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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

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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.


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(A) Colony-forming units of macrophages (CFU-M), granulocytes (CFU-G), and granulocytes, erythroid macrophages, and megakaryocytes (CFU-GEMM) after 14 days of CD34+ cell culture in Methocult. (B) The number of colonies of each type in a 35-mm dish, counted manually. (C) Fully differentiated cells of multiple lineages. (Wright-Giemsa stain, ×1,000) a)P <0.05.Abbreviations: M1, Method 1; M2, method 2; M3, Method 3.
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Figure 4: (A) Colony-forming units of macrophages (CFU-M), granulocytes (CFU-G), and granulocytes, erythroid macrophages, and megakaryocytes (CFU-GEMM) after 14 days of CD34+ cell culture in Methocult. (B) The number of colonies of each type in a 35-mm dish, counted manually. (C) Fully differentiated cells of multiple lineages. (Wright-Giemsa stain, ×1,000) a)P <0.05.Abbreviations: M1, Method 1; M2, method 2; M3, Method 3.

Mentions: An important function of HSCs is differentiation into multiple lineages of mature cells. To determine whether HSCs generated by method 3 have the differentiation potential into multilineage, we carried out colony formation assays [29]. Each CD34+ cells of all three methods sorted on day 11 and differentiated into various colonies (Fig. 4A). In line with the earlier finding that they showed enhanced induction of mesoderm formation, CD34+ cells of method 3 formed larger number of colonies than those of the other two methods after 12 days of culturing in Methocult media (Fig. 4B). In addition, fully differentiated cells―such as basophils, monocytes, neutrophils, erythrocytes, and lymphocytes― were identified by Wright-Giemsa staining (Fig. 4C).


Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method
(A) Colony-forming units of macrophages (CFU-M), granulocytes (CFU-G), and granulocytes, erythroid macrophages, and megakaryocytes (CFU-GEMM) after 14 days of CD34+ cell culture in Methocult. (B) The number of colonies of each type in a 35-mm dish, counted manually. (C) Fully differentiated cells of multiple lineages. (Wright-Giemsa stain, ×1,000) a)P <0.05.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 4: (A) Colony-forming units of macrophages (CFU-M), granulocytes (CFU-G), and granulocytes, erythroid macrophages, and megakaryocytes (CFU-GEMM) after 14 days of CD34+ cell culture in Methocult. (B) The number of colonies of each type in a 35-mm dish, counted manually. (C) Fully differentiated cells of multiple lineages. (Wright-Giemsa stain, ×1,000) a)P <0.05.Abbreviations: M1, Method 1; M2, method 2; M3, Method 3.
Mentions: An important function of HSCs is differentiation into multiple lineages of mature cells. To determine whether HSCs generated by method 3 have the differentiation potential into multilineage, we carried out colony formation assays [29]. Each CD34+ cells of all three methods sorted on day 11 and differentiated into various colonies (Fig. 4A). In line with the earlier finding that they showed enhanced induction of mesoderm formation, CD34+ cells of method 3 formed larger number of colonies than those of the other two methods after 12 days of culturing in Methocult media (Fig. 4B). In addition, fully differentiated cells―such as basophils, monocytes, neutrophils, erythrocytes, and lymphocytes― were identified by Wright-Giemsa staining (Fig. 4C).

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.


Related in: MedlinePlus