Human definitive hematopoietic specification from pluripotent stem cells is regulated by mesodermal expression of CDX4.

The generation of hematopoietic stem cells from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. Achieving this goal is complicated by our incomplete understanding of the mechanism regulating definitive hematopoietic specification. We used our stage-specific hPSC differentiation method to obtain and identify, via CD235a expression, mesoderm harboring exclusively primitive or definitive hematopoietic potential to understand the genetic regulation of definitive hematopoietic specification. Whole-transcriptome gene expression analyses on WNT-dependent KDR+CD235a- definitive hematopoietic mesoderm and WNT-independent KDR+CD235a+ primitive hematopoietic mesoderm revealed strong CDX gene expression within definitive hematopoietic mesoderm. Temporal expression analyses revealed that CDX4 was expressed exclusively within definitive hematopoietic KDR+CD235a- mesoderm in a WNT- and fibroblast growth factor-dependent manner. We found that exogenous CDX4 expression exclusively during mesoderm specification resulted in a >90% repression in primitive hematopoietic potential, but conferred fivefold greater definitive hematopoietic potential, similar to that observed following WNT stimulation. In contrast, CDX4 knockout hPSCs had intact primitive hematopoietic potential, but exhibited a fivefold decrease in multilineage definitive hematopoietic potential. Taken together, these findings indicate that CDX4 is a critical transcription factor in the regulation of human definitive hematopoietic specification, and provides a mechanistic basis for WNT-mediated definitive hematopoietic specification from hPSCs.

Mis-splicing of mitotic regulators sensitizes SF3B1-mutated human HSCs to CHK1 inhibition.

Splicing factor SF3B1 mutations are frequent somatic lesions in myeloid neoplasms that transform hematopoietic stem cells (HSCs) by inducing mis-splicing of target genes. However, the molecular and functional consequences of SF3B1 mutations in human HSCs remain unclear. Here, we identify the mis-splicing program in human HSCs as a targetable vulnerability by precise gene editing of SF3B1 K700E mutations in primary CD34+ cells. Mutant SF3B1 induced pervasive mis-splicing and reduced expression of genes regulating mitosis and genome maintenance leading to altered differentiation, delayed G2/M progression, and profound sensitivity to CHK1 inhibition (CHK1i). Mis-splicing or reduced expression of mitotic regulators BUBR1 and CDC27 delayed G2/M transit and promoted CHK1i sensitivity. Clinical CHK1i prexasertib selectively targeted SF3B1-mutant HSCs and abrogated engraftment in vivo. These findings identify mis-splicing of mitotic regulators in SF3B1-mutant HSCs as a targetable vulnerability engaged by pharmacological CHK1 inhibition.

Identification of a retinoic acid-dependent haemogenic endothelial progenitor from human pluripotent stem cells.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. During embryonic development, HSCs derive from haemogenic endothelium (HE) in a NOTCH- and retinoic acid (RA)-dependent manner. Although a WNT-dependent (WNTd) patterning of nascent hPSC mesoderm specifies clonally multipotent intra-embryonic-like HOXA+ definitive HE, this HE is functionally unresponsive to RA. Here we show that WNTd mesoderm, before HE specification, is actually composed of two distinct KDR+ CD34neg populations. CXCR4negCYP26A1+ mesoderm gives rise to HOXA+ multilineage definitive HE in an RA-independent manner, whereas CXCR4+ ALDH1A2+ mesoderm gives rise to HOXA+ multilineage definitive HE in a stage-specific, RA-dependent manner. Furthermore, both RA-independent (RAi) and RA-dependent (RAd) HE harbour transcriptional similarity to distinct populations found in the early human embryo, including HSC-competent HE. This revised model of human haematopoietic development provides essential resolution to the regulation and origins of the multiple waves of haematopoiesis. These insights provide the basis for the generation of specific haematopoietic populations, including the de novo specification of HSCs.

Potently Cytotoxic Natural Killer Cells Initially Emerge from Erythro-Myeloid Progenitors during Mammalian Development.

Natural killer (NK) cells are a critical component of the innate immune system. However, their ontogenic origin has remained unclear. Here, we report that NK cell potential first arises from Hoxaneg/low Kit+CD41+CD16/32+ hematopoietic-stem-cell (HSC)-independent erythro-myeloid progenitors (EMPs) present in the murine yolk sac. EMP-derived NK cells and primary fetal NK cells, unlike their adult counterparts, exhibit robust degranulation in response to stimulation. Parallel studies using human pluripotent stem cells (hPSCs) revealed that HOXAneg/low CD34+ progenitors give rise to NK cells that, similar to murine EMP-derived NK cells, harbor a potent cytotoxic degranulation bias. In contrast, hPSC-derived HOXA+ CD34+ progenitors, as well as human cord blood CD34+ cells, give rise to NK cells that exhibit an attenuated degranulation response but robustly produce inflammatory cytokines. Collectively, our studies identify an extra-embryonic origin of potently cytotoxic NK cells, suggesting that ontogenic origin is a relevant factor in designing hPSC-derived adoptive immunotherapies.

Genetic variation in Dip5, an amino acid permease, and Pdr5, a multiple drug transporter, regulates glyphosate resistance in S. cerevisiae.

S. cerevisiae from different environments are subject to a wide range of selective pressures, whether intentional or by happenstance. Chemicals classified by their application, such as herbicides, fungicides and antibiotics, can affect non-target organisms. First marketed as RoundUp™, glyphosate is the most widely used herbicide. In plants, glyphosate inhibits EPSPS, of the shikimate pathway, which is present in many organisms but lacking in mammals. The shikimate pathway produces chorismate which is the precursor to all the aromatic amino acids, para-aminobenzoic acid, and Coenzyme Q10. Crops engineered to be resistant to glyphosate contain a homolog of EPSPS that is not bound by glyphosate. Here, we show that S. cerevisiae has a wide-range of glyphosate resistance. Sequence comparison between the target proteins, i.e., the plant EPSPS and the yeast orthologous protein Aro1, predicted that yeast would be resistant to glyphosate. However, the growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in an ABC multiple drug transporter, Pdr5, and an amino acid permease, Dip5. Using genetic variation as a probe into glyphosate response, we uncovered mechanisms that contribute to the transportation of glyphosate in and out of the cell. Taking advantage of the natural genetic variation within yeast and measuring growth under different conditions that would change the use of the shikimate pathway, we uncovered a general transport mechanism of glyphosate into eukaryotic cells.