Summary of published reports using 3D organoids for in vitro T cell differentiation
| Gene defect . | Starting cells . | Use of the 3D organoids . | Results . | Reference . |
|---|---|---|---|---|
| Healthy donor (HD) | CD34+ | Original protocol for the generation of ATOs starting from human CD34+ cells | Establishment of a protocol for the efficient in vitro generation of mature T cells, fully functional and with a diverse TCR repertoire | Seet et al., 2017 |
| HD | iPSCs, embryonic stem cells | Original protocol for the generation of ATOs starting from human iPSCs and ES | Establishment of a protocol for the efficient in vitro generation of mature T cells, fully functional and with a diverse TCR repertoire | Montel-Hagen et al., 2019 |
| AK2, IL2RG, DGS, RAG1/2, ADA | CD34+ | Test ATOs to evaluate block in differentiation in patients with known gene defects | (1) Definition of T cell developmental block in patients with hematopoietic-intrinsic defects; (2) discrimination between hematopoietic-intrinsic and -extrinsic defects | Bosticardo et al., 2020 |
| IL2RG, TBX1, DGS, RAG1/2, ADA | CD34+ | Test 3D aggregates to evaluate block in differentiation in patients with known gene defects | (1) Definition of T cell developmental block in patients with hematopoietic-intrinsic defects; (2) discrimination between hematopoietic-intrinsic and -extrinsic defects | Bifsha et al., 2020 |
| POLD1 | CD34+ | Evaluation of T cell differentiation in vitro | POLD1-mutant CD34+ cells showed a block in differentiation, resulting in a reduced number of CD4+CD8+ DP cells and almost complete absence of TCRαβ+CD3+ cells | Nichols-Vinueza et al., 2021 |
| RAG2 | iPSCs | Test efficacy of CRISPR/Cas9-mediated gene editing (GE) approach | GE-iPSCs of a RAG2-mutant patient overcome block at CD4+CD8+ DP stage and show normal differentiation to mature TCRαβ+CD3+ cells | Gardner et al., 2021 |
| SASH3 | CD34+ | Evaluation of T cell differentiation in vitro | SASH3-mutant CD34+ cells were able reach full maturation, but frequency and absolute count of TCRαβ+CD3+ cells were significantly decreased, due to impaired cell cycle progression and increased apoptosis | Delmonte et al., 2021 |
| MAGT1 | CD34+ | Test efficacy of CRISPR/Cas9-mediated GE approach | (1) GE CD34+ cells from XMEN patients showed full T maturation; (2) restoration of NKG2D expression in CD3+ cells | Brault et al., 2021 |
| HD | CD34+ | Understanding how PNP inactivation affects T cell differentiation from iPSCs | (1) PNP inactivation causes decreased viability of CD34+ cells and accumulation in the CD4−CD8− DN stage; (2) effects of PNP inactivation were mediated through the downregulation of the dNTP tri-phosphohydrolase SAMHD1 | Abt et al., 2022 |
| HD | iPSCs | Test hypothesis for T-T–mediated selection of unconventional T cells | PLZF-expressing unconventional T cells originate from positive selection on other T cells, as opposed to conventional T cells, which are selected by cTECs | Suo et al., 2022 |
| FOXI3 | CD34+ | Evaluation of T cell differentiation in vitro | (1) CD34+ cells isolated from a patient with FOXI3 haploinsufficiency show efficient differentiation to mature TCRαβ+CD3+ cells; (2) these results support the thymic-intrinsic nature of T cell lymphopenia in FOXI3-mutant patients | Ghosh et al., 2022 |
| HD | CD34+, TSP | Evaluation the T cell differentiation potential of the different TSP (TSP1, TSP2, TSP3) | TSP1 are a more quiescent population; TSP3 cells are primed to develop much more rapidly into T cells compared with TSP1 and TSP2 cells | Cordes et al., 2022 |
| IL2RG | CD34+ | Test efficacy of lentiviral vector– and CRISPR/Cas9-mediated GE approaches | Both treatments overcome the block at the CD34+ Pro T stage and show restoration of surface expression of IL2Rgc and its mediated signaling | Brault et al., 2023 |
| TRIM37 | CD34+ | Evaluation of T cell differentiation in vitro | Normal mature TCRαβ+CD3+ cell generation, excluding an early hematopoietic-intrinsic defect of T cell development | Gazzin et al., 2023 |
| CD3D | CD34+ | Test efficacy of CRISPR/Cas9-mediated BE approach | Edited CD3D-mutant patients’ CD34+ cells differentiated to fully mature T cells, displaying diverse TCR repertoires and TCR-dependent functions | McAuley et al., 2023 |
| HD | iPSCs | Understanding how CARs affect T cell differentiation from iPSCs | iPSCs transduced with a CD19-targeted CAR showed a diversion of T cell differentiation from conventional T cell to ILC2 lineage | Li et al., 2023 |
| WAS | CD34+ | Evaluation of T cell differentiation in vitro | (1) Partial block at the positive selection checkpoint, especially on the generation of CD8+ SP cells; (2) no effects on the generation of a diverse TCR repertoire | Pille et al., 2023 |
| PAX1 | CD34+ | Evaluation of T cell differentiation in vitro | Normal mature TCRαβ+CD3+ cells generation, excluding an early hematopoietic-intrinsic defect of T cell development | Yakici et al., 2023 |
| RAG1 | CD34+ | Test efficacy of CRISPR/Cas9-mediated GE approach | (1) Integration into intron 1 of RAG1 gene achieved suboptimal correction and didn't overcome the block in differentiation at the CD4+CD8+ DP stage; (2) in-frame insertion into exon 2 led to efficient generation of mature TCRαβ+CD3+ cells | Castiello et al., 2024 |
| HD | iPSCs | Evaluation of the lymphoid differentiation potential of BMO-derived HSPC | BMO-derived HSPCs can differentiate to mature TCRαβ+CD3+ cells | Frenz-Wiessner et al., 2024 |
| PTCRA | CD34+ | Evaluation of T cell differentiation in vitro | Selective defect in TCRαβ+CD3+ cell production, directly correlating with the severity of the genetic mutation, while the ability of producing TCRγδ+CD3+ cells was preserved | Materna et al., 2024 |
| Gene defect . | Starting cells . | Use of the 3D organoids . | Results . | Reference . |
|---|---|---|---|---|
| Healthy donor (HD) | CD34+ | Original protocol for the generation of ATOs starting from human CD34+ cells | Establishment of a protocol for the efficient in vitro generation of mature T cells, fully functional and with a diverse TCR repertoire | Seet et al., 2017 |
| HD | iPSCs, embryonic stem cells | Original protocol for the generation of ATOs starting from human iPSCs and ES | Establishment of a protocol for the efficient in vitro generation of mature T cells, fully functional and with a diverse TCR repertoire | Montel-Hagen et al., 2019 |
| AK2, IL2RG, DGS, RAG1/2, ADA | CD34+ | Test ATOs to evaluate block in differentiation in patients with known gene defects | (1) Definition of T cell developmental block in patients with hematopoietic-intrinsic defects; (2) discrimination between hematopoietic-intrinsic and -extrinsic defects | Bosticardo et al., 2020 |
| IL2RG, TBX1, DGS, RAG1/2, ADA | CD34+ | Test 3D aggregates to evaluate block in differentiation in patients with known gene defects | (1) Definition of T cell developmental block in patients with hematopoietic-intrinsic defects; (2) discrimination between hematopoietic-intrinsic and -extrinsic defects | Bifsha et al., 2020 |
| POLD1 | CD34+ | Evaluation of T cell differentiation in vitro | POLD1-mutant CD34+ cells showed a block in differentiation, resulting in a reduced number of CD4+CD8+ DP cells and almost complete absence of TCRαβ+CD3+ cells | Nichols-Vinueza et al., 2021 |
| RAG2 | iPSCs | Test efficacy of CRISPR/Cas9-mediated gene editing (GE) approach | GE-iPSCs of a RAG2-mutant patient overcome block at CD4+CD8+ DP stage and show normal differentiation to mature TCRαβ+CD3+ cells | Gardner et al., 2021 |
| SASH3 | CD34+ | Evaluation of T cell differentiation in vitro | SASH3-mutant CD34+ cells were able reach full maturation, but frequency and absolute count of TCRαβ+CD3+ cells were significantly decreased, due to impaired cell cycle progression and increased apoptosis | Delmonte et al., 2021 |
| MAGT1 | CD34+ | Test efficacy of CRISPR/Cas9-mediated GE approach | (1) GE CD34+ cells from XMEN patients showed full T maturation; (2) restoration of NKG2D expression in CD3+ cells | Brault et al., 2021 |
| HD | CD34+ | Understanding how PNP inactivation affects T cell differentiation from iPSCs | (1) PNP inactivation causes decreased viability of CD34+ cells and accumulation in the CD4−CD8− DN stage; (2) effects of PNP inactivation were mediated through the downregulation of the dNTP tri-phosphohydrolase SAMHD1 | Abt et al., 2022 |
| HD | iPSCs | Test hypothesis for T-T–mediated selection of unconventional T cells | PLZF-expressing unconventional T cells originate from positive selection on other T cells, as opposed to conventional T cells, which are selected by cTECs | Suo et al., 2022 |
| FOXI3 | CD34+ | Evaluation of T cell differentiation in vitro | (1) CD34+ cells isolated from a patient with FOXI3 haploinsufficiency show efficient differentiation to mature TCRαβ+CD3+ cells; (2) these results support the thymic-intrinsic nature of T cell lymphopenia in FOXI3-mutant patients | Ghosh et al., 2022 |
| HD | CD34+, TSP | Evaluation the T cell differentiation potential of the different TSP (TSP1, TSP2, TSP3) | TSP1 are a more quiescent population; TSP3 cells are primed to develop much more rapidly into T cells compared with TSP1 and TSP2 cells | Cordes et al., 2022 |
| IL2RG | CD34+ | Test efficacy of lentiviral vector– and CRISPR/Cas9-mediated GE approaches | Both treatments overcome the block at the CD34+ Pro T stage and show restoration of surface expression of IL2Rgc and its mediated signaling | Brault et al., 2023 |
| TRIM37 | CD34+ | Evaluation of T cell differentiation in vitro | Normal mature TCRαβ+CD3+ cell generation, excluding an early hematopoietic-intrinsic defect of T cell development | Gazzin et al., 2023 |
| CD3D | CD34+ | Test efficacy of CRISPR/Cas9-mediated BE approach | Edited CD3D-mutant patients’ CD34+ cells differentiated to fully mature T cells, displaying diverse TCR repertoires and TCR-dependent functions | McAuley et al., 2023 |
| HD | iPSCs | Understanding how CARs affect T cell differentiation from iPSCs | iPSCs transduced with a CD19-targeted CAR showed a diversion of T cell differentiation from conventional T cell to ILC2 lineage | Li et al., 2023 |
| WAS | CD34+ | Evaluation of T cell differentiation in vitro | (1) Partial block at the positive selection checkpoint, especially on the generation of CD8+ SP cells; (2) no effects on the generation of a diverse TCR repertoire | Pille et al., 2023 |
| PAX1 | CD34+ | Evaluation of T cell differentiation in vitro | Normal mature TCRαβ+CD3+ cells generation, excluding an early hematopoietic-intrinsic defect of T cell development | Yakici et al., 2023 |
| RAG1 | CD34+ | Test efficacy of CRISPR/Cas9-mediated GE approach | (1) Integration into intron 1 of RAG1 gene achieved suboptimal correction and didn't overcome the block in differentiation at the CD4+CD8+ DP stage; (2) in-frame insertion into exon 2 led to efficient generation of mature TCRαβ+CD3+ cells | Castiello et al., 2024 |
| HD | iPSCs | Evaluation of the lymphoid differentiation potential of BMO-derived HSPC | BMO-derived HSPCs can differentiate to mature TCRαβ+CD3+ cells | Frenz-Wiessner et al., 2024 |
| PTCRA | CD34+ | Evaluation of T cell differentiation in vitro | Selective defect in TCRαβ+CD3+ cell production, directly correlating with the severity of the genetic mutation, while the ability of producing TCRγδ+CD3+ cells was preserved | Materna et al., 2024 |