Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with hig... more Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT -PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.
Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (... more Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could...
Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with hig... more Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT -PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.
Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either ... more Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEBderived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNc and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34 + CD45RA + precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells. Citation: Larbi A, Gombert J-M, Auvray C, l'Homme B, Magniez A, et al. (2012) The HOXB4 Homeoprotein Promotes the Ex Vivo Enrichment of Functional Human Embryonic Stem Cell-Derived NK Cells. PLoS ONE 7(6): e39514.
During human embryonic stem cell (ESC) hematopoietic differentiation, the description of the init... more During human embryonic stem cell (ESC) hematopoietic differentiation, the description of the initial steps of lymphopoiesis remains elusive. Using a two-step culture procedure, we identified two original populations of ESC-derived hematopoietic progenitor cells (HPCs) with CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) phenotypes. Bulk cultures and limiting dilution assays, culture with MS5 cells in the presence of Notch ligand Delta-like-1 (DL-1), and ex vivo colonization tests using fetal thymic organ cultures showed that although CD34(+)CD45RA(+)CD7(-) HPCs could generate cells of the three lymphoid lineages, their potential was skewed toward the B cell lineages. In contrast, CD34(+)CD45RA(+)CD7(+) HPCs predominantly exhibited a T/natural killer (NK) cell differentiation potential. Furthermore these cells could differentiate equivalently into cells of the granulo-macrophagic lineage and dendritic cells and lacked erythroid potential. Expression profiling of 18 markers by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs express genes of the lymphoid specification and that CD34(+)CD45RA(+)CD7(-) cells express B-cell-associated genes, while CD34(+)CD45RA(+)CD7(+) HPCs display a T-cell molecular profile. Altogether, these findings indicate that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs correspond to candidate multipotent early lymphoid progenitors polarized toward either the B or T/NK lineage, respectively. This work should improve our understanding of the early steps of lymphopoiesis from pluripotent stem cells and pave the way for the production of lymphocytes for cell-based immunotherapy and lymphoid development studies.
The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem ce... more The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34 ؉ hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis. STEM CELLS 2008;26:312-322
The early stages of human lymphopoiesis are poorly characterized. Here, we compared the lymphoid ... more The early stages of human lymphopoiesis are poorly characterized. Here, we compared the lymphoid potential of a novel umbilical cord blood CD34 ؉ CD45RA hi CD7 ؉ hematopoietic progenitor cell (HPC) population with that of CD34 ؉ CD45RA hi Lin ؊ CD10 ؉ HPCs, previously proposed as candidate common lymphoid progenitors. Limitingdilution and clonal analysis, fetal thymic organ cultures, and culture onto Notch ligand Delta-like-1-expressing OP9 cells, showed that although CD34 ؉ CD45RA hi CD7 ؉ HPCs could generate cells of the 3 lymphoid lineages, their potential was skewed toward the T/natural killer (T/NK) lineages. In contrast, CD34 ؉ CD45RA hi Lin ؊ CD10 ؉ HPCs predominantly exhibited a B-cell potential. Gene expression profiling with DNA microarrays confirmed that CD34 ؉ CD45RA hi CD7 ؉ HPCs selectively expressed T-lymphoid and NK lineage-committed genes while retaining expression of genes affiliated to the granulomonocytic lineage, whereas CD34 ؉ CD45RA hi Lin ؊ CD10 ؉ HPCs displayed a typical pro-B-cell transcription profile and essentially lacked genes unrelated to the B lineage. In addition, both populations could be generated in vitro from CD34 ؉ CD45RA int CD7 ؊ and CD34 ؉ CD45RA hi Lin ؊ HPCs with mixed lymphomyeloid potential, from which they emerged independently with different growth/ differentiation factor requirements. These findings indicate that CD34 ؉ CD45RA hi CD7 ؉ and CD34 ؉ CD45RA hi Lin ؊ CD10 ؉ HPCs correspond to multipotent early lymphoid progenitors polarized toward either the T/NK or B lineage, respectively. (Blood. 2004;104: 3918-3926)
Here, we identify fetal bone marrow (BM)-derived CD34 hi CD45RA hi CD7 + hematopoietic progenitor... more Here, we identify fetal bone marrow (BM)-derived CD34 hi CD45RA hi CD7 + hematopoietic progenitors as thymus-colonizing cells. This population, virtually absent from the fetal liver (FL), emerges in the BM by development weeks 8-9, where it accumulates throughout the second trimester, to finally decline around birth. Based on phenotypic, molecular, and functional criteria, we demonstrate that CD34 hi CD45RA hi CD7 + cells represent the direct precursors of the most immature CD34 hi CD1a 2 fetal thymocytes that follow a similar dynamics pattern during fetal and early postnatal development. Histological analysis of fetal thymuses further reveals that early immigrants predominantly localize in the perivascular areas of the cortex, where they form a lymphostromal complex with thymic epithelial cells (TECs) driving their rapid specification toward the T lineage. Finally, using an ex vivo xenogeneic thymus-colonization assay, we show that BMderived CD34 hi CD45RA hi CD7 + progenitors are selectively recruited into the thymus parenchyma in the absence of exogenous cytokines, where they adopt a definitive T cell fate.
Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with hig... more Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT -PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.
Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (... more Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could...
Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with hig... more Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT -PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.
Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either ... more Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEBderived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNc and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34 + CD45RA + precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells. Citation: Larbi A, Gombert J-M, Auvray C, l'Homme B, Magniez A, et al. (2012) The HOXB4 Homeoprotein Promotes the Ex Vivo Enrichment of Functional Human Embryonic Stem Cell-Derived NK Cells. PLoS ONE 7(6): e39514.
During human embryonic stem cell (ESC) hematopoietic differentiation, the description of the init... more During human embryonic stem cell (ESC) hematopoietic differentiation, the description of the initial steps of lymphopoiesis remains elusive. Using a two-step culture procedure, we identified two original populations of ESC-derived hematopoietic progenitor cells (HPCs) with CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) phenotypes. Bulk cultures and limiting dilution assays, culture with MS5 cells in the presence of Notch ligand Delta-like-1 (DL-1), and ex vivo colonization tests using fetal thymic organ cultures showed that although CD34(+)CD45RA(+)CD7(-) HPCs could generate cells of the three lymphoid lineages, their potential was skewed toward the B cell lineages. In contrast, CD34(+)CD45RA(+)CD7(+) HPCs predominantly exhibited a T/natural killer (NK) cell differentiation potential. Furthermore these cells could differentiate equivalently into cells of the granulo-macrophagic lineage and dendritic cells and lacked erythroid potential. Expression profiling of 18 markers by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs express genes of the lymphoid specification and that CD34(+)CD45RA(+)CD7(-) cells express B-cell-associated genes, while CD34(+)CD45RA(+)CD7(+) HPCs display a T-cell molecular profile. Altogether, these findings indicate that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs correspond to candidate multipotent early lymphoid progenitors polarized toward either the B or T/NK lineage, respectively. This work should improve our understanding of the early steps of lymphopoiesis from pluripotent stem cells and pave the way for the production of lymphocytes for cell-based immunotherapy and lymphoid development studies.
The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem ce... more The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34 ؉ hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis. STEM CELLS 2008;26:312-322
The early stages of human lymphopoiesis are poorly characterized. Here, we compared the lymphoid ... more The early stages of human lymphopoiesis are poorly characterized. Here, we compared the lymphoid potential of a novel umbilical cord blood CD34 ؉ CD45RA hi CD7 ؉ hematopoietic progenitor cell (HPC) population with that of CD34 ؉ CD45RA hi Lin ؊ CD10 ؉ HPCs, previously proposed as candidate common lymphoid progenitors. Limitingdilution and clonal analysis, fetal thymic organ cultures, and culture onto Notch ligand Delta-like-1-expressing OP9 cells, showed that although CD34 ؉ CD45RA hi CD7 ؉ HPCs could generate cells of the 3 lymphoid lineages, their potential was skewed toward the T/natural killer (T/NK) lineages. In contrast, CD34 ؉ CD45RA hi Lin ؊ CD10 ؉ HPCs predominantly exhibited a B-cell potential. Gene expression profiling with DNA microarrays confirmed that CD34 ؉ CD45RA hi CD7 ؉ HPCs selectively expressed T-lymphoid and NK lineage-committed genes while retaining expression of genes affiliated to the granulomonocytic lineage, whereas CD34 ؉ CD45RA hi Lin ؊ CD10 ؉ HPCs displayed a typical pro-B-cell transcription profile and essentially lacked genes unrelated to the B lineage. In addition, both populations could be generated in vitro from CD34 ؉ CD45RA int CD7 ؊ and CD34 ؉ CD45RA hi Lin ؊ HPCs with mixed lymphomyeloid potential, from which they emerged independently with different growth/ differentiation factor requirements. These findings indicate that CD34 ؉ CD45RA hi CD7 ؉ and CD34 ؉ CD45RA hi Lin ؊ CD10 ؉ HPCs correspond to multipotent early lymphoid progenitors polarized toward either the T/NK or B lineage, respectively. (Blood. 2004;104: 3918-3926)
Here, we identify fetal bone marrow (BM)-derived CD34 hi CD45RA hi CD7 + hematopoietic progenitor... more Here, we identify fetal bone marrow (BM)-derived CD34 hi CD45RA hi CD7 + hematopoietic progenitors as thymus-colonizing cells. This population, virtually absent from the fetal liver (FL), emerges in the BM by development weeks 8-9, where it accumulates throughout the second trimester, to finally decline around birth. Based on phenotypic, molecular, and functional criteria, we demonstrate that CD34 hi CD45RA hi CD7 + cells represent the direct precursors of the most immature CD34 hi CD1a 2 fetal thymocytes that follow a similar dynamics pattern during fetal and early postnatal development. Histological analysis of fetal thymuses further reveals that early immigrants predominantly localize in the perivascular areas of the cortex, where they form a lymphostromal complex with thymic epithelial cells (TECs) driving their rapid specification toward the T lineage. Finally, using an ex vivo xenogeneic thymus-colonization assay, we show that BMderived CD34 hi CD45RA hi CD7 + progenitors are selectively recruited into the thymus parenchyma in the absence of exogenous cytokines, where they adopt a definitive T cell fate.
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Papers by Rima Haddad