Scalable manufacturing of clinical‐grade differentiated cardiomyocytes derived from human‐induced pluripotent stem cells for regenerative therapy

Abstract Basic research on human pluripotent stem cell (hPSC)‐derived cardiomyocytes (CMs) for cardiac regenerative therapy is one of the most active and complex fields to achieve this alternative to heart transplantation and requires the integration of medicine, science, and engineering. Mortality in patients with heart failure remains high worldwide. Although heart transplantation is the sole strategy for treating severe heart failure, the number of donors is limited. Therefore, hPSC‐derived CM (hPSC‐CM) transplantation is expected to replace heart transplantation. To achieve this goal, for basic research, various issues should be considered, including how to induce hPSC proliferation efficiently for cardiac differentiation, induce hPSC‐CMs, eliminate residual undifferentiated hPSCs and non‐CMs, and assess for the presence of residual undifferentiated hPSCs in vitro and in vivo. In this review, we discuss the current stage of resolving these issues and future directions for realizing hPSC‐based cardiac regenerative therapy.


| INTRODUCTION
Heart disease is the leading cause of death worldwide. No cure for severe heart failure has been established other than heart transplantation, and the number of donors is insufficient. To achieve an alternative to heart transplantation, the integration of medicine, science, and engineering is needed. Therefore, basic research aimed at cardiac regenerative therapy is currently one of the most active complex fields.
To date, a lot of basic research has provided numerous clues for the development of cardiac regenerative therapy, and the underlying treatment involves replacing damaged, necrotic, or fibrotic myocardial tissue with healthy myocardium. Given a large number of differentiated cardiomyocytes (CMs) required for clinical applications, it is ideal to use human pluripotent stem cells (hPSCs) that are self-renewing and capable of differentiating any cell types of the three germ layers.
Many methods for producing large numbers of differentiated CMs from hPSCs have been reported by utilizing the metabolic characteristics of hPSCs and differentiated CMs. To prepare a large number of hPSC-derived CMs (hPSC-CMs) more efficiently, an effective method for proliferating hPSCs while maintaining their undifferentiated state is needed. Methionine (Met), one of the essential amino acids, is highly consumed to maintain pluripotency, and the Met-depleted medium induces endoderm differentiation. Recently, we reported that tryptophan supplementation promotes cell proliferation while maintaining pluripotency. 1 In addition, non-invasive methods for the purification of hPSC-CMs and elimination of residual undifferentiated hPSCs are also required for successful and safe transplantation. We previously showed that glucose and glutamine are indispensable for the survival of non-CMs, including residual undifferentiated hPSCs, and these depleted conditions enable the purification of hPSC-CMs. [2][3][4] In addition, it is necessary to establish a method to minimize the contamination of residual undifferentiated hPSCs. A major advantage of metabolic selection is that it can handle a large number of cells at once and does not require genetic modification. For instance, PluriSln, a small molecule that has screened out 52,000 candidates for eliminating hPSCs, specifically induces cell death, while progenitors and differentiated cells are sparing. 5 In addition, de novo fatty acid (FA) synthesis is crucial for hPSC survival, and blockade of FA synthesis leads to mitochondrial-mediated apoptosis of hPSCs. 6,7 These results suggest that metabolism-based methods for proliferation and purification are very advantageous for application in regenerative medicine. To realize cardiac regenerative medicine, it is also necessary to evaluate the existence of residual undifferentiated hPSCs in vitro and in vivo. To assess the residual undifferentiated hPSCs in vitro, many methods have been reported, including flow cytometry, quantitative PCR (qPCR), droplet digital PCR (ddPCR), Raman spectrometry, ELISA, and biosensors. We reviewed the detection efficiency of residual undifferentiated hPSCs using these methods. Tumorigenicity studies to assess the risk of tumorigenesis when residual undifferentiated hPSCs are injected in vivo are also needed. Finally, we reviewed the clinical applications of hPSC-based regenerative medicine ( Figure 1). Reagents used in hiPSC cultures affect the quality and safety of the cells because xeno reagents would not only increase the risk of infection but also immune rejection in hiPSC-derived cell transplantation. 8 In addition, tests against endotoxins and serious pathogenic microorganisms, such as mycoplasma and human immunodeficiency virus, are also needed to further ensure the safety for clinical applications. 9 It has also been reported that clinical-grade hiPSCs should meet the following requirements. First, parental cell donors must meet the guidelines for tissue donations. Second, the cell handling process must be performed in a GMP-controlled environment using xeno-free reagents. Third, clinical-grade hiPSCs should be integration-free and biologically safe. 10 In addition, one of the most important aspects to avoid is the integration of external genes during reprograming of hiP-SCs. Initially, hiPSCs were generated by retroviruses expressing OCT4, SOX2, KLF4, and C-MYC. Random integration could cause insertional mutagenesis, and the possible though unlikely activation of oncogene C-MYC may cause tumorigenesis. Therefore, it has previously been reported that Sendai virus, episomal vectors, minicircle DNA, mRNA, miRNA, and proteins are all integration-free reprograming methods for hiPSCs. [11][12][13][14][15][16] In 2015, clinical-grade hiPSCs were generated by integration-free Sendai virus-based reprograming under xeno-free conditions. 10 In addition, it also requires consistent operation of the whole process from the arrangements of biomaterial, culturing, and freezing of cells to quality control testing for the purpose of constantly manufacturing products of the same quality as the already established product specification. These processes are validated and executed according to the standard operating procedures (SOPs).

| GENERATION OF CLINICAL-GRADE HIPSCS
Apart from SOPs, hiPSCs have some concerns owing to their characteristics.
First, hPSCs have often been observed to contain genomic mutations and abnormal karyotypes after passages. [17][18][19] As hPSCs have low-genomic stability, long culturing and frequent thawing of hPSCs affect karyotypic changes. 20 Therefore, it is important to test them after extended passage and frequent freeze/thaw processes. Although the mechanism has not yet been elucidated, some chromosomes are commonly susceptible to genetic mutations, and some genetic aberrations have the advantage of proliferation, resistance to apoptosis, and differentiation propensity in hPSCs. 19,21,22 Furthermore, it has been reported that cultured hESCs often have supernumerary centrosomes during mitosis, which is caused by overduplication within a single-cell cycle and mitotic failure. 23 This phenomenon is considered to be one of the causes of chromosome instability in cultured hESCs.
Second, epigenetic memories in somatic cells are inherited by hiP-SCs. The epigenetic landscape, defined by histone and DNA modifications, is indispensable for maintaining cell identity. Rewriting epigenetic memories, that is, erasing epigenetic memories of somatic cells and replacing them with memories of hiPSCs, is an important aspect of inducing pluripotency. Kim et al. showed that low-passage hiPSCs, which are reprogramed from adult somatic cells, have residual DNA methylation characteristics similar to those of somatic cells of origin, and this feature favors differentiation into cells of the somatic cell lineage of the donor. 24 Another report showed that epigenetic memories are transient in early passage mouse iPSCs (miPSCs), and molecular and functional differences caused by epigenetic memories are lost through repeated passages. 25 However, a subset of hiPSCs retains their epigenetic memory even after extended passaging. 26,27 From these results, genetic and epigenetic evaluation of hiPSCs is critical for their use in clinical applications.
For genetic testing of hiPSCs, karyotyping, G-band analysis, qPCR, fluorescent in situ hybridization (FISH), microarray, wholegenome/exome sequencing, and ddPCR are used. The advantages and disadvantages of these methods are listed in Table 1. Karyotyping is the most common strategy for detecting the size and structure of chromosomes. In standard G-band analysis, it is necessary to analyze at least eight chromosomes and 20 metaphase counts. 28 qPCR is used to detect variants in regions that have already been reported. FISH relies on indirectly or directly labeled probes to detect specific target sequences with fluorescence in metaphase chromosomes. Although FISH has high sensitivity, it is only a detectable known genetic aberration and is not suitable for genome-wide applications. ddPCR provides absolute quantification and detection of rare alleles independent of the number of amplification cycles when measuring the initial amount of nucleic acid in each sample, thus providing more precise and reproducible data than qPCR. 29 It enables the detection of copy number variations and single-nucleotide variants at a reasonable cost in comparison with next-generation sequencing (NGS), F I G U R E 1 Scalable manufacturing of clinical-grade hiPSC-CMs. Tryptophan-fortified media promotes the proliferation of hiPSCs. Large numbers of hiPSC-CMs are induced in a multilayer culture plate. Cardiac differentiation efficiency is evaluated by cell count, flow cytometry, and immunostaining. Orlistat treatment selectively eliminates residual undifferentiated hPSCs. Then, hiPSC-CMs were metabolically selected with glucose-and glutamine-depleted lactate-supplemented media. After purification, hiPSC-CMs are isolated, harvested, and cryopreserved. The purity of hiPSC-CMs and the contamination rate of residual undifferentiated hPSCs are assessed. After thawing, cardiac spheroids are produced. Beating profiles of cardiac spheroids are evaluated before transplantation. Cardiac spheroids are transplanted using our developed spheroids transplantation device. FISH, and array comparative genomic hybridization. Although G-band analysis, qPCR, and ddPCR are useful for analyzing known common genetic variants, these methods cannot detect variants if they are only present in 5-10% of the cells. 30 Furthermore, genome mapping with long fluorescently labeled DNA molecules on nanochannel arrays was developed to detect whole-genome structural variation. 31 NGS has the advantage of analyzing the whole genome with high resolution and detecting most genomic abnormalities, but it is difficult to assess repeatedly due to cost, interpretation of complex results, and data analysis workload. 32 Based on these observations, a method that is inexpensive, rapid, and accurate in its analysis is needed.
hiPSCs have two solid definitions: unlimited proliferative capacity and multiple differentiation ability. To produce a large number of hiPSC-CMs for transplantation in patients suffering from severe heart failure, a large number of hiPSCs are also needed. In addition, the characteristics of hiPSC-CMs vary slightly between experiments; therefore, it is preferable to induce a large number of hiPSC-CMs at once. To achieve this, as it also requires a large amount of expensive media, it is desirable to develop a novel method to proliferate hiPSCs more efficiently and affordably without any changes in stemness or genetic mutation.
In this aspect, supplementation of biomass for cell proliferation, that is, the transition of metabolic pathways that are suitable for cell proliferation, is a feasible strategy. In particular, studies on cancer cell metabolism provide numerous insights into hPSC metabolism because cancer cells have a proliferative capacity as well as hPSCs, and these cells have a similar metabolic strategy. Many cancer cells depend on the excess uptake of glucose to survive, despite the cells being exposed to a high-oxygen environment. This phenomenon, discovered by Otto Warburg, is called the Warburg effect or aerobic glycolysis and is the hallmark of all mammalian proliferating cells. hiPSCs are also highly dependent on glycolysis and secreted glucose-derived lactate ( Figure 2A). 2 In cancer cells, abundant metabolites from aerobic glycolysis are utilized for glucose-dependent lipid synthesis and nonessential amino acid production. In addition to glucose, many cancer cell lines also prefer glutaminolysis. Glutaminolysis supplies nicotinamide adenine dinucleotide phosphate (NADP+), reduced (NADPH) required for multiple reactions: DNA synthesis, de novo FA synthesis, amino acid synthesis, and telomere maintenance. 33 Thus, hPSCs are highly dependent on glucose and glutamine metabolism as a major energy source (Figure 2A, E). 2,3 As other metabolic pathways and enzymes for generating cytosolic NADPH, oxidative pentose phosphate pathway (oxPPP) branched from glycolysis, FA oxidation, folatemediated one-carbon (1C) metabolism, malic enzyme 1 (ME1), isocitrate dehydrogenase 1 (IDH1), nicotinamide nucleotide transhydrogenase, and nicotinamide adenine dinucleotide (NAD) kinase have been recognized. The major suppliers of NADPH have been reported to be oxPPP, ME1, and 1C metabolism in proliferating and cancer cells. 34,35 A recent study revealed that oxPPP is a major producer of NADPH and loss of oxPPP by knockout of glucose-6-phosphate dehydrogenase (G6P) drastically decreases NADPH/ NADP and cell growth in cancer cells. 36 In addition, oxPPP dysfunction induces ME1 and IDH1 flux to generate NADPH from glutaminolysis, but folate-mediated 1C metabolism cannot function because of dihydrofolate reductase dysfunction in G6P knockout cancer cells. 36 These results suggest that oxPPP not only contributes to NAPDH generation but also folate-mediated 1C metabolism. hPSCs also utilize glucose as a major energy source, and oxPPP-related gene expression and metabolite levels are more abundant than hPSC-CMs. 2 With regard to the other amino acids for cancer proliferation, serine and glycine are well-known and major carbon sources of folate-mediated 1C metabolism. Serine is synthesized using 3-phosphoglyceric acid and alpha-ketoglutarate derived from glycolysis and glutaminolysis, respectively. 1C metabolism includes the folate and Met cycles; generates NADPH, nucleotides, and S-adenosyl Met (SAM); and supports cell growth, proliferation, nucleotide synthesis, redox reductive metabolism, and DNA/histone methylation ( Figure 2E). 37 In hPSCs, serine and glycine are utilized to survive, but whether these amino acids are important for proliferation has not been analyzed. We previously evaluated the consumption profiles

| PRODUCTION OF CLINICAL-GRADE HPSC-CMS
It is estimated that more than 1 Â 10 9 hiPSC-CMs per patient would be required to repair the loss of cardiomyocytes. 38  hPSCs, and it has been suggested that these hPSC-CMs show various characteristics because these methods have different points in the culture system 2D or 3D and combination of recombinant proteins and small molecules (Table 2). 4,[39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] As there is a concern about a large number of variations in hPSC-CMs for transplantation, 55 it is desirable to induce large numbers of hPSC-CMs at the same time and develop non-invasive methods to prepare hPSC-CMs only. One of the concerns with regard to mass culture in 2D culture is that there are fluctuations in differentiation efficiency, resulting from starting cell density and cell confluency. 48,49 In a 3D culture system using a bioreactor, there is no need to consider these points, although attention should be paid to the variability in the size of individual spheroids.
However, once the hPSC-CMs are aggregated, it is difficult to completely dissociate into single cells, and cell aggregates are removed by a cell strainer, 56 which may result in cell loss. To obtain a large number of hiPSC-CMs in 2D culture, we developed a method to induce hiPSC-CMs in a multilayer culture plate with active gas ventilation. 4 In this system, hiPSCs were efficiently differentiated into CMs, and these cells started beating on day 7-10. Cell viability was sustained at over 95% and the total number of hiPSC-CMs in single, 4, and 10 layers under active gas ventilation was 1.5 Â 10 8 , 6.7 Â 10 8 , and 1.5 Â 10 9 cells, respectively. We previously showed that hiPSC-derived non-CMs highly depend on glycolysis and glutaminolysis. 2,3 These cells cannot survive under glucose-and glutamine-depleted conditions ( Figure 2C). Furthermore, hPSCs hardly uptake lactate and convert lactate to pyruvate, thus, it is difficult for hPSCs to use lactate as an energy source. In contrast, hiPSC-CMs uptake lactate which is incorporated as pyruvate in the tricarboxylic acid (TCA) cycle ( Figure 2B,D). 3 These metabolic differences enable the purification of >99% hiPSC-CMs. 2,3 Applying this method to differentiation in multilayer culture plates, hPSC-CMs were metabolically selected with glucose-and glutamine-depleted lactate-supplemented media, and over 1.0 Â 10 9 hiPSC-CMs were obtained in 10-layer culture plates at the same time after purification, and immunocytochemistry data showed that almost all cells were cTNT positive. 4 Interestingly, hiPSC-CMs cultured in glucose-depleted lactatesupplemented conditions showed a metabolically and transcriptionally mature direction. 57 Genetic approaches typically knock in an inducible suicide gene in the promoter region or gene body, which is expressed only in undifferentiated hPSCs. 65,73,75 For example, it has been reported that killer red protein, which strongly induces phototoxicity by generating reactive oxygen species, induces the selective cell death of hPSCs. 65 Furthermore, to improve the safety of hPSC-based cell transplantation, hPSC lines with two drug-induced safeguards that have different functions and address different safety concerns have also been reported. 75 That is, one small molecule helps to eliminate residual undifferentiated hPSCs and the other small molecule helps to kill all of the hPSC-derivatives in case adverse events arise in vivo. In addition, strategies for genetically and pharmacologically deleting survivin to induce apoptosis in hESCs and teratomas have also been reported. 74  and showed that it can selectively and effectively eliminate residual undifferentiated hPSCs within just 1-2 h by binding to alkaline phosphatase on the surface of hPSCs. 67 Moreover, it has also been reported that salicylic diamines, which inhibit mitochondrial ATP production by decreasing the oxygen consumption rate, show selective cytotoxicity to miPSCs and hiPSCs but not to miPSC-CMs. 70 However, salicylic diamines do not have any effect on miPSC-CMs, and the exposure time allows selective elimination of miPSCs and hiPSCs. In addition, the elimination of miPSCs and hiPSCs by salicylic diamines is not complete and transplantation is compromised. 70 Among these methods, metabolismbased approaches offer some advantages in terms of cost, time, simplicity, scalability, and safety because they do not require gene modification, expensive antibodies, and time to perform cell sorting. To successfully eliminate residual undifferentiated hPSCs, it is important to understand the metabolic characteristics of hPSCs. As mentioned above, hPSCs are mainly dependent on glycolysis and oxPPP for ATP production and proliferation, thus, they cannot survive under glucosedepleted conditions ( Figure 2C). 2 In addition, glutamine is a key metabolite for the survival of hPSCs. Glutamine is utilized in some contexts as a source of nucleotide synthesis, glutathione synthesis, non-essential amino acid synthesis, and epigenetic modification. In cancer cells, glutamine contributes to FA synthesis via reductive carboxylation under hypoxia. 76 We have previously shown that glutamine not only contributes to nucleotide and glutathione syntheses, but also ATP synthesis via the latter steps of the TCA cycle, and this unique pathway is indis-  Figure 2E). 37 Met depletion induces upregulation of the p53-p38 signaling pathway, which is critical for cell cycle arrest and survival. 37 In the short-term depletion of Met, the abbreviated G1 phase, which is characteristic of hPSCs, is prolonged and finally leads to cell cycle arrest and causes the differentiation of three germ layers due to histone and DNA methylation and a decrease in NANOG expression. p53 binds to the NANOG promoter and negatively regulates mESCs. 79 82,83 Although doxorubicin is also approved by the FDA as well as orlistat, a high dose of doxorubicin can increase the risk of congestive heart failure. 84 Chour et al. showed that low-dose doxorubicin is effective in eliminating proliferative hESCs from differentiated hESC-CMs. 85 Low-dosage doxorubicin administration did not affect the gene expression and proteome profiles of hESC-CMs. 85 In the teratoma formation assay, doxorubicin-treated cells were not proliferative, and no teratomas were observed in vivo. Nevertheless, the role of apoptosis in doxorubicin-induced cardiotoxicity has been well established, and it seems unsuitable for the selective elimination of residual undifferentiated hPSCs in hiPSC-CMs. 86 In addition, using brentuximab vedotin, which is effective in eliminating CD30-positive cancer cells, hPSCs have also been used to eliminate residual undifferentiated hPSCs because they also express CD30. 62

| IN VITRO TUMORIGENICITY TESTS FOR HPSC-DERIVED PRODUCTS
For clinical applications, it is crucial to establish a system to eliminate residual undifferentiated hPSCs and for assessing the contamination of However, there is increasing recognition that the assessment of residual undifferentiated hPSCs is indispensable for the safe transplantation of hPSC derivatives. Therefore, it is important to combine methods (i.e., flow cytometry, qPCR, ddPCR, Raman spectrometry, immunocytochemistry, ELISA) considering the advantages and disadvantages of these methods.
As mentioned above, 0.025% contamination of residual undifferentiated hPSCs can be a risk factor for tumorigenesis in the transplantation of hPSC derivatives. 59 Therefore, it is crucial to develop or select highly quantitative methods and suitable factors for detection. The detection methods and efficiency are listed in Table 4. 88 88 The surface-enhanced Raman scattering-based assay showed that the detection limit of SSEA-5 and TRA-1-60 was 0.0001%, and the detection efficiency was improved dramatically. 89 These results suggest that adequate selection of hPSC-specific markers and highly sensitive methods is both necessary to accomplish the successful detection of residual undifferentiated hPSCs. Although flow cytometry has some advantages in terms of speed and quantification, it should be noted that the gating technique highly affects these results.
qPCR is simple and fast, but has the disadvantage that it is difficult to determine the exact number of residual undifferentiated hPSCs. A spike assay with hPSCs in hepatic endoderm showed that the detection limits for ESRG (Embryonic Stem Cell Related), CNMD (Chondromodulin), and SFRP (Secreted Frizzled Related Protein 2) were 0.005%, 0.025%, and 0.025%, respectively, while those for SOX2, OCT4, and NANOG were 1%, 2.5%, and 5%, respectively. 91 LIN28 was not suitable for the detection of hPSCs in the hepatic endoderm because the detection limit of LIN28 was 5%. 91 Other reports showed that the detection limits for SSEA-5 and TRA-1-60 were 0.1%-1.0% and 0.01%-0.1%, respectively. 88-90 ddPCR enables direct quantification of DNA/mRNA copies, and it can prevent the bias that comes from non-target gene amplification using qPCR. Furthermore, it enriches the template in partitioning and enables more sensitive detection of rare targets than PCR. 96 It is also used to quantify circulating fetal and maternal DNA from cell-free plasma. 96 ddPCR analysis with LIN28 probes detected as low as 0.001% residual undifferentiated hPSCs in primary CMs.
Artyuhov et al. showed that the detection limit of OCT4, TGDF, and LIN28 was 0.01%, whereas that with ddPCR was 0.002%. 94 Watanabe et al. reported a detection efficiency of 0.00002% when ddPCR was performed after enrichment of stem cell markers using magnetic beads. 97 In addition, it has been reported that the detection efficiency of hPSCs in hPSC-CMs suggests that ddPCR is more suitable than qPCR for the assessment of residual undifferentiated hPSCs.

| IN VIVO TUMORIGENICITY TESTS FOR HPSC-DERIVED PRODUCTS
Monitoring hPSC-CMs after transplantation is important, even if it is confirmed that there are no undifferentiated hPSCs remaining before transplantation. The general approach for tumorigenicity testing is based on ectopic transplantation into small animals, but the detection efficacy of tumors varies depending on the animal strain and immunosuppression. Therefore, the test animals should be deficient in cytotoxic T-lymphocyte activity. 98 To investigate the safety of transplanted cells, undifferentiated hPSCs and tumor cell lines were transplanted and the duration of tumorigenesis examined (Table 5) NOD/SCID mice develop thymic lymphomas frequently with age. 106,107 This incidence often results in shortened life span, which can confound the results of tumorigenicity testing. 99 The transplantation method needs to be considered. hPSCs are generally injected as clumps because hPSCs easily induce apoptosis in a single-cell state.
Therefore, it is difficult to transplant accurate cell numbers into animals. To resolve this issue, it has been reported that a mixture of hPSCs as a single state and mitomycin-C-treated fibroblasts with Matrigel could be engrafted into SCID and NOD/SCID mice. 59 is hoped that effective methods will become widespread and that many patients can be treated.

| CONCLUSION AND PERSPECTIVE
In this review, we present a series of steps: control of hiPSC quality, expansion of hiPSCs, mass induction of CMs from hiPSCs, purification of hiPSC-CMs, removal of residual undifferentiated hPSCs, evaluation of remaining undifferentiated hPSCs in vitro and in vivo, and evaluation of cardiac function after transplantation of hiPSC-CMs.

| Quality maintenance and mass preparation of hPSCs
First, we showed that it is necessary to maintain the quality of hPSCs, that is, to keep them in a state without significant genomic mutations; that is, it is necessary to show that the following genomic findings are extant as to rule out whether tumorigenicity could be present in the hiPSCs to be transplanted: no karyotype abnormality and no structural abnormality, including single-nucleotide variants/ single-nucleotide polymorphs or copy number abnormalities of tumor-related genes, which are referred to in the COSMIC census and Shibata list. 120 It is necessary to induce large amounts of hPSCs without mutating tumor-related genes, and so far, we found that tryptophan, an amino acid, proliferates hPSCs with high efficiency without causing genetic abnormalities. Furthermore, the addition of tryptophan to the culture medium is inexpensive, making mass maintenance possible. In addition, to realize medical treatment that provides uniform quality of treatment to patients with hiPSC products, it is important to develop a simple and inexpensive method to control the quality of hiPSCs and their evaluation method. In particular, since mass culture is necessary to achieve hiPSC-based cell therapy, it is desirable to establish a method to evaluate the quality of hPSCs for mass culture.

| Preparation of highly purified hPSC-CMs for cell therapy
It is necessary to develop a technology that can stably and efficiently induce target cells from hPSCs. It is also necessary to develop a technology that enables non-invasive detection of differentiation efficiency and eliminates residual undifferentiated hPSCs and non-target proliferating cells. hiPSC-CMs in 3D are easy to collect for quality evaluation, but it is difficult to collect cells in 2D. Therefore, an analysis method using culture supernatants would be very useful.
In addition, if the possibility of residual undifferentiated hPSCs or non-targeted proliferating cells is shown, a method for the specific removal of these cells should be established. To date, we successfully induced a large number of hPSC-CMs in a multilayer culture plate, which enabled us to induce mass differentiation in 2D; however, many issues remain to be solved, such as the stability of differentiation and how to reduce the differences in properties between other lots as mentioned above. Although we have not reached that point yet, the purification of hPSC-CMs using glucose-and glutaminedepleted lactate-supplemented medium, which utilizes the difference in metabolism between hPSC-CMs and non-CMs, has made it possible to obtain large numbers of differentiated CMs at low cost and with little effort.

| Cardiac maturation for cell therapy
To build an ideal scalable manufacturing system for clinical-grade therapy products, there have been various reports on the types of hiPSC-CMs surviving in lactate medium, and it has been reported that hiPSC-CMs survive in glucose-and glutamine-depleted lactate-supplemented medium are metabolically more mature. 57,58,121 123 Therefore, for transplantation of more mature hPSC-CMs, it is necessary to find conditions that allow survival after freezing and good engraftment during transplantation.

| Evaluation and elimination of residual undifferentiated hPSCs
Although there have been many reports on the removal of residual