Engineered T cells from induced pluripotent stem cells: from research towards clinical implementation.

Induced pluripotent stem cell (iPSC)-derived T (iT) cells represent a groundbreaking frontier in adoptive cell therapies with engineered T cells, poised to overcome pivotal limitations associated with conventional manufacturing methods. iPSCs offer an off-the-shelf source of therapeutic T cells with the potential for infinite expansion and straightforward genetic manipulation to ensure hypo-immunogenicity and introduce specific therapeutic functions, such as antigen specificity through a chimeric antigen receptor (CAR). Importantly, genetic engineering of iPSC offers the benefit of generating fully modified clonal lines that are amenable to rigorous safety assessments. Critical to harnessing the potential of iT cells is the development of a robust and clinically compatible production process. Current protocols for genetic engineering as well as differentiation protocols designed to mirror human hematopoiesis and T cell development, vary in efficiency and often contain non-compliant components, thereby rendering them unsuitable for clinical implementation. This comprehensive review centers on the remarkable progress made over the last decade in generating functional engineered T cells from iPSCs. Emphasis is placed on alignment with good manufacturing practice (GMP) standards, scalability, safety measures and quality controls, which constitute the fundamental prerequisites for clinical application. In conclusion, the focus on iPSC as a source promises standardized, scalable, clinically relevant, and potentially safer production of engineered T cells. This groundbreaking approach holds the potential to extend hope to a broader spectrum of patients and diseases, leading in a new era in adoptive T cell therapy.

Generation of an induced pluripotent stem cell line (MUSIi015-A) from a diabetic patient carrying mutations in ZYG11A (p.L475P) and GATA6 (p.E51K).

Two heterozygous mutations (p.L475P in ZYG11A and p.E51K in GATA6) were identified in a family with autosomal dominant diabetes. ZYG11A-p.L475P was proposed as a causative mutation because of the complete segregation with hyperglycemia and the proven pathogenic effect on beta-cell expansion. The modifying effect of GATA6-p.E51K was proposed owing to the earlier onset of the carriers. Herein, we establish a line of induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of a proband who carries both mutations using Sendai viral vectors. The generated iPSC line was characterized for pluripotency, chromosomal normality, and authentication.

Efficient Generation of iPSC-Derived Hematoendothelial Progenitors and Specification Toward T cell Lineage.

One of the major obstacles for adoptive cell transfer (ACT) of T cells is the loss of effector function and proliferative ability of isolated antigen-specific T cells after prolonged ex vivo expansion. To overcome this issue, induced pluripotent stem cells (iPSCs), which have unlimited proliferation and differentiation potential, can be used to generate a large number of antigen-specific T cells. Here, we describe an efficient differentiation protocol for the generation of cytotoxic CD8+ T cells from human T cell-derived iPSCs (T-iPSCs). The protocol consists of three main steps including differentiation of T-iPSCs toward hematoendothelial progenitors (HEPs), co-culture of HEPs with OP9-DL1 cells, and stimulation of T cell receptor (TCR) signaling to obtain CD8 single-positive (SP) T cells. This culture system is simple and efficient; therefore, will offer a powerful tool for studying T cell development and applications in adoptive immunotherapy.

Advances in Adoptive Cell Therapy Using Induced Pluripotent Stem Cell-Derived T Cells.

Adoptive cell therapy (ACT) using chimeric antigen receptor (CAR) T cells holds impressive clinical outcomes especially in patients who are refractory to other kinds of therapy. However, many challenges hinder its clinical applications. For example, patients who undergo chemotherapy usually have an insufficient number of autologous T cells due to lymphopenia. Long-term ex vivo expansion can result in T cell exhaustion, which reduces the effector function. There is also a batch-to-batch variation during the manufacturing process, making it difficult to standardize and validate the cell products. In addition, the process is labor-intensive and costly. Generation of universal off-the-shelf CAR T cells, which can be broadly given to any patient, prepared in advance and ready to use, would be ideal and more cost-effective. Human induced pluripotent stem cells (iPSCs) provide a renewable source of cells that can be genetically engineered and differentiated into immune cells with enhanced anti-tumor cytotoxicity. This review describes basic knowledge of T cell biology, applications in ACT, the use of iPSCs as a new source of T cells and current differentiation strategies used to generate T cells as well as recent advances in genome engineering to produce next-generation off-the-shelf T cells with improved effector functions. We also discuss challenges in the field and future perspectives toward the final universal off-the-shelf immunotherapeutic products.

Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. METHODS: Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-ß signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. RESULTS: Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-ß signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. CONCLUSION: This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

Generation of two induced pluripotent stem cell lines (MUSIi011-A and MUSIi011-B) from peripheral blood T lymphocytes of a healthy individual.

Activated T lymphocytes of a healthy individual were reprogrammed to induced pluripotent stem cells (iPSCs) using Sendai viral vectors. Two iPSC lines, MUSIi011-A and MUSIi011-B, were established and characterized for the expression of pluripotent markers. Both iPSC lines were able to differentiate into cells of three embryonic germ layers via embryoid body formation, exhibited normal karyotypes and were free of viral genome and transgenes at passage 15. These T lymphocyte-derived iPSCs (T-iPSCs) represent a useful starting cell source for developing next-generation immune cells such as chimeric antigen receptor (CAR)-engineered iPSC-derived T lymphocytes for the application in adoptive immunotherapy.

Induced pluripotent stem cell line MUSIi006-A derived from hair follicle keratinocytes as a non-invasive somatic cell source.

In this study, we used hair follicle keratinocytes for reprogramming. Collection of plucked hairs offers advantages over other somatic cells because no medical professional or operation room is required. Keratinocytes were isolated from plucked hairs of a 21-year-old healthy woman and characterized for the expression of cytokeratin 14 (CK14). Reprogramming of keratinocytes was performed using Sendai virus. Further characterization of the keratinocyte-derived iPSC line (designated as MUSIi006-A) confirmed that the cell line was pluripotent, free from Sendai viral genome and transgenes, and retained normal karyotype. Our method represents an easy, non-invasive and efficient approach for iPSC generation from hair samples.

Establishment of an integration-free induced pluripotent stem cell line (MUSIi005-A) from exfoliated renal epithelial cells.

Human induced pluripotent stem cells (iPSCs) were generated from exfoliated renal epithelial cells isolated from a urine sample of a 31-year-old healthy woman. Epithelial cells were characterized for the expression of E-cadherin and reprogrammed using non-integrating Sendai viral vectors. The urine-derived iPSC line (designated as MUSIi005-A) was karyotypically normal, expressed pluripotent markers, differentiated into cells of three embryonic germ layers, and showed no viral and transgene expressions at passage 29. Our protocol offers a non-invasive and efficient approach for iPSC generation from patients with genetic or acquired disorders.

Generation of a human induced pluripotent stem cell line (MUSIi001-A) from caesarean section scar fibroblasts using Sendai viral vectors.

We generated a human induced pluripotent stem cell (iPSC) line from caesarean section scar fibroblasts of a 33-year-old healthy woman using transgene-free Sendai viral vectors under feeder-free condition. The established iPSC line, designated as MUSIi001-A, exhibited a normal karyotype, expressed pluripotent markers, differentiated into cells of three embryonic germ layers. Further analyses showed that the Sendai viral genome was absent at passage 25. The MUSIi001-A line can serve as a control for studying developmental biology and phenotypic comparison with disease-specific iPSCs.