1-60. The iPSCs were differentiated into the keratoplasty highlight the urgent need for alternative therapeutic three germ layers and expressed ectoderm (OTX2), endoderm strategies. Deriving functional CEnCs from human induced (SOX2), and mesoderm (Brachyury) specific markers. In pluripotent stem cells (hiPSCs) offers a promising solution to address this limitation and reduce dependence on donor tissue. In differentiation cultures at D10, the pluripotency markers were thisstudy,weareoptimizingtheprotocoltoderivetheCEnCsfrom downregulated and NCC-specific markers were upregulated. On human iPSCs in a xeno-free and temporally efficient manner. D20, the iPSC-derived CEnCs showed increased expression of CEnC-specific markers. Human fibroblast-derived iPSC line was purchased from Wi Cell (Madison, WI). iPSCs were cultured on Matrigel-coated Our approach for differentiation of hiPSCs into CEnC-like cells seems optimal. Future research will focus on functional validation tissue culture dishes in mTeSR1 medium. The iPSC line was of the hiPSC-derived CEnCs in vitro and in vivo. characterized by its expression of pluripotency markers and its

Source:

Harvard / Sandhya Konar, Karin Gustafsson, David Scadden / 2025

Topics:

cell, tissue, liver, cenc, pi16, marker, ipsc, stem, human, population, disease, hipsc

Co-authors:

@ziyanliu202 , @umjilee203 , @juanmeleromartin204