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

A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells

“…We successfully scaled up the differentiation process, starting from 6 well ULA plates to shaker flasks and finally demonstrating in stirred spinner flasks as a prelude to scaling up to controlled stirred-tank bioreactors. Using the MC-hiPSC aggregates has an added advantage of generating consistent and evenly sized MC-EBs, which have been demonstrated to be scalable in suspension culture platforms (Lam et al, 2016;Sivalingam et al, 2018). Furthermore, in our spinner culture differentiation platform, by keeping the levels of lactate in culture below their inhibitory threshold, we show peak cell densities that are at least 4-fold higher (1.7 3 10 7 cells/mL) than the 4 3 10 6 cells/mL that was reported previously (Ying Wang et al, 2016).…”

“…Development of a scalable process that can eventually be transferred to large-scale stirred bioreactors would require the entire process to be performed in continuous agitation suspension culture. We have previously described means to scale up the pluripotent expansion stage by culturing hiPSCs on Laminin-521 (LN-521)-coated microcarriers (MCs) (Lam et al, 2016;Sivalingam et al, 2018). We have also shown that hiPSC-MC aggregates in suspension , undergoing hematopoietic differentiation in tissue culture plates under static conditions (monolayer static protocol) or in 6 well ULA plates under continuous agitation (suspension agitation protocol) from day 0 to 14 of the hematopoietic induction stage.…”

“…(legend continued on next page) culture can efficiently differentiate into T-Bra + and KDR + mesodermal cells (Sivalingam et al, 2018), demonstrating that hiPSC-MC aggregates could differentiate as embryoid bodies (EBs) in a scalable manner.…”

Human induced pluripotent stem cell line banking for the production of rare blood type erythrocytes

“…The major limitations for the clinical application of iPSC-derived RBCs are the inefficient RBC enucleation, difficulty of switching to adult-type globin, and the substantial number of RBCs (10 12 ) needed to generate 1 unit of transfusable RBCs. Our protocol suffered from similar limitations, therefore the use of other small molecules and cytokines should be explored to overcome these hurdles [22][23][24]. Prior to using iPSC-derived RBCs clinically, further investigations are needed including: evaluation of RBC antigen profile, additional evaluation of iPSCs for genomic mutations, testing of the RBCs in agglutination assays, and/or safety testing of the RBCs in animal models.…”

“…Hematopoietic stem cells (HSC) obtained from cord blood and bone marrow have been used to produce functional enucleated RBCs [12][13][14][15][16]. There have been numerous advances in the generation of safe erythroid cells and improvements in the efficiency of single hematopoietic cell acquisition by differentiation of CD34 + HSCs using cytokines and small molecules [9,[17][18][19][20][21][22][23][24][25][26][27][28]. As human HSCs are a limited resource and ethical concerns hinder the use of embryonic HSCs, the large-scale expansion of RBCs for transfusion purposes remains problematic.…”