Limits...
Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

Chicha L, Feki A, Boni A, Irion O, Hovatta O, Jaconi M - PLoS ONE (2011)

Bottom Line: Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors.Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types.However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Faculty of Medicine, Geneva University, Geneva, Switzerland.

ABSTRACT

Background: Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells.

Methodology/principal findings: ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors.

Conclusions/significance: Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

Show MeSH

Related in: MedlinePlus

Hematopoietic differentiation of human iPSC.(A) Oct-4 and Brachyury mRNA expression were assessed by quantitative PCR at the onset of the differentiation (d0), and 4 (d4) and 8 (d8) days later. The expression of the Gus-B housekeeping gene was used for normalization (representative data of 3 experiments). (B) CD34 and KDR expression was assessed by flow cytometry. ESC are shown in the upper panels and iPSC in the lower panels. Expression prior to differentiation (d0, left panel) and after 8 days (d8, right panel) is depicted. (C) CFU morphology was assessed upon 14 days of methylcellulose culture of iPSC-derived cells. Erythroid CFUs (upper panels) and myeloid CFUs (lower panels) were observed in these cultures and CB-derived cells are shown (initial magnification ×100).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3045374&req=5

pone-0014733-g003: Hematopoietic differentiation of human iPSC.(A) Oct-4 and Brachyury mRNA expression were assessed by quantitative PCR at the onset of the differentiation (d0), and 4 (d4) and 8 (d8) days later. The expression of the Gus-B housekeeping gene was used for normalization (representative data of 3 experiments). (B) CD34 and KDR expression was assessed by flow cytometry. ESC are shown in the upper panels and iPSC in the lower panels. Expression prior to differentiation (d0, left panel) and after 8 days (d8, right panel) is depicted. (C) CFU morphology was assessed upon 14 days of methylcellulose culture of iPSC-derived cells. Erythroid CFUs (upper panels) and myeloid CFUs (lower panels) were observed in these cultures and CB-derived cells are shown (initial magnification ×100).

Mentions: iPSC have recently been reported to recapitulate major ESC features, including the ability to differentiate in various cell lineages in vitro and to generate teratomas [12], [13], [14]. We therefore investigated whether iPSC generated in our laboratory upon reprogramming of HFF (see Figure S1 for in vitro (A-E) and in vivo (F-H) characterization, and Movie S1 for the presence of beating cardiomyocytes) behave like bona fide ESC, in particular when exposed to our protocol of hematopoietic induction. Indeed, as for ESC, iPSC rapidly down-modulated Oct-4 expression and transiently expressed Brachyury at day 4. This expression subsequently decreased when BMP-4 was substituted by early acting hematopoietic growth factors (Fig. 3A). This substitution was also associated with the emergence at day 8 of a CD34+-KDR+ population that was clearly distinguishable from its CD34−-KDR− counterpart (Fig. 3B). Sorted iPS34+ cells displayed a similar phenotype as their ESC counterpart, including expression of CD31, CD43 and CXCR4 (Fig 1C, and data not shown). After sorting and culture in clonogenic conditions, both iPS34+ and iPS34− produced hematopoietic colonies (Fig. 3C) with increased frequency of GM colonies in the iPS34+ fractions (Table 1). However, the large variability observed in these CFU experiments, including the absence of iPS34− proliferation in many methylcellulose assays, did not allow establishing a significant difference of cloning efficiency between these 2 groups (Table 1). Nevertheless, the overall cloning frequencies (erythroid plus CFU-GM) were comparable to those of ESC and approximately 150-fold lower than CB34+ cell frequencies. Interestingly, iPS34+, as with ES34+, expressed ε and γ, but not β globins (not shown), consistent with a complete reprogramming of the initial fibroblasts to an embryonic stage. They also produced in some instances, GM-colonies of very large size that had not been observed in ES34+ cultures. Altogether, these data suggest that our protocol devoid of xenogenic compounds to generate hematopoietic cells could be successfully applied to reprogrammed somatic cells.


Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

Chicha L, Feki A, Boni A, Irion O, Hovatta O, Jaconi M - PLoS ONE (2011)

Hematopoietic differentiation of human iPSC.(A) Oct-4 and Brachyury mRNA expression were assessed by quantitative PCR at the onset of the differentiation (d0), and 4 (d4) and 8 (d8) days later. The expression of the Gus-B housekeeping gene was used for normalization (representative data of 3 experiments). (B) CD34 and KDR expression was assessed by flow cytometry. ESC are shown in the upper panels and iPSC in the lower panels. Expression prior to differentiation (d0, left panel) and after 8 days (d8, right panel) is depicted. (C) CFU morphology was assessed upon 14 days of methylcellulose culture of iPSC-derived cells. Erythroid CFUs (upper panels) and myeloid CFUs (lower panels) were observed in these cultures and CB-derived cells are shown (initial magnification ×100).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045374&req=5

pone-0014733-g003: Hematopoietic differentiation of human iPSC.(A) Oct-4 and Brachyury mRNA expression were assessed by quantitative PCR at the onset of the differentiation (d0), and 4 (d4) and 8 (d8) days later. The expression of the Gus-B housekeeping gene was used for normalization (representative data of 3 experiments). (B) CD34 and KDR expression was assessed by flow cytometry. ESC are shown in the upper panels and iPSC in the lower panels. Expression prior to differentiation (d0, left panel) and after 8 days (d8, right panel) is depicted. (C) CFU morphology was assessed upon 14 days of methylcellulose culture of iPSC-derived cells. Erythroid CFUs (upper panels) and myeloid CFUs (lower panels) were observed in these cultures and CB-derived cells are shown (initial magnification ×100).
Mentions: iPSC have recently been reported to recapitulate major ESC features, including the ability to differentiate in various cell lineages in vitro and to generate teratomas [12], [13], [14]. We therefore investigated whether iPSC generated in our laboratory upon reprogramming of HFF (see Figure S1 for in vitro (A-E) and in vivo (F-H) characterization, and Movie S1 for the presence of beating cardiomyocytes) behave like bona fide ESC, in particular when exposed to our protocol of hematopoietic induction. Indeed, as for ESC, iPSC rapidly down-modulated Oct-4 expression and transiently expressed Brachyury at day 4. This expression subsequently decreased when BMP-4 was substituted by early acting hematopoietic growth factors (Fig. 3A). This substitution was also associated with the emergence at day 8 of a CD34+-KDR+ population that was clearly distinguishable from its CD34−-KDR− counterpart (Fig. 3B). Sorted iPS34+ cells displayed a similar phenotype as their ESC counterpart, including expression of CD31, CD43 and CXCR4 (Fig 1C, and data not shown). After sorting and culture in clonogenic conditions, both iPS34+ and iPS34− produced hematopoietic colonies (Fig. 3C) with increased frequency of GM colonies in the iPS34+ fractions (Table 1). However, the large variability observed in these CFU experiments, including the absence of iPS34− proliferation in many methylcellulose assays, did not allow establishing a significant difference of cloning efficiency between these 2 groups (Table 1). Nevertheless, the overall cloning frequencies (erythroid plus CFU-GM) were comparable to those of ESC and approximately 150-fold lower than CB34+ cell frequencies. Interestingly, iPS34+, as with ES34+, expressed ε and γ, but not β globins (not shown), consistent with a complete reprogramming of the initial fibroblasts to an embryonic stage. They also produced in some instances, GM-colonies of very large size that had not been observed in ES34+ cultures. Altogether, these data suggest that our protocol devoid of xenogenic compounds to generate hematopoietic cells could be successfully applied to reprogrammed somatic cells.

Bottom Line: Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors.Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types.However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Faculty of Medicine, Geneva University, Geneva, Switzerland.

ABSTRACT

Background: Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells.

Methodology/principal findings: ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors.

Conclusions/significance: Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

Show MeSH
Related in: MedlinePlus