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Original Article

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Blood Res 2017; 52(1):

Published online March 27, 2017

https://doi.org/10.5045/br.2017.52.1.37

© The Korean Society of Hematology

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

So-Jung Kim1,2, Ji-Won Jung1, Hye-Yeong Ha1, Soo Kyung Koo1, Eung-Gook Kim2, and Jung-Hyun Kim1*

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1Division of Intractable Diseases, Center for Biomedical Sciences, National Institute of Health and Korea Centers for Diseases Control and Prevention, Cheongju, Korea.

2Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, Korea.

Correspondence to : Correspondence to Jung-Hyun Kim, Ph.D. Division of Intractable Diseases, Center for Biomedical Sciences, National Institute of Health and Korea Centers for Diseases Control and Prevention, 202, Osong Health Technology Administration Complex, Osong-eup, Heungdeok-gu, Cheongju 28160, Korea. kjhcorea@korea.kr

Received: November 8, 2016; Revised: February 9, 2017; Accepted: February 9, 2017

Abstract

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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.

Keywords Pluripotent stem cell, Hematopoietic differentiation, Xeno-free protocol

Article

Original Article

Blood Res 2017; 52(1): 37-43

Published online March 27, 2017 https://doi.org/10.5045/br.2017.52.1.37

Copyright © The Korean Society of Hematology.

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

So-Jung Kim1,2, Ji-Won Jung1, Hye-Yeong Ha1, Soo Kyung Koo1, Eung-Gook Kim2, and Jung-Hyun Kim1*

1Division of Intractable Diseases, Center for Biomedical Sciences, National Institute of Health and Korea Centers for Diseases Control and Prevention, Cheongju, Korea.

2Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, Korea.

Correspondence to:Correspondence to Jung-Hyun Kim, Ph.D. Division of Intractable Diseases, Center for Biomedical Sciences, National Institute of Health and Korea Centers for Diseases Control and Prevention, 202, Osong Health Technology Administration Complex, Osong-eup, Heungdeok-gu, Cheongju 28160, Korea. kjhcorea@korea.kr

Received: November 8, 2016; Revised: February 9, 2017; Accepted: February 9, 2017

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.

Keywords: Pluripotent stem cell, Hematopoietic differentiation, Xeno-free protocol

Fig 1.

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Figure 1.

(A) Schematic representation of the optimized differentiation protocol used to generate hematopoietic precursors and progenitors from ESCs. (B–D) Relative expression of Oct4, Brachyury, and Wnt3a, respectively on days 4 and 7. (E) Immunostaining for Oct4, Brachyury, and DAPI (4',6-diamidino-2-phenylindole) during initial differentiation of colonies on days 4 and 7. a)P <0.05, b)P <0.01.

Blood Research 2017; 52: 37-43https://doi.org/10.5045/br.2017.52.1.37

Fig 2.

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Figure 2.

(A–C) Relative expression levels of Oct4, Nanog, and Brachyury, respectively on days 8 and 11. (D, E) Flow cytometric analysis of KDR+ cells after 4 days of differentiation (D) Histogram images of the KDR flow cytometry data. (E) The percentage of KDR+ cells in the total cell population. (F) Relative expression of Hoxb4 toward that in undifferentiated ESCs. a)P <0.05, b)P <0.01, c)P <0.001.

Blood Research 2017; 52: 37-43https://doi.org/10.5045/br.2017.52.1.37

Fig 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.

Blood Research 2017; 52: 37-43https://doi.org/10.5045/br.2017.52.1.37

Fig 4.

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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.

Blood Research 2017; 52: 37-43https://doi.org/10.5045/br.2017.52.1.37
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