A Feeder-Free Method To Generate Functional Cytotoxic T Cells In Vitro

INTRODUCTION Human T lymphocytes (T cells) are a key component of the immune system, expressing the CD3–T cell receptor (TCR) complex, which enables antigen recognition via specic MHC-antigen interactions. Among T cell subpopulations, cytotoxic T lymphocytes can eliminate target cells—a feature harnessed in CAR-T and TCR-T cell therapies for cancers, autoimmune disorders, and other diseases. However, these approaches face challenges such as mispairing between endogenous and introduced TCRs, which can reduce ecacy and trigger severe immune responses. Human induced pluripotent stem cells (iPSCs) oer a promising solution, allowing ecient gene editing and scalable production of genetically dened, TCR-specic, o-the-shelf T cells for clinical use. In this study, we present a feeder-free in vitro method for dierentiating human iPSCs into CD8+ single-positive T cells, providing a reproducible, clinically relevant platform for immunotherapy. Lisa Chou2 , Xing Zhang1 , Zhaolu Yan1 , Na Zhang1 , Kun Shi1 , Yuhui Cao1 , Haonan Li1 , Tianfu Zhang1 , Yueh-chun Hsieh1 1 ACROBiosystems, Beijing, China ; 2 ACROBiosystems, Newark, DE USA CD8+ Single Positive T Cell Differentiation from Human Induced Pluripotent Stem Cell with a Feeder-Free System in vitro Human T cells originate from CD34+ CD45+ hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and migrate to the thymus, where NOTCH signaling drives T-lineage commitment. Early progenitors express CD7 and CD5 and continue to dierentiation into CD4+ CD8+ double-positive T cells (DP-T). During the DP stage, TCR gene rearrangement occurs, followed by positive selection. Cells recognizing self-MHC mature into CD4+ or CD8+ single-positive T cells (SP-T). Negative selection eliminates autoreactive cells, and mature T cells then enter peripheral tissues to support adaptive immunity. T Cell Differentiation in vivo CD34+ CD45+ HSPC CD4+ CD4+ CD8+ CD8+ CD5+ CD7+ Progenitor T cells CD4+ CD8+ T cells Positive Selection NOTCH Signaling Medulla Thymus Thymus Cortex Negative Selection Feeder-free T Cell Differentiation in vitro Using a combination of selected recombinant proteins and molecules, we recapitulate key steps of T cell development in vitro. A feeder-free system enables the dierentiation of human iPSCs into HSPCs, immature T cells, and ultimately CD8+ single-positive cytotoxic T cells. Traditional methods often rely on feeder cells, such as OP9-DL1, to induce T-lineage dierentiation. However, this poses challenges in clinical applications due to variability and safety concerns. In contrast, our feeder-free approach is more dened, reproducible, and suitable for scalable, clinical-grade T cell production. Step 1- Expansion of iPSCs Human iPSCs were expanded under feeder-free conditions using dened culture systems to maintain pluripotency and ensure genetic stability. Key quality control metrics at this stage included expansion rate, expression of stemness markers, and genomic integrity. Recombinant Laminin-521 was used as the extracellular matrix to support robust and consistent growth, eliminating the need for animal-derived basement membrane extracts. GMP-grade reagents were utilized to support potential clinical translation. Step 2 – iPSC to HSPC Differentiation iPSCs were transduced with a lentiviral vector encoding a TCR-expression sequence, followed by stepwise dierentiation into HSPCs. Cells were cultured for 14 days in a medium supplemented with BMP4, VEGF, bFGF, SCF, TPO, Flt-3L, and other hematopoietic cytokines. HSPC identity was conrmed by ow cytometry analysis of CD34 and CD45 expression. Step 3 - CD4+ CD8+ Double Positive T Cell Generation Dierentiated HSPCs were cultured for 21 days in a medium containing SCF, TPO, Flt-3L, IL-7, SDF-1, and additional factors, in wells coated with DLL4, VCAM-1, and bronectin to mimic the thymic microenvironment. Expression of CD5, CD7, CD4, and CD8 was analyzed to conrm the generation of double-positive immature T cells. Figure 4. HSPC generation. (A) Embryoid bodies formed by human induced pluripotent stem cells. Scale bar, 250 μm. (B, C) CD34+ CD45+ hematopoietic cell acquisition by verified FACS. Figure 5. CD4+ CD8+ DP-T cell detection by FACS. (A, B) The expression of progenitor T cell markers, CD5 and CD7. (C, D) The expression of T cell markers, CD4 and CD8, indicates the generation of DP-T cells. Day 1 Day 2 Day 3 Day 4 OCT4 SOX2 Nanog A B C Solutions: Laminin 521 Laminin 511 Vitronectin FGF basic TGF-β Mogengel BME Solutions: BMP4 FGF-basic SCF TPO Flt-3L + Isotypes + Antibodies 250um A B C iPSC HSPC DP-T SP-T BMP4, VEGF, bFGF, SCF, TPO, Flt3L SCF, TPO, Flt3L, IL7, SDF1α OKT3, IL2, IL7, DLL4, Fibronectin VEGF Solutions: DLL4 VCAM1 Fibronectin SCF TPO Flt-3L IL-7 SDF1α Solutions: IL2 IL7 OKT-3 CONCLUSION Step 4 - CD8+ Single Positive T Cell generation To promote maturation, DP-T cells were further cultured in the presence of IL-2, IL-7, and other cytokines for 7 days. During the initial 3 days, anti-CD3 antibody (OKT3) was added to stimulate TCR signaling. Following induction, a loss of CD4 expression and an increase in CD8 single-positive cells was observed, indicating successful T cell maturation. iPSC-derived T cells oer a valuable and scalable source for cell therapy. In this study, we established a dened, feeder-free protocol that mimics in vivo T cell development. By using specic cytokines and surface-bound proteins such as DLL4 and VCAM1, we eciently guided iPSC dierentiation through HSPCs to CD4+ CD8+ double-positive and nally CD8 single-positive T cells. This approach provides a robust platform for generating functional cytotoxic T cells in vitro, supporting future applications in immunotherapy and large-scale manufacturing. Figure 6. CD8+ SP-T cell detection by FACS. (A) The expression of T cell marker, CD3. (B, C) The expression of T cell markers, CD4 and CD8. DP-T cells have lost the expression of CD4 after 7 days’ induction and turned into CD8+ SP-T cells. Figure 3. iPSC expansion process. (A) Laminin 521 used as a coating substrate for the fast expansion of single cell human PSC across 4 days. (B) Stemness markers OCT4, SOX2 and NANOG are present after several passages, showing robust self-renewal of hPSCs. (C) Karyotype analysis after 10 passages reveals a normal karyotype in iPSCs. REFERENCES 1 Albayrak, E., & Kocabas, F. (2023). 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