102 Chapter 4 Expected Outcomes The efficient expansion of functional CMs (up to a 250-fold increase of CM number within 3-5 weeks) can be obtained from multiple hiPSC lines. For the method described here, hiPSC-CMs from 3 different differentiation protocols were all suitable for expansion in cardiac expansion medium, though the expansion capacity of the hiPSC-CMs differentiated using the B27 differentiation protocol appears to be the most robust (Figure 1). Before starting the expansion steps, hiPSC-CMs microscopically should have high amounts of beating areas by d8-9 and should be used between day 11 and day 14 of differentiation. After low cell-density replating of hiPSCCMs in culture flasks, addition CHIR to the cardiac culture medium efficiently induces hiPSC-CM proliferation and subsequent passaging (Figure 2). After 3-5 days of expansion, the hiPSC-CMs will reach a near confluent monolayer. The cryopreservation steps allow for biobanking of large batches of hiPSC-CMs that can be stored before expansion or after passage 1 or 2. Thawing of cryopreserved hiPSC-CMs is efficient (74% viable hiPSC-CMs, and during subsequent expansion hiPSC-CMs exhibit a similar growth curve when compared to fresh unfrozen hiPSC-CMs (Figure 3). After passage 2, we do not recommend to biobank the expanding hiPSC-CMs due to lower viability upon recovery. In a successful differentiation batch the expression of cardiac sarcomere protein α-actinin is usually more than 80%, and expanding cells should remain α-actinin positive during subsequent passaging. Upon CHIR optimization, up to 37% of the hiPSC-CMs will be positive for proliferative cell marker Ki-67 (Figure 4). Limitations Despite high hiPSC-CM differentiation efficiency (85%–99%) in 2D monolayer cultures (85%–99%), yet, batch-to-batch and cell line-to-line variability remains an issue for stable CM production. In our protocol, we provide the possibility to robustly expand and/or further purify hiPSC-CM cultures via serial passaging and cell contact inhibition. This ultimately allows for a 250 fold expansion of day 11 hiPSC-CMs. However, we have noticed that also cell line-toline and batch-to-batch variabilities exist. Moreover, the expansion capacity decreases when cells are passaged multiple times. Another limitation of our method is that hiPSC-CMs differentiation should be at least 50% (Flow cytometry positive for α-actinin) to induce an efficient proliferative response at day 11. One explanation for the absence of massive proliferation of unpure hiPSC-CM cultures would be cell-cycle inhibiting paracrine signaling factors secreted by the other cell types. Alternatively, in low efficiency cultures (<50%) the CM are phenotypically different from efficiently differentiated hiPSC-CMs (>70%). Lastly, when it comes to cryopreservation of hiPSC-CMs the cell viability after thawing reduces from P1 to P5 (70% to 28%), which was similar to the non-expanded age-matched CMs. Therefore, cryopreservation hiPSC-CMs is recommended up to passage 2. HiPSC-CMs exceeding passage 2 are suitable for the use in direct downstream assays.
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