Integrating Human-Induced Pluripotent Stem Cell Expansion Capability and Cardiomyocyte Differentiation Potential in a Microcarrier Suspension Culture.

Human-induced pluripotent stem cells are known for their high proliferation capacity as well as their ability to differentiate to different lineages (Ban et al., Theranostics 7(7):2067-2077, 2017; Chen et al., Stem Cell Res 15(2):365-375, 2015; Serra et al., Trends Biotechnol 30(6):350-359, 2012). For stem-cell-derived cardiomyocytes to evolve into a scalable therapeutic source, a large quantity of highly pure cardiomyocytes is needed. Thus, lies the challenge of defining an efficient cardiomyocyte differentiation process. This chapter describes a method to evaluate multiple human-induced pluripotent stem cell lines for their cardiac differentiation potentials before evaluating their integrated proliferation and differentiation abilities in microcarrier cultures in a spinner culture format.

Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population.

BACKGROUND: Despite recent rapid progress in method development and biological understanding of induced pluripotent stem (iPS) cells, there has been a relative shortage of tools that monitor the early reprogramming process into human iPS cells. METHODS: We screened the in-house built fluorescent library compounds that specifically bind human iPS cells. After tertiary screening, the selected probe was analyzed for its ability to detect reprogramming cells in the time-dependent manner using high-content imaging analysis. The probe was compared with conventional dyes in different reprogramming methods, cell types, and cell culture conditions. Cell sorting was performed with the fluorescent probe to analyze the early reprogramming cells for their pluripotent characteristics and genome-wide gene expression signatures by RNA-seq. Finally, the candidate reprogramming factor identified was investigated for its ability to modulate reprogramming efficiency. RESULTS: We identified a novel BODIPY-derived fluorescent probe, BDL-E5, which detects live human iPS cells at the early reprogramming stage. BDL-E5 can recognize authentic reprogramming cells around 7 days before iPS colonies are formed and stained positive with conventional pluripotent markers. Cell sorting of reprogrammed cells with BDL-E5 allowed generation of an increased number and higher quality of iPS cells. RNA sequencing analysis of BDL-E5-positive versus negative cells revealed early reprogramming patterns of gene expression, which notably included CREB1. Reprogramming efficiency was significantly increased by overexpression of CREB1 and decreased by knockdown of CREB1. CONCLUSION: Collectively, BDL-E5 offers a valuable tool for delineating the early reprogramming pathway and clinically applicable commercial production of human iPS cells.

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor.

BACKGROUND: The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. METHODS: Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. RESULTS: Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. CONCLUSION: A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry.

Meticulous optimization of cardiomyocyte yields in a 3-stage continuous integrated agitation bioprocess.

Human pluripotent stem cells (hPSCs) can be a renewable source for generating cardiomyocyte (CM) for treating myocardial infraction. In our previous publication, we described an integrated microcarrier-based wave reactor process for the expansion and differentiation of hPSCs to CMs on a rocker based platform. However, this platform is limited in terms of linear scalability and CMs purity. The present study describes ways to overcome these limitations by the use of a stirred scalable platform and incorporation of an additional lactate based purification step which increases CM purity. Efficient CM differentiation in stirred spinners was achieved by (1) Addition of ascorbic acid (AS) during the differentiation phase which resulted in an increase of 38.42% in CM yield (0.84 ±â€¯0.03 × 106vs 1.17 ±â€¯0.07 × 106 CM/mL for cultures without AS and with AS respectively) and (2) Change of agitation regime to a shorter static intervals one (from 66 min off/6 min on (66/6) to 8 min off/1 min on (8/1)) during the first 3 days of differentiation which resulted in 22% increase in CM yield (1.50 ±â€¯0.10 × 106vs 1.23 ±â€¯0.07 × 106 CM/mL). The combination of AS addition and change in agitation regime resulted in a production yield of 1.50 ±â€¯0.10 × 106 CM/mL which is comparable to that achieved in the rocker platform as described before (1.61 ±â€¯0.36 × 106 CM/mL). Increase in CM purity was achieved by changing of culture medium to RPMI1640 (without glucose) + 5 mM lactate +0.6 mM AS at day 10 of differentiation which resulted in 44.5% increase in CM purity at day 15. The increase in purity of CMs was due to the death of the non-CM cells (~76% of cell death). It is important to note that in the absence of glucose, lactate was consumed at a rate of 0.01 mmol/106 cells/h. Addition of glucose, even in small amounts, during the purification step prevents the process of CM purification, due to the growth of the non-CM cell population. In summary, hPSC (hESC-HES3 and hiPSC-IMR90) can be efficiently differentiated to CMs in a scalable spinner process which integrates 7 days of expansion (3.01 ±â€¯0.51 × 106 to 3.50 ±â€¯0.65 × 106 cells/mL) followed by 10 days of WNT modulated CM differentiation and 5 days of lactate based purification. CM yield of 1.38 ±â€¯0.22 × 106 to 1.29 ±â€¯0.42 × 106 CM/mL with 72.5 ±â€¯8.35% to 83.12 ±â€¯8.73% cardiac troponin-T positive cells were obtained from these cultures.