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Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method

View Article: PubMed Central - PubMed

ABSTRACT

Background: Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials.

Methods: Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF.

Results: Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34+CD43+ hematopoietic progenitor cells (HPCs) and CD34+CD45+ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro.

Conclusion: In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

No MeSH data available.


HSC and progenitor cell populations among differentiated cells. (A) Dot blot images of flow cytometric analysis. Upper and lower panels show differentiated cells on days 8 and 11, respectively. (B) The proportion of CD34+CD43− cells on days 8 and 11. (C) The proportion of CD34+CD43+ cells in the total cell population on day 11. (D) The proportion of CD34+CD45+ cells on day 11. a)P <0.05, b)P <0.001.Abbreviations: M1, Method 1; M2, method 2; M3, Method 3.
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Figure 3: HSC and progenitor cell populations among differentiated cells. (A) Dot blot images of flow cytometric analysis. Upper and lower panels show differentiated cells on days 8 and 11, respectively. (B) The proportion of CD34+CD43− cells on days 8 and 11. (C) The proportion of CD34+CD43+ cells in the total cell population on day 11. (D) The proportion of CD34+CD45+ cells on day 11. a)P <0.05, b)P <0.001.Abbreviations: M1, Method 1; M2, method 2; M3, Method 3.

Mentions: Next, we measured the proportion of CD34+CD43− hemogenic endothelial cells on days 8 and 11. Compared to the other two methods, method 3 yielded 5- to 6-fold higher proportion of CD34+CD43− cells on day 8 (Fig. 3A, B). In addition, method 3 resulted in a 6- to 12-fold higher proportion of CD34+CD43+ HPCs than the other two methods on day 11 (Fig. 3C). Furthermore, method 3 yielded 3-fold higher proportion of CD34+CD45+ HPCs on day 11 compared to the other two methods (Fig. 3D).


Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method
HSC and progenitor cell populations among differentiated cells. (A) Dot blot images of flow cytometric analysis. Upper and lower panels show differentiated cells on days 8 and 11, respectively. (B) The proportion of CD34+CD43− cells on days 8 and 11. (C) The proportion of CD34+CD43+ cells in the total cell population on day 11. (D) The proportion of CD34+CD45+ cells on day 11. a)P <0.05, b)P <0.001.Abbreviations: M1, Method 1; M2, method 2; M3, Method 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383586&req=5

Figure 3: HSC and progenitor cell populations among differentiated cells. (A) Dot blot images of flow cytometric analysis. Upper and lower panels show differentiated cells on days 8 and 11, respectively. (B) The proportion of CD34+CD43− cells on days 8 and 11. (C) The proportion of CD34+CD43+ cells in the total cell population on day 11. (D) The proportion of CD34+CD45+ cells on day 11. a)P <0.05, b)P <0.001.Abbreviations: M1, Method 1; M2, method 2; M3, Method 3.
Mentions: Next, we measured the proportion of CD34+CD43− hemogenic endothelial cells on days 8 and 11. Compared to the other two methods, method 3 yielded 5- to 6-fold higher proportion of CD34+CD43− cells on day 8 (Fig. 3A, B). In addition, method 3 resulted in a 6- to 12-fold higher proportion of CD34+CD43+ HPCs than the other two methods on day 11 (Fig. 3C). Furthermore, method 3 yielded 3-fold higher proportion of CD34+CD45+ HPCs on day 11 compared to the other two methods (Fig. 3D).

View Article: PubMed Central - PubMed

ABSTRACT

Background: Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials.

Methods: Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF.

Results: Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34+CD43+ hematopoietic progenitor cells (HPCs) and CD34+CD45+ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro.

Conclusion: In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

No MeSH data available.