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Improved erythroid differentiation of multiple human pluripotent stem cell lines in microcarrier culture by modulation of Wnt/β-Catenin signaling

From left: Dr Alan Lam, Dr Steve Oh, Zac Lim Zhong Ri and Dr Jaichandran Sivalingam


Jaichandran Sivalingam1, Hong Yu Chen2, Bin-Xia Yang2, Zhong Ri Lim1, Alan Tin Lun Lam1, Tsung Liang Woo1, Allen Kuan-Liang Chen1, Shaul Reuveny1, Yuin-Han Loh2,3 and Steve Kah-Weng Oh1

1 Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore
2 Institute of Molecular and Cellular Biology, Agency for Science, Technology and Research (A*STAR), Singapore
3 Department of Biological Sciences, National University of Singapore, Singapore

Published in Haematologica 2018 103(7): e279-e283 (Online Version)



Erythroid differentiation of human pluripotent stem cells (hPSCs) has been proposed as a means for generating limitless supply of red blood cells (RBCs). For this to be a reality, scalable suspension culture differentiation methods have to be developed. We have previously demonstrated erythroid differentiation of human embryonic stem cells (hESCs) initially expanded in agitation microcarrier (MC) suspension culture using a bone morphogenetic protein-4 (BMP4)-based differentiation protocol wherein mesoderm induction and erythroblasts expansion were done in static condition. In an attempt to differentiate in the same manner human induced pluripotent stem cells (hiPSCs), we found that continuous agitation of hPSC-MC aggregates during the pluripotent expansion stage negatively impacts mesoderm induction and erythroblasts differentiation.

To overcome this limitation, we used a multifactorial design approach to identify factors for efficient mesoderm induction from hPSC-MC aggregates derived from agitation culture. Here, we report that transient activation of the Wnt/β-Catenin signaling using the small molecule, CHIR-99021 in combination with BMP4 and Activin A significantly improved mesoderm induction and hematopoietic precursors and erythroblasts differentiation from agitated hPSC-MC aggregates. We demonstrate that several O-negative hiPSC lines can be differentiated and expanded up-to 60,000 fold from hematopoietic precursors to erythroblasts within 50 days in culture. O-negative erythroblasts mainly expressed fetal hemoglobin, had oxygen binding affinity similar to hESC-differentiated erythroblasts and could be enucleated following co-culture with primary human mesenchymal stromal cells. The developed MC-suspension culture approach has potential for scaling up the expansion and mesoderm stages of differentiation of hPSCs and could be further developed for large scale generation of universal RBCs that could be used for transfusion therapy.

Figure 1. Multifactorial DoE analysis identifies CHIR-99021 as a significant factor for improved hematopoietic mesoderm induction and hematopoietic differentiation from hPSC-MC cultures of multiple cell lines initially expanded under agitation condition.

(A) hES-3-MC agitation culture: DoE analysis identified CHIR-99021 (maintained for 24 hrs only) as a significant factor for achieving higher % KDR+ cells on day 4 of differentiation (P= 2.3E-07) and (B) greater expansion of hematopoietic precursors (P= 0.01). Act=Activin; CHI=CHIR-99021 for 24 hrs, CH2= CHIR-99021 from 24-48 hr; BMP4. (C) hES-3-MC agitation culture: Correlation between high (>15%) and low (<5%) KDR expression on day 4 of differentiation and the corresponding total number of hematopoietic precursors per well derived following 14 days of culture in BGM. (D) Expansion of 9 different hPSC lines in agitated-MC culture for 7 days. Table shows: cell-MC aggregate images, cell fold-expansion, pluripotency (Oct-4, Tra1-60 and SSEA4 expression), karyotypes and average aggregate diameters. NA=Not available. (E) Corresponding RBC pellets from the different hPSC lines on day 35 of experiment.


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