This work established a robust method that successfully generated induced MKs (iMKs) from human cord blood–derived CD3+ T cells, which is a more abundant source and highlighted an optimized chemical reprogramming strategy that enables efficient conversion of T cells to MKs.
Abstract
The generation of megakaryocytes (MKs) from human somatic cells through chemical reprogramming represents a promising strategy for developing alternative platelet sources. Building on our prior chemical reprogramming protocol for converting erythroblasts to MKs, we established a robust method that successfully generated induced MKs (iMKs) from human cord blood–derived CD3+ T cells, which is a more abundant source. This method used a five–small molecule cocktail containing a reprogramming booster, AZD4205, to promote erasure of T cell identity and facilitate fate transition toward MKs. T cell–derived iMKs exhibited characteristic MK cellular and molecular signatures, demonstrating the capacity to produce proplatelets and release functional platelets. Single-cell RNA sequencing further revealed that iMKs were heterogeneous with distinct functional profiles, including cycling, immune, and thrombopoiesis-biased MKs. Our findings highlight an optimized chemical reprogramming strategy that enables efficient conversion of T cells to MKs, providing a practical and convenient approach to generating clinically relevant MKs and platelets.
Summary Direct reprogramming of human fibroblasts into hematopoietic stem cells (HSCs) offers a promising strategy for generating autologous cells to treat blood and immune disorders. Current protocols are limited by low efficiency and insufficient tools for evaluating reprogramming outcomes. Although functional assays are the standard for confirming cell identity, they require fully reprogrammed cells, limiting their utility during protocol development. To address this, we assembled a single-cell transcriptomic reference atlas of hematopoietic reprogramming and tested an algorithmically predicted transcription factor recipe for HSC induction. Long-read single-cell RNA sequencing of CD34+ reprogrammed cells revealed progressive loss of fibroblast identity alongside induction of early hematopoietic and endothelial programs, with reference-atlas benchmarking placing reprogrammed cells in an intermediate transcriptomic state between fibroblasts, endothelial cells, and HSCs. Isoform-level analysis further revealed transcriptional remodeling not captured by gene-level analyses. This experimental-computational framework offers a generalizable strategy for characterizing partially reprogrammed states and guiding optimization of reprogramming protocols.
Human pluripotent stem cells (hPSCs) are a promising cell source for producing T cells for regenerative medicine and immunotherapy. However, it is challenging to develop a scalable suspension culture system for generating functional T lymphocytes from hPSCs. Here, we developed a Matrigel-based artificial thymic organoid (Gel-ATO) system by encapsulating hPSC-derived induced hematopoietic progenitor cells (iHPCs) with MS5-hDLL4 stromal cells. The resulting Gel-ATOs produced functional iT cells capable of antigen-specific cytotoxicity in vitro and tumor suppression in vivo. Building on this system, we further established a rotating suspension culture to robustly generate iT lymphocytes with nearly 20-fold enhancement of iT cells production compared to stationary suspension culture. Importantly, these rotating suspension culture-derived iT cells were functional, exhibiting cytokine secretion, proliferation, and cytotoxicity. By addressing the dual requirements of scalability and function, our work paves the way for future clinical production of T cells for T cell-based cell therapy.
This review summarizes research with a focus on clinical translation of iPSC-derived immune cells, as well as highlights continued challenges and prospects of this field.
Luisjesus S. Cruz, Alejandro R. Castañeda, Dan S. Kaufman· Stem Cells· 0 citations
Key applications of iPSC technology in hemato-oncology are summarized, its major advantages and current limitations are discussed, and emerging directions are highlighted, including scalable iPSC-derived blood cell therapies for inherited and acquired bone marrow failure syndromes and leukemia.
Ivan Tesakov, M. Nasri, M. Klimiankou et al.· Frontiers in Immunology· 0 citations
These findings establish IRAK4 as a regulator that constrains cellular plasticity potentially by coordinating chromatin accessibility and cell cycle dynamics, which may facilitate the establishment of multi-lineage states.
Chuanshu Huang, Xiao-Yun Han, Tao Wang et al.· EMBO Reports· 0 citations
The generation of patient-specific induced pluripotent stem cells (iPSCs) from amniotic fluid cells (AFCs) carrying defined chromosomal aneuploidies provides a powerful platform for modeling genetic disorders. However, establishing a reliable and reproducible reprogramming pipeline for aneuploid AFCs remains technically challenging due to the intrinsic genomic instability and variable proliferative capacity of these cells. Here, we present a comprehensive, non-integrating method for generating aneuploid human iPSCs from primary AFCs using episomal plasmid electroporation. This protocol details the complete workflow, encompassing cell thawing and expansion with a gradual media adaptation strategy, optimized plasmid delivery via electroporation system, sequential post-electroporation culture with mesenchymal-to-epithelial transition (MET)-directed media changes, and mechanical colony picking based on defined morphological criteria. We further describe validation procedures, including immunofluorescence staining for core pluripotency markers, G-banding karyotype analysis to confirm aneuploid karyotype maintenance, and PCR-based episomal vector clearance verification. This feeder-free, integration-free protocol yields aneuploid iPSC lines suitable for disease modeling, drug screening, and studies of chromosome biology.
Unknown authors· Journal of Visualized Experi...· 0 citations
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