Verónica Ramos-mejía - Academia.edu (original) (raw)

Papers by Verónica Ramos-mejía

Cell Death and Disease, Jun 10, 2023

Clinical Immunology, Nov 1, 2012

Nature Biotechnology, 2009

Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make th... more Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal, teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced 'stemness'. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency markers at high levels and do not harbor chromosomal abnormalities by standard cytogenetic measures. We show that the two lines possess some features of neoplastic progression, including a high proliferative capacity, growth-factor independence, a 9- to 20-fold increase in frequency of tumor-initiating cells, niche independence and aberrant lineage specification, although they are not malignant. Array comparative genomic hybridization reveals an amplification at 20q11.1-11.2 in v-hESC-1 and a deletion at 5q34a-5q34b;5q3 and a mosaic gain of chromosome 12 in v-hESC-2. These results emphasize the need for functional characterization to distinguish partially transformed and normal hES cells.

Stem Cell Research, Dec 1, 2017

The Notch ligand DLL4 has key roles during embryonic development of different tissues, but most o... more The Notch ligand DLL4 has key roles during embryonic development of different tissues, but most of the data comes from animal models. Here we describe the generation and characterization of 2 human Pluripotent Stem Cell (hPSC) lines that overexpress DLL4, as well as the two corresponding control hPSC lines. DLL4 expression can be detected at the mRNA and protein level, and does not affect the pluripotency of the cells. These hPSC lines can be used to study the role of DLL4 during human embryonic development.

Stem Cell Research, Apr 1, 2017

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations i... more Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease.

Stem Cell Research, Dec 1, 2019

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder... more Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients. Resource table Unique stem cell line identifier

Stem Cell Research, Dec 1, 2017

HoxA9 is an evolutionarily conserved homeobox gene implicated in embryo development. To study the... more HoxA9 is an evolutionarily conserved homeobox gene implicated in embryo development. To study the roles of Hoxa9 during human development we generated a transgenic H9 (hESC) line that overexpresses HoxA9 and the Enhanced Green Fluorescent Protein (EGFP), and a control H9 with a stable expression of the EGFP. The resulting H9-HoxA9-EGFP and H9-EGFP cell lines allow an efficient visualization of hESCs by fluorescent microscopy, quantification by flow cytometry and cell differentiation tracking. Both transgenic cell lines maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype.

Stem Cell Research, May 1, 2016

We generated an induced pluripotent stem cell (iPSC) line from a Bernard-Soulier Syndrome (BSS) p... more We generated an induced pluripotent stem cell (iPSC) line from a Bernard-Soulier Syndrome (BSS) patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR) profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotencyassociated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

Stem Cell Research, Mar 1, 2017

Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL) is a rare... more Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL) is a rare disease with a dismal prognosis. Around 15% of patients carry the chromosomal translocation t(1;22) that originates the fusion oncogene RBM15-MKL1, which is linked to an earlier disease onset (median of 6 months of age) and arises in utero. Here we report the generation of two hPSC cell lines constitutively expressing the oncogene RBM15-MKL1, resulting in an increased expression of known RBM15-MKL1 gene targets. These cell lines represent new disease models of pediatric AMKL to study the impact of the RBM15-MKL1 oncogene on human embryonic hematopoietic development.

Stem Cell Research, Nov 1, 2015

Here we describe the generation and characterization of the human induced pluripotent stem cell (... more Here we describe the generation and characterization of the human induced pluripotent stem cell (iPSC) line PBMC1-iPS4F1 from peripheral blood mononuclear cells from a healthy female with Spanish background. We used heat sensitive, non-integrative Sendai viruses containing the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc, whose expression was silenced in the established iPSC line. Characterization of the PBMC1-iPS4F1 cell line included analysis of typical pluripotency-associated factors at mRNA and protein level, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation studies.

Cell Stem Cell, Mar 1, 2009

During human development, signals that govern lineage specification versus expansion of cells com... more During human development, signals that govern lineage specification versus expansion of cells committed to a cell fate are poorly understood. We demonstrate that activation of canonical Wnt signaling by Wnt3a promotes proliferation of human embryonic stem cells (hESCs)-precursors already committed to the hematopoietic lineage. In contrast, noncanonical Wnt signals, activated by Wnt11, control exit from the pluripotent state and entry toward mesoderm specification. Unique to embryoid body (EB) formation of hESCs, Wnt11 induces development and arrangement of cells expressing Brachyury that coexpress E-cadherin and Frizzled-7 (Fzd7). Knockdown of Fzd7 expression blocks Wnt11-dependent specification. Our study reveals an unappreciated role for noncanonical Wnt signaling in hESC specification that involves development of unique mesoderm precursors via morphogenic organization within human EBs.

Stem Cells, Sep 23, 2017

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essent... more 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 CD451 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 (CD431) 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.

Blood, Jun 27, 2013

The role of RUNX1 isoforms in hematopoietic commitment of human pluripotent stem cells In a recen... more The role of RUNX1 isoforms in hematopoietic commitment of human pluripotent stem cells In a recent issue of Blood, Ran et al 1 reported that ectopic expression of RUNX1a isoform facilitates the emergence of definitive hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells (hESCs), as well as an impressive expansion potential of the RUNX1a-hESC-derived HSPCs, eventually conferring multilineage in vivo engraftment ability. The function of RUNX1 isoforms in human blood specification remains elusive and controversial. 2 Ran et al 1 showed in bulk hESC-differentiating cultures that RUNX1b/c isoforms are higher expressed than RUNX1a very early in specification (before blood emergence), whereas RUNX1b/c expression is lower than RUNX1a at the time of or after blood emergence. However, our data using different hESC lines (H9, AND1, HS181) and both embryoid bodies-based and OP9 coculture-based differentiation systems 3 reveal that the expression of RUNX1c emerges significantly before RUNX1a and RUNX1b (Figure 1A). RUNX1c is almost the only RUNX1 isoform expressed in hESC-derived fluorescenceactivated cell sorting (FACS)-purified hemato-endothelial progenitors (CD311CD45-) and the highest expressed in FACS-purified hESCs-derived CD451 blood cells (Figure 1A). Furthermore, lentiviral-mediated expression of RUNX1c robustly accelerates and enhances the appearance of hemato-endothelial progenitors, hematopoietic cells, and colony-forming unit potential (Figure 1B). However, despite robust hematopoietic enhancement in vitro, RUNX1c does not confer engraftment potential in newborn immunodeficient SCID-IL2Rgamma(null) mice, in keeping with previous work in mouse HSPCs 4 (Figure 1C). RUNX1 is a master hematopoietic transcription factor that acts as an oncogene in several childhood leukemias, 5,6 mixed-lineagerearranged leukemias, 7 and T-cell lymphoma. 8 In addition, this laboratory has previously shown that overexpression of RUNX1a strongly contributes to leukemogenesis. 9 This occurs because RUNX1a is missing the transactivation domain essential for its normal function in hematopoiesis, 9,10 and consequently it functions as a dominant inhibitor of other RUNX1 isoforms. 9,10 This "double-edged sword" of RUNX1 11 argues whether the data reported by Ran et al 1 are a consequence of RUNX1a-mediated transformation of hESC-derived blood cells. 12 Our concern is based on the following data reported by Ran et al: (i) RUNX1a is massively (.700-fold) and nonphysiologically overexpressed; (ii) RUNX1a-hESC-derived CD451CD341 HSPCs surprisingly expand 25-fold more than the control within a short (7-day) window; (iii) RUNX1a-hESC-derived CD451CD341 HSPCs expand even more than their somatic CB-CD341 counterparts in stroma cocultures; (iv) ;80% of the CD451 hematopoietic stem cells (HSCs) remain as CD451CD341, suggestive of a lack of terminal differentiation into CD451CD34-cells; and (v) 100% of the mice analyzed displayed engraftment (CD451), which, to date, represents a major stumbling block in the field. However, when the multilineage engraftment was analyzed, the proportion of myeloid cells (CD331), B-lymphoid cells (CD191), and erythroid cells (CD361) add up to as little as 23% of the graft. The question then is: What is the phenotype of the remaining 77% of the cells within the graft?

Stem Cell Research, Sep 1, 2010

Applications of differentiated progeny generated from human embryonic stem cells (hESCs) broadly ... more Applications of differentiated progeny generated from human embryonic stem cells (hESCs) broadly span cell replacement therapies and screening studies (toxicology, disease-drug modeling). These applications require differentiation into lineage-specific cell types from hESCs that are largely dependent on several reported embryoid body (EB) formation methods. However, methodologies for in vitro EB differentiation have not been quantitatively evaluated and compared. Using the hematopoietic lineage as a test for differentiation competency, we performed multiparameter comparisons of three prevalent EB methods: (1) suspension (SP), (2) hanging drop (HD), and (3) forced aggregation (FA). Although FA improved the homogeneity between hEBs, the highest hematopoietic induction efficiencies were observed in EBs formed in SP culture independent of the presence or absence of serum. Despite the EB formation method used, EB-based hematopoietic differentiation could be potently influenced by EB size and was augmented by paracrine signaling between cocultured EBs. Our study identifies physical and physiological parameters contributing to the efficiency of hESC differentiation in EB formats and reveals that EB methods are best tailored to specific applications unique to cell replacement vs small molecule screening or early human development.

Cell Research, Sep 7, 2010

Here, we provide data suggesting that the absence of silencing of the ectopic reprogramming facto... more Here, we provide data suggesting that the absence of silencing of the ectopic reprogramming factors used to reprogram somatic cells to induced pluripotent stem cells (iPSCs) may predispose iPSCs to genomic instability. We encourage stem cell scientists to undertake an extensive characterization and standardization of much larger cohorts of iPSC lines in order to set up rigorous criteria to define safe and stable bona fide iPSCs.

Trends in Molecular Medicine, May 1, 2012

Reprogramming and oncogenic transformation are stepwise processes that share many similarities, a... more Reprogramming and oncogenic transformation are stepwise processes that share many similarities, and induced pluripotent stem cells (iPSCs) generated from cancer cells could illuminate molecular mechanisms underlying the pathogenesis of human cancer. Deciphering the barriers underlying the reprogramming process of primary cancer cells could reveal information on the links between pluripotency and oncogenic transformation that would be instrumental for therapy development.

Stem Cells Translational Medicine, Feb 19, 2020

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenera... more Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenerative medicine and for autoimmune/inflammatory diseases. However, a main hurdle for MSCs-based therapies is the loss of their proliferative potential in vitro. Here we report that glycoprotein A repetitions predominant (GARP) is required for the proliferation and survival of adipose-derived MSCs (ASCs) via its regulation of transforming growth factor-β (TGF-β) activation. Silencing of GARP in human ASCs increased their activation of TGF-β which augmented the levels of mitochondrial reactive oxygen species (mtROS), resulting in DNA damage, a block in proliferation and apoptosis. Inhibition of TGF-β signaling reduced the levels of mtROS and DNA damage and restored the ability of GARP −/low ASCs to proliferate. In contrast, overexpression of GARP in ASCs increased their proliferative capacity and rendered them more resistant to etoposide-induced DNA damage and apoptosis, in a TGF-β-dependent manner. In summary, our data show that the presence or absence of GARP on ASCs gives rise to distinct TGF-β responses with diametrically opposing effects on ASC proliferation and survival.

Stem Cells International, 2017

Experimental Hematology, Sep 1, 2016

Cell Death and Disease, Jun 10, 2023

Clinical Immunology, Nov 1, 2012

Nature Biotechnology, 2009

Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make th... more Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal, teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced 'stemness'. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency markers at high levels and do not harbor chromosomal abnormalities by standard cytogenetic measures. We show that the two lines possess some features of neoplastic progression, including a high proliferative capacity, growth-factor independence, a 9- to 20-fold increase in frequency of tumor-initiating cells, niche independence and aberrant lineage specification, although they are not malignant. Array comparative genomic hybridization reveals an amplification at 20q11.1-11.2 in v-hESC-1 and a deletion at 5q34a-5q34b;5q3 and a mosaic gain of chromosome 12 in v-hESC-2. These results emphasize the need for functional characterization to distinguish partially transformed and normal hES cells.

Stem Cell Research, Dec 1, 2017

The Notch ligand DLL4 has key roles during embryonic development of different tissues, but most o... more The Notch ligand DLL4 has key roles during embryonic development of different tissues, but most of the data comes from animal models. Here we describe the generation and characterization of 2 human Pluripotent Stem Cell (hPSC) lines that overexpress DLL4, as well as the two corresponding control hPSC lines. DLL4 expression can be detected at the mRNA and protein level, and does not affect the pluripotency of the cells. These hPSC lines can be used to study the role of DLL4 during human embryonic development.

Stem Cell Research, Apr 1, 2017

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations i... more Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease.

Stem Cell Research, Dec 1, 2019

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder... more Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients. Resource table Unique stem cell line identifier

Stem Cell Research, Dec 1, 2017

HoxA9 is an evolutionarily conserved homeobox gene implicated in embryo development. To study the... more HoxA9 is an evolutionarily conserved homeobox gene implicated in embryo development. To study the roles of Hoxa9 during human development we generated a transgenic H9 (hESC) line that overexpresses HoxA9 and the Enhanced Green Fluorescent Protein (EGFP), and a control H9 with a stable expression of the EGFP. The resulting H9-HoxA9-EGFP and H9-EGFP cell lines allow an efficient visualization of hESCs by fluorescent microscopy, quantification by flow cytometry and cell differentiation tracking. Both transgenic cell lines maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype.

Stem Cell Research, May 1, 2016

We generated an induced pluripotent stem cell (iPSC) line from a Bernard-Soulier Syndrome (BSS) p... more We generated an induced pluripotent stem cell (iPSC) line from a Bernard-Soulier Syndrome (BSS) patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR) profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotencyassociated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

Stem Cell Research, Mar 1, 2017

Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL) is a rare... more Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL) is a rare disease with a dismal prognosis. Around 15% of patients carry the chromosomal translocation t(1;22) that originates the fusion oncogene RBM15-MKL1, which is linked to an earlier disease onset (median of 6 months of age) and arises in utero. Here we report the generation of two hPSC cell lines constitutively expressing the oncogene RBM15-MKL1, resulting in an increased expression of known RBM15-MKL1 gene targets. These cell lines represent new disease models of pediatric AMKL to study the impact of the RBM15-MKL1 oncogene on human embryonic hematopoietic development.

Stem Cell Research, Nov 1, 2015

Here we describe the generation and characterization of the human induced pluripotent stem cell (... more Here we describe the generation and characterization of the human induced pluripotent stem cell (iPSC) line PBMC1-iPS4F1 from peripheral blood mononuclear cells from a healthy female with Spanish background. We used heat sensitive, non-integrative Sendai viruses containing the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc, whose expression was silenced in the established iPSC line. Characterization of the PBMC1-iPS4F1 cell line included analysis of typical pluripotency-associated factors at mRNA and protein level, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation studies.

Cell Stem Cell, Mar 1, 2009

During human development, signals that govern lineage specification versus expansion of cells com... more During human development, signals that govern lineage specification versus expansion of cells committed to a cell fate are poorly understood. We demonstrate that activation of canonical Wnt signaling by Wnt3a promotes proliferation of human embryonic stem cells (hESCs)-precursors already committed to the hematopoietic lineage. In contrast, noncanonical Wnt signals, activated by Wnt11, control exit from the pluripotent state and entry toward mesoderm specification. Unique to embryoid body (EB) formation of hESCs, Wnt11 induces development and arrangement of cells expressing Brachyury that coexpress E-cadherin and Frizzled-7 (Fzd7). Knockdown of Fzd7 expression blocks Wnt11-dependent specification. Our study reveals an unappreciated role for noncanonical Wnt signaling in hESC specification that involves development of unique mesoderm precursors via morphogenic organization within human EBs.

Stem Cells, Sep 23, 2017

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essent... more 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 CD451 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 (CD431) 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.

Blood, Jun 27, 2013

The role of RUNX1 isoforms in hematopoietic commitment of human pluripotent stem cells In a recen... more The role of RUNX1 isoforms in hematopoietic commitment of human pluripotent stem cells In a recent issue of Blood, Ran et al 1 reported that ectopic expression of RUNX1a isoform facilitates the emergence of definitive hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells (hESCs), as well as an impressive expansion potential of the RUNX1a-hESC-derived HSPCs, eventually conferring multilineage in vivo engraftment ability. The function of RUNX1 isoforms in human blood specification remains elusive and controversial. 2 Ran et al 1 showed in bulk hESC-differentiating cultures that RUNX1b/c isoforms are higher expressed than RUNX1a very early in specification (before blood emergence), whereas RUNX1b/c expression is lower than RUNX1a at the time of or after blood emergence. However, our data using different hESC lines (H9, AND1, HS181) and both embryoid bodies-based and OP9 coculture-based differentiation systems 3 reveal that the expression of RUNX1c emerges significantly before RUNX1a and RUNX1b (Figure 1A). RUNX1c is almost the only RUNX1 isoform expressed in hESC-derived fluorescenceactivated cell sorting (FACS)-purified hemato-endothelial progenitors (CD311CD45-) and the highest expressed in FACS-purified hESCs-derived CD451 blood cells (Figure 1A). Furthermore, lentiviral-mediated expression of RUNX1c robustly accelerates and enhances the appearance of hemato-endothelial progenitors, hematopoietic cells, and colony-forming unit potential (Figure 1B). However, despite robust hematopoietic enhancement in vitro, RUNX1c does not confer engraftment potential in newborn immunodeficient SCID-IL2Rgamma(null) mice, in keeping with previous work in mouse HSPCs 4 (Figure 1C). RUNX1 is a master hematopoietic transcription factor that acts as an oncogene in several childhood leukemias, 5,6 mixed-lineagerearranged leukemias, 7 and T-cell lymphoma. 8 In addition, this laboratory has previously shown that overexpression of RUNX1a strongly contributes to leukemogenesis. 9 This occurs because RUNX1a is missing the transactivation domain essential for its normal function in hematopoiesis, 9,10 and consequently it functions as a dominant inhibitor of other RUNX1 isoforms. 9,10 This "double-edged sword" of RUNX1 11 argues whether the data reported by Ran et al 1 are a consequence of RUNX1a-mediated transformation of hESC-derived blood cells. 12 Our concern is based on the following data reported by Ran et al: (i) RUNX1a is massively (.700-fold) and nonphysiologically overexpressed; (ii) RUNX1a-hESC-derived CD451CD341 HSPCs surprisingly expand 25-fold more than the control within a short (7-day) window; (iii) RUNX1a-hESC-derived CD451CD341 HSPCs expand even more than their somatic CB-CD341 counterparts in stroma cocultures; (iv) ;80% of the CD451 hematopoietic stem cells (HSCs) remain as CD451CD341, suggestive of a lack of terminal differentiation into CD451CD34-cells; and (v) 100% of the mice analyzed displayed engraftment (CD451), which, to date, represents a major stumbling block in the field. However, when the multilineage engraftment was analyzed, the proportion of myeloid cells (CD331), B-lymphoid cells (CD191), and erythroid cells (CD361) add up to as little as 23% of the graft. The question then is: What is the phenotype of the remaining 77% of the cells within the graft?

Stem Cell Research, Sep 1, 2010

Applications of differentiated progeny generated from human embryonic stem cells (hESCs) broadly ... more Applications of differentiated progeny generated from human embryonic stem cells (hESCs) broadly span cell replacement therapies and screening studies (toxicology, disease-drug modeling). These applications require differentiation into lineage-specific cell types from hESCs that are largely dependent on several reported embryoid body (EB) formation methods. However, methodologies for in vitro EB differentiation have not been quantitatively evaluated and compared. Using the hematopoietic lineage as a test for differentiation competency, we performed multiparameter comparisons of three prevalent EB methods: (1) suspension (SP), (2) hanging drop (HD), and (3) forced aggregation (FA). Although FA improved the homogeneity between hEBs, the highest hematopoietic induction efficiencies were observed in EBs formed in SP culture independent of the presence or absence of serum. Despite the EB formation method used, EB-based hematopoietic differentiation could be potently influenced by EB size and was augmented by paracrine signaling between cocultured EBs. Our study identifies physical and physiological parameters contributing to the efficiency of hESC differentiation in EB formats and reveals that EB methods are best tailored to specific applications unique to cell replacement vs small molecule screening or early human development.

Cell Research, Sep 7, 2010

Here, we provide data suggesting that the absence of silencing of the ectopic reprogramming facto... more Here, we provide data suggesting that the absence of silencing of the ectopic reprogramming factors used to reprogram somatic cells to induced pluripotent stem cells (iPSCs) may predispose iPSCs to genomic instability. We encourage stem cell scientists to undertake an extensive characterization and standardization of much larger cohorts of iPSC lines in order to set up rigorous criteria to define safe and stable bona fide iPSCs.

Trends in Molecular Medicine, May 1, 2012

Reprogramming and oncogenic transformation are stepwise processes that share many similarities, a... more Reprogramming and oncogenic transformation are stepwise processes that share many similarities, and induced pluripotent stem cells (iPSCs) generated from cancer cells could illuminate molecular mechanisms underlying the pathogenesis of human cancer. Deciphering the barriers underlying the reprogramming process of primary cancer cells could reveal information on the links between pluripotency and oncogenic transformation that would be instrumental for therapy development.

Stem Cells Translational Medicine, Feb 19, 2020

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenera... more Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenerative medicine and for autoimmune/inflammatory diseases. However, a main hurdle for MSCs-based therapies is the loss of their proliferative potential in vitro. Here we report that glycoprotein A repetitions predominant (GARP) is required for the proliferation and survival of adipose-derived MSCs (ASCs) via its regulation of transforming growth factor-β (TGF-β) activation. Silencing of GARP in human ASCs increased their activation of TGF-β which augmented the levels of mitochondrial reactive oxygen species (mtROS), resulting in DNA damage, a block in proliferation and apoptosis. Inhibition of TGF-β signaling reduced the levels of mtROS and DNA damage and restored the ability of GARP −/low ASCs to proliferate. In contrast, overexpression of GARP in ASCs increased their proliferative capacity and rendered them more resistant to etoposide-induced DNA damage and apoptosis, in a TGF-β-dependent manner. In summary, our data show that the presence or absence of GARP on ASCs gives rise to distinct TGF-β responses with diametrically opposing effects on ASC proliferation and survival.

Stem Cells International, 2017

Experimental Hematology, Sep 1, 2016