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CD4 + T cells induced from human iPSCs (iCD4 + T cells) offer a therapeutic opportunity for overcoming immune pathologies arising from hematopoietic stem cell transplantation. However, most iCD4 + T cells are conventional helper T cells, which secrete inflammatory cytokines. We induced high-level expression of FOXP3, a master transcription factor of regulatory T cells, in iCD4 + T cells. Human iPSC-derived, FOXP3-induced CD4 + T (iCD4 + Treg-like) cells did not secrete inflammatory cytokines upon activation. Moreover, they showed demethylation of the Treg-specific demethylation region, suggesting successful conversion to immunosuppressive iCD4 + Treg-like cells. We further assessed these iCD4 + Treg-like cells for CAR-mediated immunosuppressive ability. HLA-A2 CAR-transduced iCD4 + Treg-like cells inhibited CD8 + cytotoxic T cell (CTL) division in a mixed lymphocyte reaction assay with A2 + allogeneic CTLs and suppressed xenogeneic graft-versus-host disease (GVHD) in NSG mice treated with A2 + human PBMCs. In most cases, these cells suppressed the xenogeneic GvHD progression as much as natural CD25 + CD127 - Tregs did., Competing Interests: Declaration of interests S.K. is a director and shareholder at Shinobi Tx and received research funding from Shinobi Tx, Takeda Pharmaceutical, and Astellas. H.K received research funding from Chugai, Kyowa Kirin, Zenyaku Kogyo, Sumitomo Pharma, Eisai, Daiichi Sankyo, Otsuka Pharmaceutical, Perseus Proteomics, CURED, Astellas Pharma, Asahi Kasei, AbbVie, Nippon Shinyaku, JCR Pharmaceuticals, and Takeda Pharmaceutical. S.K. has patent applications related to the research (patent no. WO/2023/182328). T. Sato., T. Shinohara., K.K., A.M., K.N., M.S., and Y.K. are employees of Takeda Pharmaceutical Co. Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro . Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models., Competing Interests: S.K. is a founder, shareholder, and chief scientific officer at Thyas, Co., Ltd., and received research funding from Takeda Pharmaceutical, Co., Ltd., Kirin Holdings, Co., Ltd., Astellas, Co., Ltd, Terumo Co., Ltd., Tosoh, Co., Ltd., and Thyas, Co., Ltd., (© 2021 The Authors.)

In this chapter, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from non-T cells, followed by the differentiation of the T-cell lineage. Derivation of T cells from PSCs involves three steps: induction of PSCs to hematopoietic progenitor cells (HPCs), differentiation of HPCs into progenitor T cells, and maturation of progenitor T cells into mature T cells (CD8 single-positive (SP) or CD4 SP).

In this chapter, we describe a protocol for hematopoietic differentiation of nonhuman primate (NHP)-induced pluripotent stem cells (iPSCs) derived from T cells and generation of T cells. Derivation of T cells from PSCs involves three steps: induction of PSCs to hematopoietic progenitor cells (HPCs), differentiation of HPCs into progenitor T cells, and maturation of progenitor T cells into mature T cells, in particular CD8 single-positive (SP) T cells.

Highly active antiretroviral therapy (HAART) has markedly prolonged the prognosis of HIV-1 patients. However, lifelong dependency on HAART is a continuing challenge, and an effective therapeutic is much desired. Recently, introduction of short hairpin RNA (shRNA) targeting the HIV-1 promoter was found to suppress HIV-1 replication via transcriptional gene silencing (TGS). The technology is expected to be applied with hemato-lymphopoietic cell transplantation of HIV patients to suppress HIV transcription in transplanted hemato-lymphopoietic cells. Combination of the TGS technology with new cell transplantation strategy with induced pluripotent stem cell (iPSC)-derived hemato-lymphopoietic cells might contribute to new gene therapy in the HIV field. In this study, we evaluated iPSC-derived macrophage functions and feasibility of TGS technology in macrophages. Human iPSCs were transduced with shRNAs targeting the HIV-1 promoter region (shPromA) by using a lentiviral vector. The shPromA-transfected iPSCs were successfully differentiated into functional macrophages, and they exhibited strong protection against HIV-1 replication with alteration in the histone structure of the HIV-1 promoter region to induce heterochromatin formation. These results indicated that iPS-derived macrophage is a useful tool to investigate HIV infection and protection, and that the TGS technology targeting the HIV promoter is a potential candidate of new gene therapy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Johne disease is ruminant chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). The domestic animals infected with this pathogen present severe weight loss due to chronic diarrhea and a reduction in lactation yield. These result in enormous economic loss since the affected animals are subsequently subject to artificial selections and disinfection of the environment are absolutely necessary. Furthermore, MAP has been suspected to have pathological relationship to Crohn's disease, human chronic granulomatous enteritis. The bacterium grows slower on solid culture and its colony becomes visible after two months of culture. In Japan, there has been almost no investigation on pasteurization temperature of commercial milk using MAP. It comes from the fact that the growth rate of MAP is very slow and that MAP is a related species to Mycobacterium tuberculosis, which pasteurization condition has been well defined. The studies on the pasteurization conditions of commercial milk have been mainly targeted to reduce the risk of infection to Coxiella and Mycobacterium tuberculosis. However, there has been a concern about the possibility that MAP is remained in pasteurized milk because MAPs form an aggregate and the bacterium at its center may not receive enough heat to get pasteurized. From these reasons, the present study aims to investigate validity of the current pasteurization conditions of commercial milk by implementing experimental pasteurization at various pasteurization temperatures using milk experimentally infected with MAP, and to clarify if MAP is eliminated at these temperatures in order to achieve smooth enforcement of the current ministry order. We conducted plant pasteurization experiment at four pasteurization conditions (high temperature, short time (HTST); 82, 77, 72 degrees C for 15 seconds and low temperature, long time (LTLT); 63 degrees C for 30 minutes) using two MAP strains, ATCC19698 and OKY-20. In conclusion, there appeared no colony of the two MAP strains formed from the milk pasteurized at the four pasteurization conditions examined.