Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood.

Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.

A target discovery pipeline identified ILT3 as a target for immunotherapy of multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.

Lipid droplet-associated lncRNA LIPTER preserves cardiac lipid metabolism.

Lipid droplets (LDs) are cellular organelles critical for lipid homeostasis, with intramyocyte LD accumulation implicated in metabolic disorder-associated heart diseases. Here we identify a human long non-coding RNA, Lipid-Droplet Transporter (LIPTER), essential for LD transport in human cardiomyocytes. LIPTER binds phosphatidic acid and phosphatidylinositol 4-phosphate on LD surface membranes and the MYH10 protein, connecting LDs to the MYH10-ACTIN cytoskeleton and facilitating LD transport. LIPTER and MYH10 deficiencies impair LD trafficking, mitochondrial function and survival of human induced pluripotent stem cell-derived cardiomyocytes. Conditional Myh10 deletion in mouse cardiomyocytes leads to LD accumulation, reduced fatty acid oxidation and compromised cardiac function. We identify NKX2.5 as the primary regulator of cardiomyocyte-specific LIPTER transcription. Notably, LIPTER transgenic expression mitigates cardiac lipotoxicity, preserves cardiac function and alleviates cardiomyopathies in high-fat-diet-fed and Leprdb/db mice. Our findings unveil a molecular connector role of LIPTER in intramyocyte LD transport, crucial for lipid metabolism of the human heart, and hold significant clinical implications for treating metabolic syndrome-associated heart diseases.

Experimental Models of Mouse and Human Hematopoietic Stem Cell Transplantation.

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time points post-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6 × BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.

Transient regulation of RNA methylation in human hematopoietic stem cells promotes their homing and engraftment.

Enhancing the efficiency of hematopoietic stem cell (HSC) homing and engraftment is critical for cord blood (CB) hematopoietic cell transplantation (HCT). Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of mRNAs that are critical for stem cell function by influencing their stability. Here, we demonstrate that inhibition of RNA decay by regulation of RNA methylation, enhances the expression of the homing receptor chemokine C-X-C receptor-4 (CXCR4) in HSCs. We show that YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), a m6A reader and FTO α-ketoglutarate dependent dioxygenase (FTO), a m6A eraser play an opposite role in this process. Through screening, we identified several FDA-approved compounds that regulate the expression of YTHDF2 and FTO in CB CD34+ cells. We show that transient downregulation of YTHDF2 or activation of FTO by using these compounds inhibits CXCR4 decay in CB HSCs and promotes their homing and engraftment. Our results demonstrate a novel regulation strategy to enhance the function of CB HSCs and provide a translational approach to enhance the clinical efficacy of HCT.

Ex Vivo Expansion and Homing of Human Cord Blood Hematopoietic Stem Cells.

Cord blood (CB) has been proven to be an alternative source of haematopoietic stem cells (HSCs) for clinical transplantation and has multiple advantages, including but not limited to greater HLA compatibility, lower incidence of graft-versus-host disease (GvHD), higher survival rates and lower relapse rates among patients with minimal residual disease. However, the limited number of HSCs in a single CB unit limits the wider use of CB in clinical treatment. Many efforts have been made to enhance the efficacy of CB HSC transplantation, particularly by ex vivo expansion or enhancing the homing efficiency of HSCs. In this chapter, we will document the major advances regarding human HSC ex vivo expansion and homing and will also discuss the possibility of clinical translation of such laboratory work.

Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy.

Neutrophils, the most abundant white blood cells in circulation, are closely related to cancer development and progression. Healthy primary neutrophils present potent cytotoxicity against various cancer cell lines through direct contact and via generation of reactive oxygen species. However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with chimeric antigen receptors (CARs) to enhance their antitumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells with synthetic CARs and differentiated them into functional neutrophils by implementing a chemically defined platform. The resulting CAR neutrophils present superior and specific cytotoxicity against tumor cells both in vitro and in vivo. Collectively, we established a robust platform for massive production of CAR neutrophils, paving the way to myeloid cell-based therapeutic strategies that would boost current cancer-treatment approaches.

Physioxia-induced downregulation of Tet2 in hematopoietic stem cells contributes to enhanced self-renewal.

Hematopoietic stem cells (HSCs) manifest impaired recovery and self-renewal with a concomitant increase in differentiation when exposed to ambient air as opposed to physioxia. Mechanism(s) behind this distinction are poorly understood but have the potential to improve stem cell transplantation. Single-cell RNA sequencing of HSCs in physioxia revealed upregulation of HSC self-renewal genes and downregulation of genes involved in inflammatory pathways and HSC differentiation. HSCs under physioxia also exhibited downregulation of the epigenetic modifier Tet2. Tet2 is α-ketoglutarate, iron- and oxygen-dependent dioxygenase that converts 5-methylcytosine to 5-hydroxymethylcytosine, thereby promoting active transcription. We evaluated whether loss of Tet2 affects the number and function of HSCs and hematopoietic progenitor cells (HPCs) under physioxia and ambient air. In contrast to wild-type HSCs (WT HSCs), a complete nonresponsiveness of Tet2-/- HSCs and HPCs to changes in oxygen tension was observed. Unlike WT HSCs, Tet2-/- HSCs and HPCs exhibited similar numbers and function in either physioxia or ambient air. The lack of response to changes in oxygen tension in Tet2-/- HSCs was associated with similar changes in self-renewal and quiescence genes among WT HSC-physioxia, Tet2-/- HSC-physioxia and Tet2-/- HSC-air. We define a novel molecular program involving Tet2 in regulating HSCs under physioxia.

Expansion of Human Megakaryocyte-Lineage Progeny via Aryl Hydrocarbon Receptor Antagonism with CH223191.

Aryl hydrocarbon receptor (AhR) antagonism is known to expand human hematopoietic stem cells (HSCs). However, its regulatory effect on the lineage-skewed differentiation of HSCs has not been sufficiently studied. Here, we investigate the effect of the AhR-selective antagonist CH223191 on the regulation of HSC differentiation. Consistent with the well-known effects of AhR antagonists, CH223191 treatment increase phenotypic HSCs (Lin-CD34 + CD38-CD90 + CD45RA-) and preserves their functionality. On the other hand, CH223191 leads to an overall expansion of megakaryocyte (MK)-lineage populations, such as MK progenitors (MKps, CD34 + CD41 +), immature MKs (CD41 + CD42b-), and mature MKs (CD41 + CD42b +), and it also activates MK/platelet-associated signaling pathways. Furthermore, CH223191 expands MKps, mature MKs, and p-selectin (CD62p)-positive platelet-like particles in immune thrombocytopenia (ITP) patient bone marrow (BM). These results highlight the numerical expansion of human MK-lineage progeny through AhR antagonism with CH223191. This approach using CH2231291 may be applicable in the development of auxiliary treatment regimens for patients with abnormal thrombopoiesis.

Chemically-defined generation of human hemogenic endothelium and definitive hematopoietic progenitor cells.

Human hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFß inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.

Leukemia Inhibitory Factor Promotes Survival of Hematopoietic Progenitors Ex Vivo and Is Post-Translationally Regulated by DPP4.

Hematopoietic cells are regulated in part by extracellular cues from cytokines. Leukemia inhibitory factor (LIF) promotes survival, self-renewal, and pluripotency of mouse embryonic stem cells (mESC). While genetic deletion of LIF affects hematopoietic progenitor cells (HPCs), the direct effect of LIF protein exposure on HPC survival is not known. Furthermore, post-translational modifications (PTM) of LIF and their effects on its function have not been evaluated. We demonstrate that treatment with recombinant LIF preserves mouse and human HPC numbers in stressed conditions when growth factor addition is delayed ex vivo. We show that Lif is upregulated in response to irradiation-induced stress. We reveal novel PTM of LIF where it is cleaved twice by dipeptidyl peptidase 4 (DPP4) protease so that it loses its 4 N-terminal amino acids. This truncation of LIF down-modulates LIF's ability to preserve functional HPC numbers ex vivo following delayed growth factor addition. DPP4-truncated LIF blocks the ability of full-length LIF to preserve functional HPC numbers. This LIF role and its novel regulation by DPP4 have important implications for normal and stress hematopoiesis, as well as for other cellular contexts in which LIF and DPP4 are implicated.

CaMKK2 Knockout Bone Marrow Cells Collected/Processed in Low Oxygen (Physioxia) Suggests CaMKK2 as a Hematopoietic Stem to Progenitor Differentiation Fate Determinant.

Little is known about a regulatory role of CaMKK2 for hematopoietic stem (HSC) and progenitor (HPC) cell function. To assess this, we used Camkk2-/- and wild type (WT) control mouse bone marrow (BM) cells. BM cells were collected/processed and compared under hypoxia (3% oxygen; physioxia) vs. ambient air (~21% oxygen). Subjecting cells collected to ambient air, even for a few minutes, causes a stress that we termed Extra Physiological Shock/Stress (EPHOSS) that causes differentiation of HSCs and HPCs. We consider physioxia collection/processing a more relevant way to assess HSC/HPC numbers and function, as the cells remain in an oxygen tension closer physiologic conditions. Camkk2-/- cells collected/processed at 3% oxygen had positive and negative effects respectively on HSCs (by engraftment using competitive transplantation with congenic donor and competitor cells and lethally irradiated congenic recipient mice), and HPCs (by colony forming assays of CFU-GM, BFU-E, and CFU-GEMM) compared to WT cells processed in ambient air. Thus, with cells collected/processed under physioxia, and therefore never exposed and naïve to ambient air conditions, CaMKK2 not only appears to act as an HSC to HPC differentiation fate determinant, but as we found for other intracellular mediators, the Camkk-/- mouse BM cells were relatively resistant to effects of EPHOSS. This information is of potential use for modulation of WT BM HSCs and HPCs for future clinical advantage.

Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity.

Preclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O2. Here, we demonstrate that tumors collected, processed, and propagated at physiologic O2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.

Chromatin remodeling subunit BRM and valine regulate hematopoietic stem/progenitor cell function and self-renewal via intrinsic and extrinsic effects.

Little is known of hematopoietic stem (HSC) and progenitor (HPC) cell self-renewal. The role of Brahma (BRM), a chromatin remodeler, in HSC function is unknown. Bone marrow (BM) from Brm-/- mice manifested increased numbers of long- and short-term HSCs, GMPs, and increased numbers and cycling of functional HPCs. However, increased Brm-/- BM HSC numbers had decreased secondary and tertiary engraftment, suggesting BRM enhances HSC self-renewal. Valine was elevated in lineage negative Brm-/- BM cells, linking intracellular valine with Brm expression. Valine enhanced HPC colony formation, replating of human cord blood (CB) HPC-derived colonies, mouse BM and human CB HPC survival in vitro, and ex vivo expansion of normal mouse BM HSCs and HPCs. Valine increased oxygen consumption rates of WT cells. BRM through CD98 was linked to regulated import of branched chain amino acids, such as valine, in HPCs. Brm-/- LSK cells exhibited upregulated interferon response/cell cycle gene programs. Effects of BRM depletion are less apparent on isolated HSCs compared to HSCs in the presence of HPCs, suggesting cell extrinsic effects on HSCs. Thus, intracellular valine is regulated by BRM expression in HPCs, and the BRM/valine axis regulates HSC and HPC self-renewal, proliferation, and possibly differentiation fate decisions.

Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.

Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

CXCL15/Lungkine has suppressive activity on proliferation and expansion of multi-potential, erythroid, granulocyte and macrophage progenitors in S-phase specific manner.

Cytokines/chemokines regulate hematopoiesis, most having multiple cell actions. Numerous but not all chemokine family members act as negative regulators of hematopoietic progenitor cell (HPC) proliferation, but very little is known about such effects of the chemokine, CXCL15/Lungkine. We found that CXCL15/Lungkine-/- mice have greatly increased cycling of multi cytokine-stimulated bone marrow and spleen hematopoietic progenitor cells (HPCs: CFU-GM, BFU-E, and CFU-GEMM) and CXCL15 is expressed in many bone marrow progenitor and other cell types. This suggests that CXCL15/Lungkine acts as a negative regulator of the cell cycling of these HPCs in vivo. Recombinant murine CXCL15/Lungkine, decreased numbers of functional HPCs during cytokine-enhanced ex-vivo culture of lineage negative mouse bone marrow cells. Moreover, CXCL15/Lungkine, through S-Phase specific actions, was able to suppress in vitro colony formation of normal wildtype mouse bone marrow CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-GEMM. This clearly identifies the negative regulatory activity of CXCL15/Lungkine on proliferation of multiple types of mouse HPCs.

Bmi1 Regulates Wnt Signaling in Hematopoietic Stem and Progenitor Cells.

Polycomb group protein Bmi1 is essential for hematopoietic stem cell (HSC) self-renewal and terminal differentiation. However, its target genes in hematopoietic stem and progenitor cells are largely unknown. We performed gene expression profiling assays and found that genes of the Wnt signaling pathway are significantly elevated in Bmi1 null hematopoietic stem and progenitor cells (HSPCs). Bmi1 is associated with several genes of the Wnt signaling pathway in hematopoietic cells. Further, we found that Bmi1 represses Wnt gene expression in HSPCs. Importantly, loss of ß-catenin, which reduces Wnt activation, partially rescues the HSC self-renewal and differentiation defects seen in the Bmi1 null mice. Thus, we have identified Bmi1 as a novel regulator of Wnt signaling pathway in HSPCs. Given that Wnt signaling pathway plays an important role in hematopoiesis, our studies suggest that modulating Wnt signaling may hold potential for enhancing HSC self-renewal, thereby improving the outcomes of HSC transplantation.

All in for nuclear PFKP-induced CXCR4 metastasis: a T cell acute lymphoblastic leukemia prognostic marker.

Phosphofructokinase 1 (PFK1) is expressed in T cell acute lymphoblastic leukemia (T-ALL), where its upregulation is linked with cancer progression. While PFK1 functions in the glycolysis pathway within the cytoplasm, it is also present in the nucleus where it regulates gene transcription. In this issue of the JCI, Xueliang Gao, Shenghui Qin, et al. focus their mechanism-based investigation on the nucleocytoplasmic shuttling aspect of the PFK1 platelet isoform, PFKP. Functional nuclear export and localization sequences stimulated CXC chemokine receptor type 4 (CXCR4) expression to promote T-ALL invasion that involved cyclin D3/CDK6, c-Myc, and importin-9. Since the presence of nuclear PFKP is associated with poor survival in T-ALL, nuclear PFKP-induced CXCR4 expression might serve as a prognostic marker for T-ALL. More promising, though, are the mechanistic insights suggesting that approaches to dampening metastatic migration may have application to benefit patients with T-ALL.