Generation of a heterozygous GAPDH-Luciferase human ESC line (HVRDe008-A-1) for in vivo monitoring of stem cells and their differentiated progeny

Gerace, Dario; Boulanger, Kyle; Kenty, Jennifer Hyoje-Ryu; Melton, Douglas A.

doi:10.1016/j.scr.2021.102371

Cited by 8 publications

(16 citation statements)

References 5 publications

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“…The sequence of the covalently attached peptide (VMAPRTLLL) in the peptide::B2M::HLA-E fusion is derived from the HLA-Cw7 molecule (Kaiser et al, 2005). hESCs were nucleofected with the GAP-PD or GAP-BEC targeting plasmids and GAPDH-targeting RNP as previously described (Gerace et al, 2021). For GAP-B2P and GAP-BEC lines, a polyclonal population of puromycin-selected cells was subsequently nucleofected with B2M-targeting RNP, and single-colonies were picked for expansion after FACS sorting as described above.…”

Section: Methodsmentioning

confidence: 99%

“…The existing GAPluc (WT) hESC line served as starting material for the generation of HLA-deficient hESCs (Gerace et al, 2021). To generate HLA-deficient hESCs, 1x10 6 WT hESCs were nucleofected using the 4D-Nucleofector (Lonza) with RNP complexed with 120 pmol B2M gRNA (5'-GCTACTCTCTCTTTCTGGCC'3) (Mandal et al, 2014) (IDT) and 104 pmol Alt-R® S.p.…”

Section: Generation Of Immune-evasive Hescsmentioning

confidence: 99%

“…In vivo NK cell cytotoxicity 2.5 x 10 5 WT and B2M -/-SC-islet cell clusters were resuspended either alone or with 7.5 x 10 5 primary human NK cells (pre-treated with 0.5 ng/ml rhIL-2 for 12 h) in phenol-free Matrigel (Corning,356237) and transplanted subcutaneously in Scid/beige mice (n = 5). Bioluminescence was measured 10 min after i.p injection of 10 µL/g D-luciferin (15mg/ml) on days 1 and 5 following cell transplantation on the IVIS Spectrum (PerkinElmer, 124262) as previously described (Gerace et al, 2021).…”

Section: In Vitro Nk Cell Cytotoxicity Assaysmentioning

confidence: 99%

“…To address the issue of transgene silencing during differentiation into SC-islet cells, we employ a transgene targeting strategy that makes use of the constitutively expressed GAPDH gene in primary human islets and SC-islet cells (Gerace et al, 2021; Sintov et al, 2021). We generate hypoimmunogenic SC-islet cells that over-express PD-L1 and the HLA-E long-chain fusion in both HLA-competent and deficient settings.…”

Section: Introductionmentioning

confidence: 99%

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Secreted cytokines provide local immune tolerance for human stem cell-derived islets

Gerace

Zhou

Kenty

et al. 2022

Preprint

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SummaryImmunological protection of transplanted stem cell-derived islet (SC-islet) cells is yet to be achieved without chronic immunosuppression or encapsulation. Existing genetic engineering approaches to produce hypoimmunogenic SC-islet cells have so far shown variable results. Here, we show that targeting the human leukocyte antigens (HLAs) and PD-L1 alone do not sufficiently protect SC-islet cells from xeno- or allo-rejection. As an addition to these approaches, we genetically engineered SC-islet cells to secrete the cytokines IL-10, TGF-β and modified IL-2 such that they promote a tolerogenic local microenvironment by activating and expanding regulatory T cells (Tregs). These cytokine-secreting human SC-islet cells prevented xeno-rejection for up to 9 weeks post-transplantation in B6/albino mice. Thus, hESCs engineered to induce a tolerogenic local microenvironment may represent a source of replacement SC-islet cells that do not require encapsulation or immunosuppression for diabetes cell replacement therapy.

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Section: Methodsmentioning

confidence: 99%

Section: Generation Of Immune-evasive Hescsmentioning

confidence: 99%

Section: In Vitro Nk Cell Cytotoxicity Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Secreted cytokines provide local immune tolerance for human stem cell-derived islets

Gerace

Zhou

Kenty

et al. 2022

Preprint

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“…HES3 and MEL1 hESC lines encoding a copy of GFP in the INS locus ( INS GFP/wt ) are useful to trace INS+ cells through the differentiation process [ 114 ]. hESC expressing luciferase ( GAPDH luc/wt ) can be used to monitor in vivo transplanted cells and organoids [ 115 ]. Some of the examples generated to date include inducible Cas9 for CRISPR screenings [ 116 ] and a transcriptional transactivator for gain-of-function studies [ 117 ].…”

Section: Models To Study the Genetic Basis Of β Cell Dysfunctionmentioning

confidence: 99%

Stem Cell-Derived β Cells: A Versatile Research Platform to Interrogate the Genetic Basis of β Cell Dysfunction

Bartolomé

2022

IJMS

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Pancreatic β cell dysfunction is a central component of diabetes progression. During the last decades, the genetic basis of several monogenic forms of diabetes has been recognized. Genome-wide association studies (GWAS) have also facilitated the identification of common genetic variants associated with an increased risk of diabetes. These studies highlight the importance of impaired β cell function in all forms of diabetes. However, how most of these risk variants confer disease risk, remains unanswered. Understanding the specific contribution of genetic variants and the precise role of their molecular effectors is the next step toward developing treatments that target β cell dysfunction in the era of personalized medicine. Protocols that allow derivation of β cells from pluripotent stem cells, represent a powerful research tool that allows modeling of human development and versatile experimental designs that can be used to shed some light on diabetes pathophysiology. This article reviews different models to study the genetic basis of β cell dysfunction, focusing on the recent advances made possible by stem cell applications in the field of diabetes research.

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Transplantation of committed pre-adipocytes from brown adipose tissue improves whole-body glucose homeostasis

Dewal,

Yang,

Baer

et al. 2024

iScience

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Generation of a heterozygous GAPDH-Luciferase human ESC line (HVRDe008-A-1) for in vivo monitoring of stem cells and their differentiated progeny

Cited by 8 publications

References 5 publications

Secreted cytokines provide local immune tolerance for human stem cell-derived islets

Secreted cytokines provide local immune tolerance for human stem cell-derived islets

Stem Cell-Derived β Cells: A Versatile Research Platform to Interrogate the Genetic Basis of β Cell Dysfunction

Transplantation of committed pre-adipocytes from brown adipose tissue improves whole-body glucose homeostasis

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