Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes.

Deletion of master regulators of the B cell lineage reprograms B cells into T cells. Here we found that the transcription factor Hoxb5, which is expressed in uncommitted hematopoietic progenitor cells but is not present in cells committed to the B cell or T cell lineage, was able to reprogram pro-pre-B cells into functional early T cell lineage progenitors. This reprogramming started in the bone marrow and was completed in the thymus and gave rise to T lymphocytes with transcriptomes, hierarchical differentiation, tissue distribution and immunological functions that closely resembled those of their natural counterparts. Hoxb5 repressed B cell 'master genes', activated regulators of T cells and regulated crucial chromatin modifiers in pro-pre-B cells and ultimately drove the B cell fate-to-T cell fate conversion. Our results provide a de novo paradigm for the generation of functional T cells through reprogramming in vivo.

Publisher Correction: Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes.

In the version of this article initially published, some identification of the supplementary information was incorrect. The items originally called Supplementary Tables 1, 2, 3, 4 and 5 should be Source Data Figures 1, 2, 4, 5 and 7, respectively; those originally called Supplementary Tables 6, 7 and 8 should be Supplementary Tables 1, 2 and 3, respectively; and those originally called Source Data Figures 1, 2, 4, 5 and 7 should be Supplementary Tables 4, 5, 6, 7 and 8, respectively. The errors have been corrected in the HTML version of the article.

Loss of Nupr1 promotes engraftment by tuning the quiescence threshold of hematopoietic stem cell repository via regulating p53-checkpoint pathway.

Hematopoietic stem cells (HSCs) are dominantly quiescent under homeostasis, which is a key mechanism of maintaining the HSC pool for life-long hematopoiesis. Dormant HSCs poise to be immediately activated on urgent conditions and can return to quiescence after regaining homeostasis. To date, the molecular networks of regulating the threshold of HSC dormancy, if exist, remain largely unknown. Here, we unveiled that deletion of Nupr1, a gene preferentially expressed in HSCs, activated the quiescence HSCs under homeostatic status, which conferred engraftment competitive advantage on HSCs without compromising their stemness and multi-lineage differentiation abilities in serial transplantation settings. Following an expansion protocol, the Nupr1-/- HSCs proliferate more robustly than their wild type counterparts in vitro. Nupr1 inhibits the expression of p53 and the rescue of which offsets the engraftment advantage. Our data unveil the de novo role of Nupr1 as an HSC quiescence-regulator, which provides insights into accelerating the engraftment efficacy of HSC transplantation by targeting the HSC quiescence-controlling network.

Transcription factor Hoxb5 reveals the unidirectional hierarchy of hematopoietic stem cell pool.

Hoxb5 exhibits preferential expression in hematopoietic stem cells (HSCs) and uniquely marks the long-term HSCs (LT-HSCs). Previous studies have demonstrated the remarkable capability of Hoxb5 to alter cell fates when enforced expression in blood progenitors, such as B cell progenitors and multipotent progenitors. Additionally, Hoxb5 deficiency does not hinder the generation of LT-HSCs. However, the specific impact of Hoxb5 deletion on LT-HSCs has remained unexplored. To address this, we developed a conditional Hoxb5 knockout-reporter mouse model, wherein Hoxb5 was knock out by the Vav-cre recombinase, and the endogenous Hoxb5 promoter drove the expression of the blue fluorescent protein (BFP). Our findings revealed that the primary recipients, who transplanted with HSCs indicating Hoxb5 deficiency by the presence of BFP (BFP-positive HSCs), exhibited comparable levels of donor chimerism and lineage chimerism to recipients transplanted with HSCs that spontaneously did not express Hoxb5 and thus lacked BFP expression (BFP-negative HSCs). However, during the secondary transplantation, recipients receiving total bone marrow (BM) from the primary recipients with BFP-positive HSCs showed significantly higher levels of donor chimerism and more robust multi-lineage chimerism compared to those receiving total BM from the primary recipients with BFP-negative HSCs. Our results indicate that deleting Hoxb5 in LT-HSCs transiently influences their lineage differentiation bias without compromising their long-term self-renewal capacity. These findings highlight the primary role of Hoxb5 in regulating lineage commitment decisions in LT-HSCs, while emphasizing that its presence is not indispensable for the maintenance of long-term self-renewal capacity.

Mesothelin CAR-engineered NK cells derived from human embryonic stem cells suppress the progression of human ovarian cancer in animals.

CAR-NK cell therapy does not require HLA matching and has minimal side effects. However, traditional methods of engineering CARs into human tissue-derived NK cells exhibit heterogeneity, low transduction efficiency, and high manufacturing costs. Here, we provide a reliable approach for generating large-scale and cryopreserved mesothelin (MSLN) CAR-NK cells from human embryonic stem cells (hESCs) as an alternative cell source. We first constructed MSLN CAR-expressing hESCs to reduce CAR engineering costs and subsequently differentiated these stem cells into MSLN CAR-NK cells via an efficient organoid induction system. The MSLN CAR-NK cells exhibit the typical expression patterns of activating receptors, inhibitory receptors, and effector molecules of NK cells. In the presence of tumour cells, the MSLN CAR-NK cells show increased secretion of IFN-γ and TNF-α, as well as elevated CD107a expression level compared with induced NK cells. We cryopreserved the MSLN CAR-NK cells in liquid nitrogen using a clinical-grade freezing medium (CS10) for more than 6 months to mimic an off-the-shelf CAR-NK cell product. The thawed MSLN CAR-NK cells immediately recovered after 48-72-h culture and effectively eliminated ovarian tumour cells, including human primary ovarian tumour cells from patients. The thawed MSLN CAR-NK cells efficiently suppressed ovarian tumour development in vivo and prolonged the survival of tumour-bearing mice. Our study provides insights into the clinical translation of hESC-derived MSLN CAR-NK cells as a promising off-the-shelf cell product.

Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti-tumour activity.

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is emerging as a promising cancer treatment, with notable safety and source diversity benefits over CAR-T cells. This study focused on optimizing CAR constructs for NK cells to maximize their therapeutic potential. We designed seven CD19 CAR constructs and expressed them in NK cells using a retroviral system, assessing their tumour-killing efficacy and persistence. Results showed all constructs enhanced tumour-killing and prolonged survival in tumour-bearing mice. In particular, CAR1 (CD8 TMD-CD3ζ SD)-NK cells showed superior efficacy in treating tumour-bearing animals and exhibited enhanced persistence when combined with OX40 co-stimulatory domain. Of note, CAR1-NK cells were most effective at lower effector-to-target ratios, while CAR4 (CD8 TMD-OX40 CD- FcεRIγ SD) compromised NK cell expansion ability. Superior survival rates were noted in mice treated with CAR1-, CAR2 (CD8 TMD- FcεRIγ SD)-, CAR3 (CD8 TMD-OX40 CD- CD3ζ SD)- and CAR4-NK cells over those treated with CAR5 (CD28 TMD- FcεRIγ SD)-, CAR6 (CD8 TMD-4-1BB CD-CD3ζ 1-ITAM SD)- and CAR7 (CD8 TMD-OX40 CD-CD3ζ 1-ITAM SD)-NK cells, with CAR5-NK cells showing the weakest anti-tumour activity. Increased expression of exhaustion markers, especially in CAR7-NK cells, suggests that combining CAR-NK cells with immune checkpoint inhibitors might improve anti-tumour outcomes. These findings provide crucial insights for developing CAR-NK cell products for clinical applications.

Requirements for human natural killer cells.

'Requirements for Human Natural Killer Cells' is the latest set of guidelines on human NK cells in China, jointly drafted and agreed upon by experts from the Standards Committee of Chinese Society for Cell Biology. This standard specifies requirements for the human natural killer (NK) cells, including the technical requirements, test methods, test regulations, instructions for use, labeling requirements, packaging requirements, storage and transportation requirements, and waste disposal requirements of NK cells. This standard is applicable for the quality control of NK cells, derived from human tissues, or differentiated/transdifferentiated from stem cells. It was originally released by the Chinese Society for Cell Biology on 30 August, 2022. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human NK cells for applications.

Antigen-specific TCR-T cells from Rag2 gene-deleted pluripotent stem cells impede solid tumour growth in a mouse model.

The technology of adoptive transfer of T-cell receptor (TCR) engineered T cells is wildly investigated as it has the potential to treat solid cancers. However, the therapeutic application of TCR-T cells is hampered by the poor quality derived mainly from patients' peripheral blood, as well as heterogeneous TCRs caused by the mismatch between transgenic and endogenous TCRs. To improve the homogeneity, antigen-specificity and reduce possible autoreactivity, here we developed a technique to generate antigen-specific T cells from Rag2 gene-deleted pluripotent stem cells (PSCs) and further measured their anti-tumour efficacy. PSCs were first targeted with OT1 TCR into the Rag2 locus to prevent TCR rearrangement during T-cell development. The engineered PSCs were then differentiated through a two-step strategy, in vitro generation of haematopoietic progenitor cells, and in vivo development and maturation of TCR-T cells. Finally, the response to tumour cells was assessed in vitro and in vivo. The regenerated OT1-iT displayed monoclonal antigen-specific TCR expression, and phonotypic normalities in the spleen and lymph node tissues. Importantly, the OT1-iT cells eliminated tumour cells while releasing specific cytokines in vitro. Furthermore, adoptive transfer of OT1-iT cells suppresses solid tumour growth in tumour-bearing animals. Our study presents a novel and straightforward strategy for producing antigen-specific TCR-T cells in vivo from PSCs, allowing for allogeneic transplantation and therapy of solid tumours.

Prolonged generation of multi-lineage blood cells in wild-type animals from pluripotent stem cells.

Regenerating prolonged multi-lineage hematopoiesis from pluripotent stem cells (PSCs), an unlimited cell source, is a crucial aim of regenerative hematology. In this study, we used a gene-edited PSC line and revealed that simultaneous expression of three transcription factors, Runx1, Hoxa9, and Hoxa10, drove the robust emergence of induced hematopoietic progenitor cells (iHPCs). The iHPCs engrafted successfully in wild-type animals and repopulated abundant and complete myeloid-, B-, and T-lineage mature cells. The generative multi-lineage hematopoiesis distributed normally in multiple organs, persisted over 6 months, and eventually declined over time with no leukemogenesis. Transcriptome characterization of generative myeloid, B, and T cells at the single-cell resolution further projected their identities to natural cell counterparts. Thus, we provide evidence that co-expression of exogenous Runx1, Hoxa9, and Hoxa10 simultaneously leads to long-term reconstitution of myeloid, B, and T lineages using PSC-derived iHPCs as the cell source.

Regeneration of immunocompetent B lymphopoiesis from pluripotent stem cells guided by transcription factors.

Regeneration of functional B lymphopoiesis from pluripotent stem cells (PSCs) is challenging, and reliable methods have not been developed. Here, we unveiled the guiding role of three essential factors, Lhx2, Hoxa9, and Runx1, the simultaneous expression of which preferentially drives B lineage fate commitment and in vivo B lymphopoiesis using PSCs as a cell source. In the presence of Lhx2, Hoxa9, and Runx1 expression, PSC-derived induced hematopoietic progenitors (iHPCs) immediately gave rise to pro/pre-B cells in recipient bone marrow, which were able to further differentiate into entire B cell lineages, including innate B-1a, B-1b, and marginal zone B cells, as well as adaptive follicular B cells. In particular, the regenerative B cells produced adaptive humoral immune responses, sustained antigen-specific antibody production, and formed immune memory in response to antigen challenges. The regenerative B cells showed natural B cell development patterns of immunoglobulin chain switching and hypermutation via cross-talk with host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study exhibits de novo evidence that B lymphopoiesis can be regenerated from PSCs via an HSC-independent approach, which provides insights into treating B cell-related deficiencies using PSCs as an unlimited cell resource.

Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-induced NK (iNK) cells are a source of off-the-shelf cell products for universal immune therapy. Conventional methods for iNK cell regeneration from hPSCs include embryoid body (EB) formation and feeder-based expansion steps, which are time-consuming and cause instability and high costs of manufacturing. Here, we develop an EB-free, organoid aggregate method for NK cell regeneration from hPSCs. In a short time-window of 27-day induction, millions of hPSC input can output over billions of iNK cells without the necessity of NK cell expansion feeders. The iNK cells highly express classical toxic granule proteins, apoptosis-inducing ligands, as well as abundant activating and inhibitory receptors. Functionally, the iNK cells eradicate human tumor cells via mechanisms of direct cytotoxicity, apoptosis, and antibody-dependent cellular cytotoxicity. This study provides a reliable scale-up method for regenerating human NK cells from hPSCs, which promotes the universal availability of NK cell products for immune therapy.

Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors.

Achievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells (PSCs) is a central aim in T cell regenerative medicine. To date, preferentially reconstituting T lymphopoiesis in vivo from PSCs remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial-to-hematopoietic transition and hematopoietic maturation stages in a PSC differentiation scheme (iR9-PSC) in vitro induced preferential generation of engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSCs, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The induced T cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαß repertoire. The regenerative T lymphopoiesis restored immune surveillance in immunodeficient mice. Furthermore, gene-edited iR9-PSCs produced tumor-specific T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T cells from the unlimited and editable PSC source.

Mesenchymal stem cells suppress leukemia via macrophage-mediated functional restoration of bone marrow microenvironment.

Bone marrow (BM) mesenchymal stem cells (MSCs) are critical components of the BM microenvironment and play an essential role in supporting hematopoiesis. Dysfunction of MSCs is associated with the impaired BM microenvironment that promotes leukemia development. However, whether and how restoration of the impaired BM microenvironment can inhibit leukemia development remain unknown. Using an established leukemia model and the RNA-Seq analysis, we discovered functional degeneration of MSCs during leukemia progression. Importantly, intra-BM instead of systemic transfusion of donor healthy MSCs restored the BM microenvironment, demonstrated by functional recovery of host MSCs, improvement of thrombopoiesis, and rebalance of myelopoiesis. Consequently, intra-BM MSC treatment reduced tumor burden and prolonged survival of the leukemia-bearing mice. Mechanistically, donor MSC treatment restored the function of host MSCs and reprogrammed host macrophages into arginase 1 positive phenotype with tissue-repair features. Transfusion of MSC-reprogrammed macrophages largely recapitulated the therapeutic effects of MSCs. Taken together, our study reveals that donor MSCs reprogram host macrophages to restore the BM microenvironment and inhibit leukemia development.

Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition.

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

Trim27 confers myeloid hematopoiesis competitiveness by up-regulating myeloid master genes.

Trim27 (Zinc finger protein RFP) is a potential regulator of hematopoietic stem cells (HSC), yet its role in hematopoiesis remains elusive. Here, we investigated the roles of Trim27 in hematopoiesis by enforcing its expression in mouse and human hematopoietic stem and progenitor cells (HSPC). Ectopic expression of Trim27 in mouse fetal liver (FL) HSPC confers repopulating advantage with myeloid dominance. However, the number of HSC from Trim27 group was comparable with empty vector control group, indicating that overexpression of Trim27 unlikely expanded HSC. Transcriptome analysis of Trim27-overexpressing myeloid progenitor cells (MP) indicated that Trim27 up-regulated essential regulators of myelopoiesis, including Spi1 and Cebpg, up-regulated myeloid proliferation-related signaling genes Nras, Runx1, and Cbfb, up-regulated JAK/STAT signaling inhibitors Socs2, Socs3, and Cish, and up-regulated myeloid maturation-related genes Adam8 and Dek. Moreover, the myeloproliferative advantage caused by overexpressing Trim27/TRIM27 is conserved between mouse and human hematopoiesis. To our knowledge, this is the first study showing that Trim27 confers competitive hematopoiesis by promoting myeloid bias differentiation of HSPC, but not by expanding HSC.

Pyrimidoindole derivative UM171 enhances derivation of hematopoietic progenitor cells from human pluripotent stem cells.

In the field of hematopoietic regeneration, deriving hematopoietic stem cells (HSCs) from pluripotent stem cells with engraftment potential is the central mission. Unstable hematopoietic differentiation protocol due to variation factors such as serums and feeder cells, remains a major technical issue impeding the screening of key factors for the derivation of HSCs. In combination with hematopoietic cytokines, UM171 has the capacity to facilitate the maintenance and expansion of human primary HSCs in vitro. Here, using a serum-free, feeder-free, and chemically defined induction protocol, we observed that UM171 enhanced hematopoietic derivation through the entire process of hematopoietic induction in vitro. UM171 facilitated generation of robust CD34+CD45+ derivatives that formed more and larger sized CFU-GM as well as larger sized CFU-Mix. In our protocol, the derived hematopoietic progenitors failed to engraft in NOG mice, indicating the absence of long-term HSC from these progenitors. In combination with other factors and protocols, UM171 might be broadly used for hematopoietic derivation from human pluripotent stem cells in vitro.

Oncogenic NRAS hyper-activates multiple pathways in human cord blood stem/progenitor cells and promotes myelomonocytic proliferation in vivo.

Oncogenic NRAS mutations are prevalent in human myeloid leukemia, especially in chronic myelomonocytic leukemia (CMML). NrasG12D mutation at its endogenous locus in murine hematopoietic stem cells (HSCs) leads to CMML and acute T-cell lymphoblastic lymphoma/leukemia in a dose-dependent manner. Hyper-activated MAPK and STAT5 pathways by oncogenic Nras contribute to the leukemogenesis in vivo. However, it is unclear whether these conclusions remain true in a more human relevant model. Here, we evaluated the effects of NRASG12D on human hematopoiesis and leukemogenesis in vitro and in vivo by ectopically expressing NRASG12D in human cord blood stem/progenitor cells (hSPCs). NRASG12D expressing hSPCs preferentially differentiated into myelomonocytic lineage cells, demonstrated by forming more colony forming unit-macrophages than control hSPCs in cultures. Transplantation of NRASG12D expressing hSPCs initiated myeloproliferative neoplasm in immune deficiency mice. All the recipient mice died of myeloid tumor burdens in spleens and bone marrows and none developed lymphoid leukemia. Phospho-flow analysis of CD34(+) CD38(-) hSPCs confirmed that NRASG12D hyper-activated MAPK, AKT and STAT5 pathways. Our study provides the strong evidence that NRASG12D mutation mainly targets monocytic lineage cells and leads to myelomonocytic proliferation in vivo in a highly human relevant context.

OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo.

Generating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However, the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study, we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly, the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore, the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo.