Derivation of induced pluripotent stem cells line (RCPCMi007-A-1) with inactivation of the beta-2-microglobulin gene by CRISPR/Cas9 genome editing

Bogomiakova, Margarita E.; Sekretova, Elizaveta K.; Eremeev, A V; Shuvalova, L.D.; Bobrovsky, Pavel A.; Zerkalenkova, Elena; Lebedeva, Olga; Lagarkova, Maria A.

doi:10.1016/j.scr.2021.102451

Cited by 8 publications

(7 citation statements)

References 3 publications

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“…In this paper, we use designations according to the donor: iPSC-A and iPSC-B. The iPSC-A and iPSC-B were characterized according to standard criteria [ 20 , 47 ]. Using CRISPR/Cas9 genome editing for the B2M gene knockout, we obtained ΔiPSC-A and ΔiPSC-B lines lacking HLA-I expression.…”

Section: Resultsmentioning

confidence: 99%

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iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

et al. 2023

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Background Dozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming. Methods We established an isogeneic cell model consisting of parental dermal fibroblasts, fibroblast-like iPSC-derivatives (iPS-fibro) and iPS-fibro lacking beta-2-microglobulin (B2M). Using the cells obtained from two patients, we analyzed the activation of autologous and allogeneic T-lymphocytes and NK-cells co-cultured with target cells. Results Here we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that pretreatment of iPS-fibro with proinflammatory cytokine IFNγ restored the ligand imbalance, thereby reducing the degranulation and cytotoxicity of NK-cells. Conclusion In summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies. Trial registration Not applicable.

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Section: Resultsmentioning

confidence: 99%

“…Using CRISPR/Cas9 genome editing for the B2M gene knockout, we obtained ΔiPSC-A and ΔiPSC-B lines lacking HLA-I expression. A detailed description of ΔiPSC-A was previously published [ 20 ]. The iPSC-A and its subclone ΔiPSC-A are registered in hPSCreg database [ 48 ].…”

Section: Resultsmentioning

confidence: 99%

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

et al. 2023

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“…These manufacturing processes are robust and reproducible with the GMPc hESC line and could be used to produce ATMP with a huge industrial scale up perspective [ 35 ]. Finally, the hPSC cell line could be developed from specific donors to obtained haplobanks [ 36 ] or could be genetically manipulated to evade the immune system [ 37 ] in order to obtain “universal graftable” engineered composite skins avoiding tissue rejection. Altogether, this strategy could lead to a reduction in the cost in the engineered skin production in order to offer a treatment to the greatest number.…”

Section: Discussionmentioning

confidence: 99%

Clinical Grade Human Pluripotent Stem Cell-Derived Engineered Skin Substitutes Promote Keratinocytes Wound Closure In Vitro

Domingues

Darle

Masson

et al. 2022

Cells

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Chronic wounds, such as leg ulcers associated with sickle cell disease, occur as a consequence of a prolonged inflammatory phase during the healing process. They are extremely hard to heal and persist as a significant health care problem due to the absence of effective treatment and the uprising number of patients. Indeed, there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Development in skin engineering leads to a small catalogue of available substitutes manufactured in Good Manufacturing Practices compliant (GMPc) conditions. Those substitutes are produced using primary cells that could limit their use due to restricted sourcing. Here, we propose GMPc protocols to produce functional populations of keratinocytes and fibroblasts derived from pluripotent stem cells to reconstruct the associated dermo-epidermal substitute with plasma-based fibrin matrix. In addition, this manufactured composite skin is biologically active and enhances in vitro wounding of keratinocytes. The proposed composite skin opens new perspectives for skin replacement using allogeneic substitute.

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“…Using CRISPR/Cas9 genome editing for the B2M gene knockout, we obtained ΔiPSC-A and ΔiPSC-B lines lacking HLA-I expression. A detailed description of ΔiPSC-A was previously published [20]. The iPSC-A and its subclone ΔiPSC-A are registered in hPSCreg database [47].…”

Section: Generation Of Ips-bro and δIps-bromentioning

confidence: 99%

“…The diversity of the HLA phenotypes is the main reason for histoincompatibility. As such, HLA-editing is the most common approach to creating hypoimmunogenic hPSCs lines, with the most common modi cation -the knockout of a light chain of HLA-I dimer encoded by the beta-2-microglobulin (B2M) gene [15][16][17][18][19][20][21]. In some studies, individual genes of the HLA-I locus were edited [22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

Bogomiakova

Sekretova

Anufrieva

et al. 2022

Preprint

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Background: Dozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming. Methods: We established an isogeneic cell model consisting of parental dermal fibroblasts, fibroblast-like iPSC-derivatives (iPS-fibro) and iPS-fibro lacking beta-2-microglobulin (B2M). Using the cells obtained from two patients, we analyzed the activation of autologous and allogeneic T-lymphocytes and NK-cells cocultured with target cells. Results: Here we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that the ligand imbalance could be reversed to normal. Longer cultivation and passaging of iPS-fibro or pretreatment with proinflammatory cytokines boosted HLA class I expression, which reduced the degranulation and cytotoxicity of NK-cells. Conclusion: In summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies. Trial registration: Not applicable.

show abstract

Derivation of induced pluripotent stem cells line (RCPCMi007-A-1) with inactivation of the beta-2-microglobulin gene by CRISPR/Cas9 genome editing

Cited by 8 publications

References 3 publications

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

Clinical Grade Human Pluripotent Stem Cell-Derived Engineered Skin Substitutes Promote Keratinocytes Wound Closure In Vitro

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

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