Cristina Prieto

The Notch ligand DLL4 specifically marks human hematoendothelial progenitors and regulates their hematopoietic fate

Notch signaling is essential for definitive hematopoiesis, but its role in human embryonic hematopoiesis is largely unknown. We show that in hESCs the expression of the Notch ligand DLL4 is induced during hematopoietic differentiation. We found that DLL4 is only expressed in a sub-population of bipotent hematoendothelial progenitors (HEPs) and segregates their hematopoietic versus endothelial potential. We demonstrate at the clonal level and through transcriptome analyses that DLL4high HEPs are enriched in endothelial potential, whereas DLL4low/– HEPs are committed to the hematopoietic lineage, albeit both populations still contain bipotent cells. Moreover, DLL4 stimulation enhances hematopoietic differentiation of HEPs and increases the amount of clonogenic hematopoietic progenitors. Confocal microscopy analysis of whole differentiating embryoid bodies revealed that DLL4high HEPs are located close to DLL4low/– HEPs, and at the base of clusters of CD45+ cells, resembling intra-aortic hematopoietic clusters found in mouse embryos. We propose a model for human embryonic hematopoiesis in which DLL4low/– cells within hemogenic endothelium receive Notch-activating signals from DLL4high cells, resulting in an endothelial-to-hematopoietic transition and their differentiation into CD45+ hematopoietic cells.

Activated KRAS Cooperates with MLL-AF4 to Promote Extramedullary Engraftment and Migration of Cord Blood CD34+ HSPC But Is Insufficient to Initiate Leukemia.

The MLL-AF4 (MA4) fusion gene is the genetic hallmark of an aggressive infant pro-B-acute lymphoblastic leukemia (B-ALL). Our understanding of MA4-mediated transformation is very limited. Whole-genome sequencing studies revealed a silent mutational landscape, which contradicts the aggressive clinical outcome of this hematologic malignancy. Only RAS mutations were recurrently detected in patients and found to be associated with poorer outcome. The absence of MA4-driven B-ALL models further questions whether MA4 acts as a single oncogenic driver or requires cooperating mutations to manifest a malignant phenotype. We explored whether KRAS activation cooperates with MA4 to initiate leukemia in cord blood-derived CD34(+) hematopoietic stem/progenitor cells (HSPC). Clonogenic and differentiation/proliferation assays demonstrated that KRAS activation does not cooperate with MA4 to immortalize CD34(+) HSPCs. Intrabone marrow transplantation into immunodeficient mice further showed that MA4 and KRAS(G12V) alone or in combination enhanced hematopoietic repopulation without impairing myeloid-lymphoid differentiation, and that mutated KRAS did not cooperate with MA4 to initiate leukemia. However, KRAS activation enhanced extramedullary hematopoiesis of MA4-expressing cell lines and CD34(+) HSPCs that was associated with leukocytosis and central nervous system infiltration, both hallmarks of infant t(4;11)(+) B-ALL. Transcriptional profiling of MA4-expressing patients supported a cell migration gene signature underlying the mutant KRAS-mediated phenotype. Collectively, our findings demonstrate that KRAS affects the homeostasis of MA4-expressing HSPCs, suggesting that KRAS activation in MA4(+) B-ALL is important for tumor maintenance rather than initiation. Cancer Res; 76(8); 2478-89. ©2016 AACR.

Ligand-independent FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform cord blood CD34+ cells.

MLL-AF4 fusion is hallmark in high-risk infant pro-B-acute lymphoblastic leukemia (pro-B-ALL). Our limited understanding of MLL-AF4-mediated transformation reflects the absence of human models reproducing this leukemia. Hematopoietic stem/progenitor cells (HSPCs) constitute likely targets for transformation. We previously reported that MLL-AF4 enhanced hematopoietic engraftment and clonogenic potential in cord blood (CB)-derived CD34+ HSPCs but was not sufficient for leukemogenesis, suggesting that additional oncogenic lesions are required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL display enormous levels of FLT3, and occasionally FLT3-activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. We have explored whether FLT3.TKD (tyrosine kinase domain) mutation or increased expression of FLT3.WT (wild type) cooperates with MLL-AF4 to immortalize/transform CB-CD34+ HSPCs. In vivo, FLT3.TKD/FLT3.WT alone, or in combination with MLL-AF4, enhances hematopoietic repopulating function of CB-CD34+ HSPCs without impairing migration or hematopoietic differentiation. None of the animals transplanted with MLL-AF4+FLT3.TKD/WT-CD34+ HSPCs showed any sign of disease after 16 weeks. In vitro, enforced expression of FLT3.TKD/FLT3.WT conveys a transient overexpansion of MLL-AF4-expressing CD34+ HSPCs associated to higher proportion of cycling cells coupled to lower apoptotic levels, but does not augment clonogenic potential nor confer stable replating. Together, FLT3 activation does not suffice to immortalize/transform MLL-AF4-expressing CB-CD34+ HSPCs, suggesting the need of alternative (epi)-genetic cooperating oncogenic lesions.

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

Summary Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency

Coenzyme Q10 (CoQ10) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype–phenotype association, mainly affecting tissues with high‐energy demand including brain and skeletal muscle (SkM). Here, we report a four‐year‐old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patients fibroblasts showed a decrease in [CoQ10], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4‐iPSCs) were generated, characterized and genetically edited using the CRISPR‐Cas9 system (CQ4ed‐iPSCs). Extensive differentiation and metabolic assays of control‐iPSCs, CQ4‐iPSCs and CQ4ed‐iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency‐associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687–1703

Genetic rescue of Mitochondrial and Skeletal Muscle Impairment in an IPSCs Model of Coenzyme Q10 Deficiency

Coenzyme Q10 (CoQ10) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype–phenotype association, mainly affecting tissues with high‐energy demand including brain and skeletal muscle (SkM). Here, we report a four‐year‐old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patients fibroblasts showed a decrease in [CoQ10], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4‐iPSCs) were generated, characterized and genetically edited using the CRISPR‐Cas9 system (CQ4ed‐iPSCs). Extensive differentiation and metabolic assays of control‐iPSCs, CQ4‐iPSCs and CQ4ed‐iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency‐associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687–1703

The AF4-MLL fusion transiently augments multilineage hematopoietic engraftment but is not sufficient to initiate leukemia in cord blood CD34 + cells

The translocation t(4;11)(q21;q23) is the hallmark genetic abnormality associated with infant pro-B acute lymphoblastic leukemia (B-ALL) and has the highest frequency of rearrangement in Mixed-lineage leukemia (MLL) leukemias. Unlike other MLL translocations, MLL-AF4-induced proB-ALL is exceptionally difficult to model in mice/humans. Previous work has investigated the relevance of the reciprocal translocation fusion protein AF4-MLL for t(4;11) leukemia, finding that AF4-MLL is capable of inducing proB-ALL without requirement for MLL-AF4 when expressed in murine hematopoietic stem/progenitor cells (HSPCs). Therefore, AF4-MLL might represent a key genetic lesion contributing to t(4;11)-driven leukemogenesis. Here, we aimed to establish a humanized mouse model by using AF4-MLL to analyze its transformation potential in human cord blood-derived CD34+ HSPCs. We show that AF4-MLL-expressing human CD34+ HSPCs provide enhanced long-term hematopoietic reconstitution in primary immunodeficient recipients but are not endowed with subsequent self-renewal ability upon serial transplantation. Importantly, expression of AF4-MLL in primary neonatal CD34+ HSPCs failed to render any phenotypic or hematological sign of disease, and was therefore not sufficient to initiate leukemia within a 36-week follow-up. Species-specific (epi)-genetic intrinsic determinants may underlie the different outcome observed when AF4-MLL is expressed in murine or human HSPCs.The translocation t(4;11)(q21;q23) is the hallmark genetic abnormality associated with infant pro-B acute lymphoblastic leukemia (B-ALL) and has the highest frequency of rearrangement in Mixed-lineage leukemia (MLL) leukemias. Unlike other MLL translocations, MLL-AF4-induced proB-ALL is exceptionally difficult to model in mice/humans. Previous work has investigated the relevance of the reciprocal translocation fusion protein AF4-MLL for t(4;11) leukemia, finding that AF4-MLL is capable of inducing proB-ALL without requirement for MLL-AF4 when expressed in murine hematopoietic stem/progenitor cells (HSPCs). Therefore, AF4-MLL might represent a key genetic lesion contributing to t(4;11)-driven leukemogenesis. Here, we aimed to establish a humanized mouse model by using AF4-MLL to analyze its transformation potential in human cord blood-derived CD34+ HSPCs. We show that AF4-MLL-expressing human CD34+ HSPCs provide enhanced long-term hematopoietic reconstitution in primary immunodeficient recipients but are not endowed with subsequent self-renewal ability upon serial transplantation. Importantly, expression of AF4-MLL in primary neonatal CD34+ HSPCs failed to render any phenotypic or hematological sign of disease, and was therefore not sufficient to initiate leukemia within a 36-week follow-up. Species-specific (epi)-genetic intrinsic determinants may underlie the different outcome observed when AF4-MLL is expressed in murine or human HSPCs.

Intra-Bone Marrow Transplantation Confers Superior Multilineage Engraftment of Murine Aorta-Gonad Mesonephros Cells Over Intravenous Transplantation

Hematopoietic stem cell (HSC) engraftment has been achieved using single-cell transplantation of prospectively highly purified adult HSC populations. However, bulk transplants are still performed when assessing the HSC potential of early embryonic hematopoietic tissues such as the aorta-gonad mesonephros (AGM) due to very low HSC activity content early in development. Intra-bone marrow transplantation (IBMT) has emerged as a superior administration route over intravenous (IV) transplantation for assessing the reconstituting ability of human HSCs in the xenotransplant setting since it bypasses the requirement for homing to the BM. In this study, we compared the ability of IBMT and IV administration of embryonic day 11.5 AGM-derived cells to reconstitute the hematopoietic system of myeloablated recipients. IBMT resulted in higher levels of AGM HSC long-term multilineage engraftment in the peripheral blood, BM, spleen, and thymus of primary and secondary recipients, and in limiting dilution experiments. The administration route did not skew the multilineage contribution pattern, but IBMT conferred higher Lineage(-)Sca-1(+)c-kit(+) long-term engraftment, in line with the superior IBMT reconstitution. Therefore, IBMT represents a superior administration route to detect HSC activity from developmentally early sources with limited HSC activity content, such as the AGM.

Epigenome-wide analysis reveals specific DNA hypermethylation of T cells during human hematopoietic differentiation

AIM Epigenetic regulation plays an important role in cellular development and differentiation. A detailed map of the DNA methylation dynamics that occur during cell differentiation would contribute to decipher the molecular networks governing cell fate commitment. METHODS Illumina MethylationEPIC BeadChip platform was used to describe the genome-wide DNA methylation changes observed throughout hematopoietic maturation by analyzing multiple myeloid and lymphoid hematopoietic cell types. RESULTS We identified a plethora of DNA methylation changes that occur during human hematopoietic differentiation. We observed that T lymphocytes display substantial enhancement of de novo CpG hypermethylation as compared with other hematopoietic cell populations. T-cell-specific hypermethylated regions were strongly associated with open chromatin marks and enhancer elements, as well as binding sites of specific key transcription factors involved in hematopoietic differentiation, such as PU.1 and TAL1. CONCLUSION These results provide novel insights into the role of DNA methylation at enhancer elements in T-cell development.