Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation

Reviewer #1 (Public Review):

Summary:
The authors have presented data showing that there is a greater amount of spontaneous differentiation in human pluripotent cells cultured in suspension vs static and have used PKCβ and Wnt signaling pathway inhibitors to decrease the amount of differentiation in suspension culture.

Strengths:
This is a very comprehensive study that uses a number of different rector designs and scales in addition to a number of unbiased outcomes to determine how suspension impacts the behaviour of the cells and in turn how the addition of inhibitors counteracts this effect. Furthermore, the authors were also able to derive new hiPSC lines in suspension with this adapted protocol.

Weaknesses:
The main weakness of this study is the lack of optimization with each bioreactor change. It has been shown multiple times in the literature that the expansion and behaviour of pluripotent cells can be dramatically impacted by impeller shape, RPM, reactor design, and multiple other factors. It remains unclear to me how much of the results the authors observed (e.g. increased spontaneous differentiation) was due to not having an optimized bioreactor protocol in place (per bioreactor vessel type). For instance - was the starting seeding density, RPM, impeller shape, feeding schedule, and/or any other aspect optimized for any of the reactors used in the study, and if not, how were the values used in the study determined?

Reviewer #2 (Public Review):

This study by Matsuo-Takasaki et al. reported the development of a novel suspension culture system for hiPSC maintenance using Wnt/PKC inhibitors. The authors showed elegantly that inhibition of the Wnt and PKC signaling pathways would repress spontaneous differentiation into neuroectoderm and mesendoderm in hiPSCs, thereby maintaining cell pluripotency in suspension culture. This is a solid study with substantial data to demonstrate the quality of the hiPSC maintained in the suspension culture system, including long-term maintenance in >10 passages, robust effect in multiple hiPSC lines, and a panel of conventional hiPSC QC assays. Notably, large-scale expansion of a clinical grade hiPSC using a bioreactor was also demonstrated, which highlighted the translational value of the findings here. In addition, the author demonstrated a wide range of applications for the IWR1+LY suspension culture system, including support for freezing/thawing and PBMC-iPSC generation in suspension culture format. The novel suspension culture system reported here is exciting, with significant implications in simplifying the current culture method of iPSC and upscaling iPSC manufacturing.

Another potential advantage that perhaps wasn't well discussed in the manuscript is the reported suspension culture system does not require additional ECM to provide biophysical support for iPSC, which differentiates from previous studies using hydrogel and this should further simplify the hiPSC culture protocol.

Interestingly, although several hiPSC suspension media are currently available commercially, the content of these suspension media remained proprietary, as such the signaling that represses differentiation/maintains pluripotency in hiPSC suspension culture remained unclear. This study provided clear evidence that inhibition of the Wnt/PKC pathways is critical to repress spontaneous differentiation in hiPSC suspension culture.

I have several concerns that the authors should address, in particular, it is important to benchmark the reported suspension system with the current conventional culture system (eg adherent feeder-free culture), which will be important to evaluate the usefulness of the reported suspension system. Also, the manuscript lacks a clear description of a consistent robust effect in hiPSC maintenance across multiple cell lines. There are also several minor comments that should be addressed to improve readability, including some modifications to the wording to better reflect the results and conclusions.

Reviewer #3 (Public Review):

In the current manuscript, Matsuo-Takasaki et al. have demonstrated that the addition of PKCβ and WNT signaling pathway inhibitors to the suspension cultures of iPSCs suppresses spontaneous differentiation. These conditions are suitable for large-scale expansion of iPSCs. The authors have shown that they can perform single-cell cloning, direct cryopreservation, and iPSC derivation from PBMCs in these conditions. Moreover, the authors have performed a thorough characterization of iPSCs cultured in these conditions, including an assessment of undifferentiated stem cell markers and genetic stability. The authors have elegantly shown that iPSCs cultured in these conditions can be differentiated into derivatives of three germ layers. By differentiating iPSCs into dopaminergic neural progenitors, cardiomyocytes, and hepatocytes they have shown that differentiation is comparable to adherent cultures. This new method of expanding iPSCs will benefit the clinical applications of iPSCs.

Recently, multiple protocols have been optimized for culturing human pluripotent stem cells in suspension conditions and their expansion. Additionally, a variety of commercially available media for suspension cultures are also accessible. However, the authors have not adequately justified why their conditions are superior to previously published protocols (indicated in Table 1) and commercially available media. They have not conducted direct comparisons. Additionally, the authors have not adequately addressed the observed variability among iPSC lines. While they claim in the Materials and Methods section to have tested multiple pluripotent stem cell lines, they do not clarify in the Results section which line they used for specific experiments and the rationale behind their choices. There is a lack of comparison among the different cell lines. It would also be beneficial to include testing with human embryonic stem cell lines. Additionally, there is a lack of information regarding the specific role of the two small molecules in these conditions. The authors have not attempted to elucidate the underlying mechanism other than RNA expression analysis.

For these reasons some aspects of the manuscript need to be extended:

1. It is crucial for authors to specify the culture media used for suspension cultures. In the Materials and Methods section, the authors mentioned that cells in suspension were cultured in either StemFit AK02N medium, 415 StemFit AK03N (Cat# AK03N, Ajinomoto, Co., Ltd., Tokyo, Japan), or StemScale PSC416 suspension medium (A4965001, Thermo Fisher Scientific, MA, USA). The authors should clarify in the text which medium was used for suspension cultures and whether they observed any differences among these media.

2. In the Materials and Methods section, the authors mentioned that they used multiple cell lines for this study. However, it is not clear in the text which cell lines were used for various experiments. Since there is considerable variation among iPSC lines, I suggest that the authors simultaneously compare 2 to 3 pluripotent stem cell lines for expansion, differentiation, etc.

3. Single-cell sorting can be confusing. Can iPSCs grown in suspensions be single-cell sorted? Additionally, what was the cloning efficiency? The cloning efficiency should be compared with adherent cultures.

4. The authors have not addressed the naïve pluripotent state in their suspension cultures, even though PKC inhibition has been shown to drive cells toward this state. I suggest the authors measure the expression of a few naïve pluripotent state markers and compare them with adherent cultures