TY - JOUR
T1 - Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells
AU - Netsrithong, Ratchapong
AU - Suwanpitak, Siriwal
AU - Boonkaew, Bootsakorn
AU - Trakarnsanga, Kongtana
AU - Chang, Lung Ji
AU - Tipgomut, Chartsiam
AU - Vatanashevanopakorn, Chinnavuth
AU - Pattanapanyasat, Kovit
AU - Wattanapanitch, Methichit
N1 - Funding Information: We would like to thank the Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, for performing HPLC globin typing. Funding Information: This study was supported by grants from the Thailand Research Fund (grant no. RSA6280090 to MW and DPG5980001 to KP); the Siriraj Research Fund, Faculty of Medicine Siriraj Hospital, Mahidol University (grant number (IO) R016234002 to MW); the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (grant no. 9573 to MW); and Science and Technology Planning Technical Research Project of Shenzhen (grant no. JCYJ20170413154349187, JCYJ20170817172416991, JCYJ20170817172541842, and KQTD20140630143254906 to LJC). RN is supported by the Development and Promotion of Science and Technology Talents Project. KT, CV, and MW are supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University. Acknowledgements Publisher Copyright: © 2020, The Author(s).
PY - 2020/12
Y1 - 2020/12
N2 - Background: Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. Methods: Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. Results: Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. Conclusion: This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.
AB - Background: Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. Methods: Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. Results: Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. Conclusion: This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.
KW - Endothelial cells
KW - Erythroid cells
KW - Hematoendothelial progenitors
KW - Hematopoietic differentiation
KW - Induced pluripotent stem cells
KW - T lymphocytes
UR - http://www.scopus.com/inward/record.url?scp=85095818441&partnerID=8YFLogxK
U2 - https://doi.org/10.1186/s13287-020-01997-w
DO - https://doi.org/10.1186/s13287-020-01997-w
M3 - Article
C2 - 33176890
SN - 1757-6512
VL - 11
JO - Stem Cell Research and Therapy
JF - Stem Cell Research and Therapy
IS - 1
M1 - 481
ER -