Mitochondria and stem cell function: from somatic cells to iPSC-based disease modeling

[eng] Homeostasis of the hematopoietic stem/progenitor cell pool relies on a finely tuned balance between self-renewal, differentiation and proliferation. Recent work has revealed the importance of mitochondria during stem cell differentiation; however, it remains unclear whether mitochondrial content/function affects human hematopoietic stem versus progenitor function. We sorted cord blood-derived CD34+ cells on the basis of mitochondrial mass and examined their homeostasis and clonogenic potential in vitro and hematopoietic repopulation potential in vivo. CD34+ cells with high mitochondrial mass contained and expressed 2-fold high ATP levels and mitochondrial-specific genes than cells with low mitochondrial mass, however, HIF-1α and MEIS1 were high in the CD34+ cells with low mitochondria. We found that CD34+ cells with low mitochondrial content were enriched for hematopoietic stem cell function as demonstrated by significantly higher hematopoietic reconstitution potential in immunodeficient mice. By contrast, CD34+ cells with high mitochondrial content were enriched for hematopoietic progenitor function with high in vitro clonogenic capacity. Coenzyme Q10 (CoQ10) plays a critical role in mitochondria as an electron carrier within the electron transport chain (ETC) 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). A four-year-old girl was identified with a heterozygous mutation (c.483G>C; E161D) in COQ4, associated with a reduction in [CoQ10], CoQ10 biosynthesis and ETC activity affecting complexes I/II+III. Bona fide induced pluripotent stem cell (iPSC) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically corrected using CRISPR/Cas9 genome-editing (CQ4ed-iPSCs). Comprehensive differentiation and metabolic analysis of control-iPSCs, CQ4-iPSCs and CQ4ed-iPSCs faithfully reproduced the disease phenotype. Accordingly, the COQ4 mutation in iPSCs was associated with CoQ10 deficiency, metabolic dysfunction and impaired differentiation into SkM. Remarkably, differentiation of CQ4-iPSCs into 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

Generation of heterozygous SAMD9 CRISPR/Cas9-edited iPSC line (ESi086-A-3), carrying I1567M mutation

Germline SAMD9 mutations are one of the most common alterations that predispose to pediatric myelodysplastic syndrome (MDS), a clonal disorder characterized by ineffective hematopoiesis, increasing the risk of developing acute myeloid leukemia (AML). Up to date, a disease model to study the role of SAMD9 mutation in MDS is still lacking. Here, we have generated a human induced pluripotent stem cell (hiPSC) line carrying SAMD9mut (p.I1567M), taking advantage of CRISPR/Cas9 system. As a result, the genetic engineered hiPSC line represent a new in vitro disease model to understand the impact of SAMD9 mutation at molecular and cellular level during hematopoiesis

Generation of neurons from somatic cells of healthy individuals and neurological patients through induced pluripotency or direct conversion

Access to healthy or diseased human neural tissue is a daunting task and represents a barrierfor advancing our understanding about the cellular, genetic, and molecular mechanisms underly-ing neurogenesis and neurodegeneration. Reprogramming of somatic cells to pluripotency bytransient expression of transcription factors was achieved a few years ago. Induced pluripotentstem cells (iPSC) from both healthy individuals and patients suffering from debilitating, life-threatening neurological diseases have been differentiated into several specific neuronal sub-types. An alternative emerging approach is the direct conversion of somatic cells (i.e., fibro-blasts, blood cells, or glial cells) into neuron-like cells. However, to what extent neuronal directconversion of diseased somatic cells can be achieved remains an open question. Optimizationof current expansion and differentiation approaches is highly demanded to increase the differ-entiation efficiency of specific phenotypes of functional neurons from iPSCs or through somaticcell direct conversion. The realization of the full potential of iPSCs relies on the ability to pre-cisely modify specific genome sequences. Genome editing technologies including zinc fingernucleases, transcription activator-like effector nucleases, and clustered regularly interspacedshort palindromic repeat/CAS9 RNA-guided nucleases have progressed very fast over the lastyears. The combination of genome-editing strategies and patient-specific iPSC biology will offera unique platform for in vitro generation of diseased and corrected neural derivatives for per-sonalized therapies, disease modeling and drug screening

GATA2 deficiency and MDS/AML: experimental strategies for disease modelling and future therapeutic prospects

The importance of predisposition to leukaemia in clinical practice is being increasingly recognized. This is emphasized by the establishment of a novel WHO disease category in 2016 called 'myeloid neoplasms with germline predisposition'. A major syndrome within this group is GATA2 deficiency, a heterogeneous immunodeficiency syndrome with a very high lifetime risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). GATA2 deficiency has been identified as the most common hereditary cause of MDS in adolescents with monosomy 7. Allogenic haematopoietic stem cell transplantation is the only curative option; however, chances of survival decrease with progression of immunodeficiency and MDS evolution. Penetrance and expressivity within families carrying GATA2 mutations is often variable, suggesting that co-operating extrinsic events are required to trigger the disease. Predictive tools are lacking, and intrafamilial heterogeneity is poorly understood; hence there is a clear unmet medical need. On behalf of the ERAPerMed GATA2 HuMo consortium, in this review we describe the genetic, clinical, and biological aspects of familial GATA2-related MDS, highlighting the importance of developing robust disease preclinical models to improve early detection and clinical decision-making of GATA2 carriers

Cord blood-derived CD34+ hematopoietic cells with low mitochondrial mass are enriched in hematopoietic repopulating stem cell function

This work was funded by the CICE/FEDER (P08-CTS-3678) de la Junta de Andalucia with a grant to PM, the FIS/FEDER (PI10/00449 to PM and PI11/00119 to CB), the MICINN (Fondo Especial del Estado para Dinamizacion de la Economia y Empleo/PLE-2009-0111) with a grant to PM, and the Foundation "Spanish Association Against Cancer"/Junta Provincial de Albacete (CI110023 to PM). DRM (PFIS scholarship FI11/0511), RM and CB (CP07/0059) are supported by the ISCIII. ON-M was supported by the Health of Department of the Junta de Andalucia.The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understanding the role of mitochondria during stem cell differentiation, it remains unclear whether the mitochondrial content/function affects human hematopoietic stem versus progenitor function. We found that mitochondrial mass correlates strongly with mitochondrial membrane potential in CD34(+) hematopoietic stem/progenitor cells. We, therefore, sorted cord blood CD34(+) cells on the basis of their mitochondrial mass and analyzed the in vitro homeostasis and clonogenic potential as well as the in vivo repopulating potential of CD34(+) cells with high (CD34(+) Mito(High)) versus low (CD34(+) Mito(Low)) mitochondrial mass. The CD34(+) Mito(Low) fraction contained 6-fold more CD34(+)CD38(-) primitive cells and was enriched in hematopoietic stem cell function, as demonstrated by its significantly greater hematopoietic reconstitution potential in immuno deficient mice. In contrast, the CD34(+) Mito(High) fraction was more enriched in hematopoietic progenitor function with higher in vitro clonogenic capacity. In vitro differentiation of CD34(+) Mito(Low) cells was significantly delayed as compared to that of CD34(+) Mito(High) cells. The eventual complete differentiation of CD34(+) Mito(Low) cells, which coincided with a robust expansion of the CD34(-) differentiated progeny, was accompanied by mitochondrial adaptation, as shown by significant increases in ATP production and expression of the mitochondrial genes ND1 and COX2. In conclusion, cord blood CD34(+) cells with low levels of mitochondrial mass are enriched in hematopoietic repopulating stem cell function whereas high levels of mitochondrial mass identify hematopoietic progenitors. A mitochondrial response underlies hematopoietic stem/progenitor cell differentiation and proliferation of lineage-committed CD34(-) cells.Junta de Andalucia P08-CTS-3678European Union (EU) Instituto de Salud Carlos III PI10/00449 PI11/00119MICINN (Fondo Especial del Estado para Dinamizacion de la Economia y Empleo) PLE-2009-0111Foundation "Spanish Association Against Cancer"/Junta Provincial de Albacete CI110023Instituto de Salud Carlos III FI11/0511 CP07/0059ICRE

Generation of two heterozygous GATA2 CRISPR/Cas9-edited iPSC lines, R398W and R396Q, for modeling GATA2 deficiency

Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors

Generation, genome edition and characterization of iPSC lines from a patient with coenzyme Q10 deficiency harboring a heterozygous mutation in COQ4 gene

We report the generation, CRISPR/Cas9-edition and characterization of induced pluripotent stem cell (iPSC) lines from a patient with coenzyme Q deficiency harboring the heterozygous mutation c.483G > C in the COQ4 gene. iPSCs were generated using non-integrative Sendai Viruses containing the reprogramming factors OCT4, SOX2, KLF4 and C-MYC. The iPSC lines carried the c.483G > C COQ4 mutation, silenced the OKSM expression and were mycoplasma-free. They were bona fide pluripotent cells as characterized by morphology, immunophenotype/gene expression for pluripotent-associated markers/genes, NANOG and OCT4 promoter demethylation, karyotype and teratoma formation. The COQ4 mutation was CRISPR/Cas9 edited resulting in isogenic, diploid and off-target free COQ4-corrected iPSCs.Peer Reviewe

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.This work was supportedbythe European Research Council CoG to P.M., the Spanish Ministry of Economy and Competitiveness (ISCIII/FEDER PI14/01119 to C.B and SAF2013-43065R to P.M), the Spanish Association Against Cancer to P.M. and C.B., the Fundación Inocente Inocente to PM, a Marie Curie Career Integration Grant to A.S-P (FP7-PEOPLE-2013-CIG-631171), and the Josep Carreras Dutch Leukemia Fund to P.M and ASP. C.B. is supported by a Miguel Servet II contract (CPII13/00011). C.P. is supported by PFIS scholarship (FI12/00468). P.M. also acknowledges the financial support from The Obra Social La Caixa-Fundació Josep Carreras and The Generalitat de Catalunya (SGR330)

RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling.

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266