ALBERT

Description: Two of the unsolved, important questions about epigenetics are: do histone arginine demethylases exist, and is the removal of histone tails by proteolysis a major epigenetic modification process? Here, we report that two orphan Jumonji C domain (JmjC)-containing proteins, JMJD5 and JMJD7, have divalent cation-dependent protease activities that preferentially cleave the tails of histones 2, 3, or 4 containing methylated arginines. After the initial specific cleavage, JMJD5 and JMJD7, acting as aminopeptidases, progressively digest the C-terminal products. JMJD5-deficient fibroblasts exhibit dramatically increased levels of methylated arginines and histones. Furthermore, depletion of JMJD7 in breast cancer cells greatly decreases cell proliferation. The protease activities of JMJD5 and JMJD7 represent a mechanism for removal of histone tails bearing methylated arginine residues and define a potential mechanism of transcription regulation.

Description: Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.

Description: Hematopoietic development during mammalian embryogenesis is comprised of a restricted primitive program of primitive erythroid, megakaryocytic, and macrophage lineages, and a definitive program of definitive erythroid, myeloid and lymphoid potential emerging from hematopoietic stem cell (HSC)-independent and dependent processes. Interestingly, progenitors of natural killer (NK) cells, but not B- or T-cells, have been found in the early human yolk sac, suggesting that NK cells may arise from HSC-independent sources. NK cells recognize and kill virally infected cells and tumor cells, making them a highly desirable cell-type for adoptive immunotherapy. To bypass donor-related issues, human pluripotent stem cell (hPSC)-derived NK cells offer the possibility of uniform activity in a renewable "off-the-shelf" cell product. As the differentiation of hPSCs recapitulates early developmental processes, we sought to characterize the developmental origin of hPSC-derived NK cells. Studies in mice indicate that NK cells in the adult are derived from hematopoietic stem cells (HSCs) that commit to a lymphoid differentiation pathway. However, while NK cells, like HSCs, have been found in the fetal liver, the developmental origin of the fetal NK cell lineage remains poorly understood. We have developed a stage-specific hPSC differentiation method that separates WNT-independent (WNTi) hematopoietic progenitors that harbor "primitive" hematopoietic potential from WNT-dependent (WNTd) erythro-myeloid-(T-)lymphoid "definitive" hematopoietic progenitors. Using this system, we find that CD34+ cells from both populations harbor NK cell potential. NK cells from hPSC WNTi progenitors (WNTi-NK cells) mature rapidly, are significantly more granular, and express very high levels of CD16 in comparison to their hPSC WNTd counterparts (WNTd-NK cells) and cord blood-derived NK (cbNK) cells. Further, WNTi CD34+ progenitors always gave rise to a granulocyte population alongside NK cells, suggesting they may be derived from a myeloid progenitor. Both WNTi-NK and WNTd-NK cells robustly respond to tumor targets, antibody-dependent cell-mediated cytotoxicity (ADCC), and PMA/ionomycin stimulation in comparison to cbNK cells. In all cases, WNTi-NK cells exhibited a strong bias for cytolytic degranulation over cytokine production, while WNTd-NK cells were biased for IFNg secretion. Similarly, WNTi-NK cells exhibit superior ADCC-mediated cell killing of Raji cells. We then turned to the well-characterized murine embryo to determine whether HSC-independent NK cell progenitors are developmentally conserved. Assessing NK cell potential via explant culture, we found that as early as E7.5, yolk sac explants give rise to NK cells, as well as primitive and definitive erythroid progenitors. Further, we find that murine E9.5 yolk sac kit+CD41+CD16/32+ erythro-myeloid progenitors (EMP) give rise to NK cells ex vivo. Similar to hPSC WNTi-NK cells, EMP-derived NK cells were larger and more granular, and emerged alongside a granulocyte population in explant culture. Thus, the murine yolk sac harbors unique NK cell potential, from a committed myeloid progenitor, prior to HSC emergence. Collectively, these studies suggest that ontological origin is an unexpectedly important consideration in the design of hPSC-derived NK cell-based therapeutics, and raise new questions regarding the potential of early hematopoietic progenitors in the mammalian embryo. Disclosures Fehniger: Celgene: Research Funding; Cyto-Sen Therapeutics: Consultancy; Altor BioScience: Research Funding; Affimed: Research Funding; NIH/NCI: Other: R01 CA205239, P50CA171963. Palis:Rubies Therapeutics: Consultancy.

Description: The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine, as it will provide an unlimited source of these cells for transplantation, and a unique platform for the study of both normal and disease hematopoietic processes. To reproducibly achieve this goal in all hPSC lines, we must first fully understand hematopoietic ontogeny. Understanding hematopoietic development is complicated by the existence of at least two distinct programs during development that are difficult to distinguish: a transient "primitive" extra-embryonic hematopoietic program that does not give rise to HSCs, and a "definitive" program that gives rise to HSCs and all hematopoietic lineages. We have recently developed a novel method to obtain and identify, from hPSCs, mesoderm harboring exclusively primitive or exclusively definitive hematopoietic potential, by the differential expression of CD235a within KDR+ mesoderm. With this, we were able to determine that stage-specific canonical Wnt signaling specified the definitive hematopoietic program, while simultaneously repressing the primitive hematopoietic program. Further, we have now found that definitive hematopoietic specification is also dependent on bFGF signaling during this same window of time. We then asked if this tractable system could help us understand the genetic regulation of definitive hematopoietic specification from hPSCs. We isolated Wnt-dependent KDR+CD235a- definitive hematopoietic mesoderm and Wnt-independent KDR+CD235a+ primitive hematopoietic mesoderm and performed whole-transcriptome gene expression analysis, which revealed strong CDX and HOX gene enrichment exclusively within KDR+CD235a- definitive hematopoietic mesoderm. Monitoring CDX expression over time in the differentiation cultures revealed that both CDX1 and CDX2 were expressed in a bFGF-independent manner, prior to Wnt-dependent definitive hematopoietic KDR+CD235a- mesoderm specification. In contrast, CDX4 was expressed exclusively within definitive hematopoietic KDR+CD235a- mesoderm in a Wnt- and bFGF-dependent manner. This expression pattern suggested that CDX4 expression is specific to definitive, but not primitive, hematopoietic specification. To determine whether CDX4 expression plays a role in definitive hematopoietic specification, we generated an inducible CDX4 expression hPSC line using the "safe-harbor" AAVS1 locus. We manipulated exogenous CDX4 expression during the same stage of the differentiation culture that Wnt signaling is critical for definitive hematopoietic specification. Interestingly, in the absence of Wnt stimulation, exogenous CDX4 expression caused a 〉90% repression in primitive hematopoietic potential. Critically, CDX4 expression during this same time conferred 10-fold greater definitive hematopoietic potential within CD34+CD73-CD184- hemogenic endothelium, giving rise to definitive erythroid-myeloid-lymphoid multilineage progenitors. This is consistent with CDX4 being the transcriptional effector of Wnt signaling during early hematopoietic specification within mesoderm. We next generated a CDX4 knockout hPSC line by CRISPR/Cas9, and a CDX4 knockdown hPSC line via shRNA expression from the AAVS1 locus. As expected, the absence of CDX4 expression did not reduce primitive hematopoietic potential in comparison to control lines. However, when Wnt signaling was stimulated to specify definitive hematopoiesis, hPSCs lacking CDX4 expression exhibited a 10-fold decrease in definitive CD34+CD73-CD184- hemogenic endothelium specification, indicating its expression is critical for definitive hematopoietic specification. Taken together, these findings indicate that CDX4 is the earliest identified transcription factor that is a critical regulator of human definitive hematopoietic specification, and provide a mechanistic basis for Wnt-mediated definitive hematopoietic specification from hPSCs. By understanding the genetic regulation of early definitive hematopoietic specification from hPSCs, we can now identify the additional signal pathways required for efficient HSC specification from hPSCs. Disclosures No relevant conflicts of interest to declare.

Description: Natural killer (NK) cells are innate immune cells that target and kill virally infected and malignant cells, making them an attractive target for adoptive immunotherapies. An alternative to donor-derived NK cells is the use of human pluripotent stem cell (hPSC)-derived NK cells, as a renewable "off the shelf" product. Previous studies have identified hPSC-derived NK cells as potently cytotoxic, compared to donor-derived NK cells. As the differentiation of hPSCs mimics early embryonic development, this raises the possibility that hPSC-derived NK cells are ontogenically distinct from adult NK cells. NK cells are present during embryonic hematopoiesis, but their ontogenic origins are poorly understood. NK cells are thought to arise from a common lymphoid progenitor (CLP), lying downstream of hematopoietic stem cells (HSCs), but evidence exists that NK cells may arise from HSC-independent progenitors as NK cells are found in the early murine fetal liver, and NK cell progenitors are found in the early human yolk sac (YS). In this study, we investigated the emergence of NK cells during murine and human embryonic hematopoietic development. During murine embryogenesis, overlapping HSC-independent waves of hematopoietic progenitors occur in the YS that give rise to hematopoietic cells prior to HSC emergence at E10.5. The "primitive" wave occurs at E7.5, followed by an "erythro-myeloid progenitor" (EMP) wave at E8.5. To study NK cell potential during murine YS hematopoiesis, we cultured total YS and sorted hematopoietic progenitors under NK cell promoting conditions. Strikingly, we found that the YS contains NK cell potential. Further, sorted E8.5 kit+CD41+CD16/32+ EMP progenitors, but not primitive hematopoietic progenitors, contain robust NK cell potential. EMP-derived NK (EMP-NK) cells were larger and more granular than adult CLP-derived NK cells. Additionally, NK cells from the E15.5 fetal liver were larger and more granular than NK cells from the adult spleen. Both EMP-NK cells and E15.5 fetal liver NK cells had a more robust degranulation response than their HSC-derived counterparts. Together, these data support the concept that EMP in the YS serve as an initial source of physiologically relevant, functional embryonic NK cells that are phenotypically and functionally distinct from adult NK cells. As hPSC-derived NK cells were described as potently cytotoxic, and we observed that murine HSC-independent NK cells robustly degranulate, we next asked whether NK cell development from hPSCs recapitulates that found in the murine embryo. We have demonstrated previously, using a stage-specific WNT signal manipulation approach that specifies ontogenically distinct hematopoietic progenitors, that hPSC-derived NK cell progenitors can be obtained from two distinct progenitors in vitro. In this study, we sought to better understand the development and function of these two NK cell populations. Stage-specific WNT inhibition (WNTi) during hPSC mesodermal patterning yielded extra-embryonic-like HOXA-/low CD34+ populations that possessed erythroid, myeloid and NK cell potential, but lacked T cell potential. The CD56+ NK cells in these cultures co-emerged with CD15+ granulocytes, indicating that these NK cells may arise from a committed myeloid progenitor. In contrast, HOXA+ CD34+ cells, obtained in a WNT-dependent (WNTd) manner, harbored erythro-myelo-lymphoid multi-lineage potential, including NK cell potential. Phenotypically, WNTi-NK cells were larger, more granular and more mature, compared to WNTd-NK and cord blood (CB)-derived NK cells, reminiscent of murine EMP-NK cells. Further, following multiple stimulation assays, WNTi-NK and WNTd-NK cells had different effector biases. WNTi-NK cells are biased for potent cytotoxic degranulation and exhibited superior cell killing in an ADCC assay. In contrast, WNTd-NK and CB-NK had an attenuated degranulation response, but robustly produced inflammatory cytokines. Finally, RNA-seq analysis demonstrated that WNTd-NK cells were most similar to CB-NK cells. Collectively, these studies identify for the first time that the murine EMP harbor NK cell potential, and these NK cells are functionally unique. These observations raise new questions regarding which ontogenic origin of NK cells should be used in future hPSC-derived adoptive immunotherapy strategies. Disclosures Fehniger: Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse). Palis:Rubius Therapeutics: Consultancy.

Description: Key Points CDX genes are differentially expressed in mesoderm harboring definitive hematopoietic potential in a WNT-dependent manner. CDX4 expression during mesoderm specification regulates human definitive hematopoietic specification.

Description: The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a− mesodermal population that gives rise to a clonally multipotent HOXA+ definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes and is functionally unresponsive to RA treatment. Thus, the specification of an RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNA-seq (scRNA-seq) analyses, we identified that 2 distinct KDR+CD235a− populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, KDR+CD235a−CXCR4− mesoderm expressed CYP26A1, an RA degrading enzyme, and harbored definitive hematopoietic potential within hPSC differentiation cultures in the absence of RA signaling, indicating the HE specified from CXCR4− mesoderm as RA-independent (RAi). In sharp contrast, KDR+CD235a−CXCR4+ mesoderm exclusively expressed ALDH1A2, the key enzyme in the synthesis of RA, but lacked hematopoietic potential under the same culture conditions. However, the stage-specific application of RA signaling to CXCR4+ mesoderm resulted in the robust specification of CD34+HOXA+ HE with definitive erythroid, myeloid, and lymphoid hematopoietic potential, establishing this HE as RA-dependent (RAd). Furthermore, while RAi HE entirely failed to persist following murine hematopoietic xenografts, RAd HE transiently persisted within the peripheral blood and bone marrow of murine hosts. To assess whether these functionally distinct hPSC mesodermal progenitors are physiologically relevant to human embryonic development, we integrated scRNA-seq datasets from the hPSC mesodermal cultures and a gastrulating human embryo. These analyses revealed that in vivo, distinct KDR+CXCR4−CYP26A1+ and KDR+CXCR4+ALDH1A2+ populations can be found at the stage of emergent mesoderm, following patterning of nascent mesoderm. Additional comparison to later stage human embryos demonstrated that RAd HE has a more fetal-like HOXA expression pattern than RAi HE. Scoring of single fetal HE cells against hPSC-derived HE revealed that while some early fetal HE cells were similar to RAi HE, the late fetal HE cells, which are hypothesized to give rise to HSCs, were more similar to RAd HE. Lastly, as HSC-competent HE is expected to express arterial genes, we found a subset of late fetal HE with this phenotype that were exclusively similar to RAd HE. Collectively, these data represent the first ever characterization of RA-dependent hPSC-derived definitive hematopoiesis and its mesodermal progenitor. Additionally, we provide evidence for in vivo mesodermal and HE correlates for both RAi and RAd hematopoietic programs within human embryos. This novel insight into human hematopoietic development will serve as an important tool for modeling development and ultimately provide the basis for de novo specification of HSCs. Disclosures No relevant conflicts of interest to declare.