Skip to main content
Download PDF

Generation and purification of iPSC-derived cardiomyocytes for clinical applications

Stem Cell Research & Therapy volume 16, Article number: 189 (2025) Cite this article

Abstract

Background

Over the past decade, the field of cell therapy has rapidly expanded with the aim to replace and repair damaged cells and/or tissue. Depending on the disease many different cell types can be used as part of such a therapy. Here we focused on the potential treatment of myocardial infarction, where currently available treatment options are not able to regenerate the loss of healthy heart tissue.

Method

We generated good manufacturing practice (GMP)-compatible cardiomyocytes (iCMs) from transgene- and xenofree induced pluripotent stem cells (iPSCs) that can be seamless adapted for clinical applications. Further protocols were established for replating and freezing/thawing iCMs under xenofree conditions.

Results

iCMs showed a cardiac phenotype, with the expression of specific cardiac markers and absence of pluripotency markers at RNA and protein level. To ensure a pure iCMs population for in vivo applications, we minimized risks of iPSC contamination using RNA-switch technology to ensure safety.

Conclusion

We describe the generation and further processing of xeno- and transgene-free iCMs. The use of GMP-compliant differentiation protocols ab initio facilitates the clinical translation of this project in later stages.

Background

Despite significant advances in its medical management, cardiovascular disease (CVD) remains the leading cause of morbidity and mortality, accounting for more than 17 million deaths per year worldwide [1]. In fact, myocardial infarction (MI), also known as heart attack, is the most common form of CVD and is caused by decreased or complete cessation of blood flow to the heart. In the adult mammalian heart, the tissue damage following ischemia or occlusion of coronary arteries results in cardiomyocyte necrosis that triggers an inflammatory response and is quickly replaced with dense fibrotic scar tissue [2]. These processes also contribute to additional heart failure development, such as congestive heart failure or dilated cardiomyopathy. Furthermore, patients with MI show in most cases comorbidity, for example, anaemia, chronic kidney disease, or chronic obstructive pulmonary disease [3].

Different techniques are available in order to restore blood supply to the infarcted myocardium, including thrombolytic drugs, balloon angioplasty, stent placement, and bypass surgery depending on the severity of the blockages. However, these therapies do not aim to regenerate the already damaged myocardium. Pluripotent stem cell (PSC) therapy, which includes both embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), has emerged as an alternative strategy for the treatment of CVD [4, 5]. With the ability to differentiate into any cell type within the human body, it is the goal of researchers to be able to isolate somatic cells from a patient and to generate ‘personalized’ cells, such as cardiomyocytes, for tissue regeneration. The potential access to patient specific cells, thus maintain the host genetic profile and their ability to be differentiated into any cell type, makes them an ideal cell source to investigate CVD resulting from genetic or non-genetic pathologies, hence allow for the development of new diagnostic and therapeutic approaches [4, 5]. Implantation of PSC derived-CM into different animal models, such as mouse, rat, guinea pig, pig and non-human primate have shown cell survival and functional improvement of the injured heart [6,7,8,9]. In general, the areas of application of iPSC-derived cells are diverse, for example in the field of drug testing, bioengineering or disease modelling (Fig. 1).

Fig. 1
figure 1

Schematic overview of the workflow to produce purified transgene- and xenofree human iPSC-derived cardiomyocytes (iCMs). First somatic cells from a patient are reprogrammed into induced pluripotent stem cells (iPSCs). These iPSCs are differentiated into cardiomyocytes, followed by purification. Human iCMs are either replated or frozen for further applications, such as tissue engineering or disease modelling

Full size image

However, there is a lack of standardization for differentiation protocols with subsequent purification of cells of interest. Further improvement and homogenization in production is essential for future clinical trials. Standardization helps to guarantee that the cell lines are properly characterized and meet defined criteria in terms of genetic stability, differentiation efficiency, and functionality. Regulatory bodies such as the FDA (Food and Drug Administration) and EMA (European Medicines Agency) require that iPSC-derived products, including cardiomyocytes, meet stringent standards for quality and consistency [10]. Therefore, transgene- and xenofree iCMs were generated and tested in vivo. Furthermore, xenofree protocols for replating and freezing are a valuable tool for a successful clinical translation.

Methods

Isolation and reprogramming of PBMCs

Human peripheral blood was collected with written informed consent according to the permission from the cantonal ethics commission of Zurich, Switzerland [KEK-ZH-2014-0430]. Peripheral blood mononuclear cells (PBMCs) were isolated according to standard protocols using the Ficoll-Paque method and cultured in Stem Pro-34 SFM Medium (Life technologies) supplemented with SCF, FLT-3 (100ng/ml), IL-3 and IL-6 (20ng/ml) for four days (Sigma). Then, PBMCs were reprogrammed using the CytoTune-iPSC 2.1 Sendai Reprogrammig Kit (ThermoFisher) following the manufacture’s protocol [11]. From day 20, emerging colonies were manually picked based on their morphology, and subsequently cultured and passaged as iPSCs thereafter (Fig. 2A). The generated iPSCs were cultured on iMatrix-511 (Takara) coated plates with iPSC StemBrew medium (Miltenyi Biotec). The culture medium was changed every day.

Fig. 2
figure 2

Human blood derived-iPSCs show the pluripotent nature of generated iPSCs. (A) Schematic representation of the timeline and experimental setup. (B) Morphology of an iPSC colony in culture. Immunostaining for pluripotency markers OCT4, SOX2, TRA-1-60, TRA-1-81 (all Alexa 594) and Alkaline phosphatase staining of cultured iPSCs. DAPI is shown in blue. (C) Reprogrammed cells show a normal karyotype. (D) Quantitative qRT-PCR assay for expression analyses of pluripotency markers OCT4, SOX2, NANOG, REX1, and DNTM3 in iPSCs. Data are normalized with GAPDH and relative to the original PBMC. (E) Differentiation of iPSCs into three germ layers. Differentiated cells were analyzed by qRT-PCR for the expression of endodermal (FOXA2 and CXCR4), mesodermal (MSX1 and Hand1) and ectodermal (PAX6 and OTX2) lineage markers. Data are normalized with GAPDH and relative to undifferentiated iPSCs. Quantitative data are presented as mean ± standard deviation (n = 3). One representative clone DW4 is shown in Fig. 2. (Scale bars: 200 μm)

Full size image

Differentiation of iPSC into cardiomyocytes

Cardiac differentiation from iPSCs was performed following the instructions based on provided protocol of the StemMACS™ CardioDiff Kit XF (Miltenyi Biotec). In short, iPSCs were seeded on iMatrix-511 coated dishes and cultured for 24 h in mesoderm induction media (MIM), followed by 24 h in cardiomyocyte maintenance media (CMM) and 24 h in cardiac induction media (CIM). Then cells were cultured in cardiomyocyte maintenance media until day 10 or 17. Media was changed every day. Human iCMs were purified before further processing.

Purification of human iCMs

The RNA switch was prepared as previously described [12,13,14]. Briefly, template DNA for in vitro transcription of normal mRNA was amplified by PCR from a vector encoding for either Barnase, Barstar, or puromycin using appropriate primers with the T7 promoter and poly(A) tail. We either used miR-302a-5p to target directly iPSC or miR-1 for iCMs. All template DNAs were purified using the MinElute PCR Purification Kit (Qiagen). The RNAs were transcribed using MEGAScript T7 Transcription Kit (ThermoFisher) using 1-Methylpseudouridine-5’-Triphosphate and Anti Reverse Cap Analog, ARCA. Next, the transcribed RNA was purified by RNeasy MinElute Cleanup Kit (QIAGEN). The concentration was determined by Qubit microRNA Assay Kit.

First iCMs were seeded and mRNA was transfected using Lipofectamin RNAiMAX (ThermoFisher) Transfection Reagent at the following day. The transfection complex was added to the cells in a drop-wise manner and the plate was agitated before being placed into the incubator, followed by a medium change after 4 h. To remove non-transfected cells, puromycin selection was carried out by treatment with 2 µg/ml puromycin for 24 h. Cells were finally analyzed by flow cytometry. When using the miR-1, the transfection was performed in suspension and the tube was inverted every 10 min. The cells were then treated with 4 µg/ml puromycin for 24 h.

Characterization of human PBMC-derived iPSCs and iCMs

Three different iPSC lines (DW4, DW5, DW8) were characterized as followed:

  • Quantitative Real-Time PCR (qPCR)

Total RNA was obtained using the RNeasy Mini Kit (Qiagen) and reverse-transcribed to cDNA using iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s protocol. qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and standard conditions on a QuantStudio 7 flex real-time PCR system (Applied Biosystems). Primers are listed in Supp. Data Table 1. The gene expression level analysis was normalized versus GAPDH and conducted in triplicate.

  • Alkaline Phosphatase (AP) activity:

Alkaline phosphatase activity was assessed using Alkaline phosphatase detection kit (Merck) according to manufacturer’s instructions. Briefly, cells were fixed with 4% Paraformaldehyde/4% Sucrose (Sigma) for 2 min and washed once with TBS-T (Tween 0.05%). Then staining solution (2:1:1 Fast Red Violet: AS-BI phosphate solution: water) was applied for 15 min protected from light at RT. Reaction was stopped by washing with TBS-T (Tween 0.05%) and DPBS.

  • Immunofluorescence

Cells were fixed in 4% paraformaldehyde/4% Sucrose in PBS for 20 min at RT. After washing, cells were blocked and permeabilized in 10% goat serum (Sigma) with 0.1% Triton X-100 (Sigma) for 1 h and incubated with primary antibodies overnight at 4 °C (PBS with 3% goat serum and 0.1% Triton X-100). Followed by the secondary antibodies staining for 2 h at RT. Nuclei were counterstained with DAPI for 20 min. The primary and secondary antibodies are listed in Supp. Data Tables 2 and 3.

  • Differentiation into three germ layers

The iPSCs were plated and differentiated according the StemMACS Trilineage Differentiation Kit protocol (Miltenyi Biotec). On day 7, cells were harvested for RNA extraction and analyzed by qPCR (Supp. Data Table 1).

  • Karyotyping

Proliferating iPSCs were arrested at metaphase by treatment with 80 ng/ml colcemid for 1 h at 37 °C. Subsequently, cells were dissociated with accutase, treated with hypotonic solution (0.075 M KCL) and fixed with methanol-acetic solution (3:1). Metaphase spreads were stained by Quinacrine (Sigma), examined with Zeiss Axioskop HBO 50 fluorescent microscope (Zeiss) and arranged in Ikaros Software (MetaSystems).

  • Mycoplasma detection

The absence of mycoplasma in the culture medium was analyzed on a regular basis by sending samples to eurofins.

  • Flow cytometric analysis

Cells were dissociated with accutase (Sigma) and incubated in fluorescence activated cell sorting (FACS) buffer (0.1% BSA in PBS) containing antibodies for extracellular proteins and Live/Dead Fixable Near-IR Dead Cell stain (Thermofisher) for 30 min at 4 °C. Subsequently, cells were fixed in 1% paraformaldehyde for 20 min at RT and permeabilized by FACS buffer and 0.5% Saponin for 20 min at 4 °C. Then, intracellular proteins were stained with corresponding antibodies for 30 min at 4 °C, followed by 3 washes. The antibodies are listed in Supp. Data Table 4. Cells were acquired using a LSR Fortessa (BD Bioscience) and the data were analyzed by FlowJo software (Tree Star).

  • In vivo safety assay

The veterinary office of the Canton Zurich, Switzerland approved all animal experiments (ZH174/2020). The work described in this paper has been reported in line with the ARRIVE guideline 2.0.

Either 1,000,000 purified iCMs or 200,000 undifferentiated hiPSCs were injected subcutaneously into one dorsal flank of NOD.CB17-Prkdcscid mice in order to investigate the risk of tumor formation. Anaesthesia was not required for the almost painless injection procedure. Tumor growth was assessed non-invasively by a sliding caliper 3 times per week for 12 weeks. First tumor formations were detected after about 7–8 weeks. After the follow-up time of 12 weeks (84 days), if end points have not been reached earlier, all animals will be euthanized by Carbon dioxide (CO2).

Detaching iCMs for replating and freezing

Cell detachment was performed using a 1:1 trypsin and Stempro accutase mixture, followed by collagenase diluted in RPME 1640 for cell dissociation. Then, cells were either seeded in cardio attachment medium (RPME 1640 with Glutamax, B27 Supplement, 20% KO Serum Replacement, Y27632 (10 μm)) or frozen down (KO Serum Replacement and 10% DMSO). After replating medium was replaced to cardiac maintenance medium II (CMMII, cardio attachment medium without Y27632) 48 h later. Cell culture plates were coated with iMatrix-511.

Results

Characterization of xeno-free and transgene-free HiPSCs derived from a blood sample

After blood collection, the PBMCs were isolated from the blood by the Ficoll-Paque method. Subsequently, the cells were reprogrammed according to the protocol using Sendai Virus Kit an efficient and integration-free technique [11]. In total, three different clones (DW4, DW5 and DW8) were selected, expanded and further characterized (Fig. 2 and Supp. Figure 1). The main Figs. 2, 3, 4 and 5 show DW4, in contrast results of DW5 and DW8 are presented in the supplementary Figs. 1–5. Besides Oct4 and Sox2, human specific pluripotency markers TRA-1-80 and TRA-1-60 were expressed in the hiPSC colonies of clone DW4 (Fig. 2B). All colonies showed high alkaline phosphatase (AP) activity (Fig. 2B). qRT-PCR showed an upregulated expression of the pluripotency associated transcription factors OCT4, SOX2, NANOG, REX1, and DNTM3 compared to the original, non-reprogrammed PBMCs (Fig. 2D). To assess the pluripotent potential of the generated hiPSCs, cells were differentiated in vitro and then further analyzed. The expression of early differentiation markers revealed differentiation into all three germ layers: endoderm (FOXA2 and CXCR4), ectoderm (PAX6 and OTX2) as well as mesoderm (MSX1 and HAND1) (Fig. 2D). In contrast, the pluripotency markers OCT4, SOX2 and DNMT3 were downregulated in comparison to their expression before differentiation. Furthermore, karyotyping was performed to detect structural and numerical abnormalities. Reprogrammed cells retained a normal karyotype (Fig. 2C). All results confirmed the pluripotent nature of the transgene and xenofree generated human iPSCs.

Fig. 3
figure 3

Human iPSC-derived cardiomyocytes show a cardiac phenotype. (A) Schematic representation of the timeline and experimental setup. (B) After differentiation of iPSC into cardiomyocytes immunostaining was performed for following markers: alpha smooth muscle actin (αSMA), cTNT, Myl2, Oct4, Ki67, Nanog and Dapi. (C) Flow cytometry dot plot of Oct4, cTnT and SIRPA expression for iCMs DW4 and undifferentiated iPSCs. (D) Quantitative RT-PCR assay for expression analyses of mesodermal, cardiac and pluripotent markers at day10 and day17 after differentiation. Data were normalized with GAPDH and relative to the undifferentiated iPSCs. Quantitative data are presented as mean ± standard deviation. One representative clone DW4 is shown. MIM: Mesoderm Induction Media, CMM: Cardiac Maintenance Media, CIM: Cardiac Induction Media, CIM II: Cardiac Induction Media II. (Scale bars: 100 μm)

Full size image
Fig. 4
figure 4

Purified human iPSC-derived cardiomyocytes are a safe cell source for in vivo application. (A) Schematic representation of the experimental setup. B + C) Flow cytrometry dot plot of iCMs and iPSC before (upper row) and after (lower row) purification. The proportion of artificially mixed iPS cells in the iCMs is 10% (B) and 50% (C) respectively. Pie charts show percentage of iCMs (red, cTNT positive) and iPSC (blue, Oct4 positive) for the respective marker. (D) Measurement of the volume (mm3) over time after injecting subcutaneously matrigel containing either iCMs (1,000,000 cells) or iPSCs (200,000 cells) using an immunodeficient NSG mouse model. (n = 5) (E) Summary of the tumor formation after subcutaneous injection of iCMs and iPSCs. One representative clone is shown in Fig. 4 (DW4)

Full size image
Fig. 5
figure 5

Human iPSC-derived cardiomyocytes can be replated and frozen/thawed for later applications without phenotype change. (A) Quantitative RT-PCR assay for expression analyses of mesodermal, cardiac and pluripotent markers at day10 after differentiation, after replating (P1) and after freeze/thawing (F/T). Data were normalized with GAPDH and relative to the undifferentiated iPSCs. Quantitative data are presented as mean ± standard deviation. (B + C) Immunostaining for different markers (alpha smooth muscle actin (αSMA), Nkx2.5, cTNT, Myl2, Nanog, Ki67, Actinin, Oct4, and Dapi) of cultured iCMs after replating or freeze/thawing. (D) Pie charts of analyzed flow cytometry show percentage of iCM (red, cTNT positive) and iPSC (blue, Oct4 positive) for the respective marker after replating or freeze/thawing. One representative clone DW4 is shown

Full size image

Characterization of human xenofree iPSC-derived cardiomyocytes

After reprogramming human PBMCs into iPSCs, 3 different hiPSC clones were differentiated into iCMs and further characterized (Fig. 3 and Supp. Figure 2). Generated iCMs showed a beating phenotype around day 7 and cells were analyzed on day 10 or day 17 (Supp. Data Movie 1).

Human iCMs of clone DW4 lost the expression of pluripotency genes (OCT4 and SOX2) and started to express mesoderm associated (ISL1 and GATA4) and cardiac specific genes (cTNT, IRX4, MLC2v, HAND1, CX40, NPPA, NPPB, TBX5, MYL7, PITX2 and TBX18) (Fig. 3B). Moreover, human iCMs expressed aSMA, cTNT and MYL2 at protein level, whereas OCT4 and NANOG could not be detected (Fig. 3B). Interestingly, the proliferation marker Ki67 is still expressed after differentiation. Flow cytometry of cTNT, SIRPA and OCT4 was performed to evaluate the differentiation efficiency (Fig. 3C). About 90% of all alive cells were positive for the cardiac marker cTNT. Only 0.65% of cells were positive for OCT4 at day 10 and 2.55% at day 17.

Generation of pure iCMs for safe in vivo applications

Even few iPSC can lead to a tumor formation in vivo [15]. Therefore, purification of cells after differentiation is essential, in particular for clinical applications. We used the microRNA (miRNA)-sensing synthetic mRNA method, called RNA-switch. Despite not being widely known, this unique method allows for a highly efficient purification of iPSCs [12]. Human iPSCs can be specifically targeted and eliminated based on miRNA expressed miR-302a-5p only in iPSC [13]. miRNAs are small non-coding RNAs that regulate gene expression by either repressing translation or promoting mRNA degradation. Depending on the activity of the target miRNA, the expression of the selection marker protein can be either downregulated or remain unaffected. This mechanism has been used to express apoptotic regulatory proteins, allowing for the selection of cell types based on their miRNA activity (Fig. 4a).

The differentiation efficiency of our lines was already high; therefore, we simulated a lower efficiency by adding iPSC to the iCMs culture (Fig. 4 and Supp. Figure 4). Human iPSCs were directly targeted by using the miR-302a-5p [13]. An iPSC-content of about 10% was reduced to less than 1% (OCT4 positive cells measured by flow cytometry) (Fig. 4B). Interestingly, even a concentration of about 50% OCT4 positive cells were eliminated and less than 1% were detected after purification (Fig. 4C). In a next step, we injected either 200,000 iPSCs or 1 million purified iCMs subcutaneously into NOD-SCID mice. After about 7 weeks, an increase of the tumor volume was detected and further tracked until a maximum of 1000 mm3 (Fig. 4D). In total, injection of iPSCs lead in all cases to tumor formation (n = 5 per clone). In contrast, no tumor was detected after injecting a 5-times higher amount of purified iCMs in comparison to the iPSCs (Fig. 4E). In addition, also iCMs can be directly targeted by using miR-1 (Supp. Figure 3C + D) [14]. Taken together, the RNA switch technology is an efficient method to purify iCMs which are contaminated by up to 50% iPSCs without tumor formation in vivo.

Re-plating and freezing/thawing of human iCMs for later applications

The possibility to re-plate and freezing/thawing human iCMs efficiently without affecting their normal phenotype is of high value and facilitates their further applications. Human iCMs were detached and further processed at day 10 after differentiation. Approximately 3 days after re-plating or thawing, the iCMs started to beat again (Supp. Data Movie 2). Cardiac markers (cTNT, IRX4, MLC2v, HAND1, CX40, NPPA, NPPB, TBX5, MYL7, PITX2, and TBX18) were expressed at a similar level compared to the initial differentiated iCMs (Fig. 5A). Immunohistochemistry confirmed that further processing did not compromise the expression of aSMA, cTNT, ACTININ, NKX2.5, OCT4, NANOG and Ki67 (Fig. 5B + C). A comparable amount of cTNT-positive and OCT4-positive cells were detected after replating (96.6% iCMs, 1.38% iPSC) or thawing (91% iCMs, 0.74% iPSC) (96.6% iCMs, 1.38% iPSC) the iCMs (Fig. 5D). Overall, the phenotype of replated or cryopreserved human iCMs are similar to their freshly-derived cells.

Discussion

Areas of application of human iPSCs ranges from basic research to clinical applications such as disease modeling, drug screening or cell therapy. However, the impact of generating xeno- and transgene-free purified iPSC and iPSC-derived cells is still underestimated. On one hand, this is crucial for patient safety, as even 0.2% iPSC can result in tumor formation in vivo [15, 16]. On the other hand, it holds significant impportance for quality assurance in diagnostic applications, cell banking and research (e.g. omics technology). To address these needs, we have established a workflow for producing purified, xeno- and transgene-free human iCMs for both clinical and research settings [17, 18]. Generated human iPSCs showed a pluripotent nature and were further differentiated into iCMs, followed by a purification step using an RNA-based technique. Human iCMs showed a cardiac phenotype with expression of cardiac specific markers and a beating phenotype. Furthermore, xenofree protocols have been established to replate and cryopreserve human iCMs without affecting their phenotype.

However, with regard to the clinical application of these cells, additional factors must be considered, including large-scale production. About 1 billion cardiomyocytes die and do not recover after MI [19]. Though the regenerative capacity of the human adult heart is very limited, as the turnover of cardiomyocyte is less than 1% and decreases with increasing age [20]. Therefore, a considerable amount of cardiomyocytes is needed for patients after MI. Several research groups have been developed and further improved protocols to increase efficiency and scalability and remove animal-derived products [21] (Suppl. Table 5). Initial protocols were based on a combination of different formats, such as embryoid body (EB) culture in suspension with subsequent digestion and plating of a monolayer as well as uncontrolled and spontaneous differentiation [22]. Subsequent work aimed to develop more controllable protocols and an important step in this was the introduction of RPMI/B27 [23,24,25]. Burridge at el. systematically compared different conditions and established a protocol based on a synthetic coating, metabolic selection and subsequent cryopreservation of iCM [26]. The efficiency was very high (85–95%) but nevertheless this protocol was still partially based on animal-derived products. In general, over the last years iCM differentiation protocols have become more controllable and simpler [27, 28].

Another way for upscaling is the use of stirred bioreactors. It has been shown that large quantities of iCMs can be produced easily and quickly in this way [29,30,31]. Zweigerdt et al. showed a purity of approximately 85% iCMs in bioreactors underlining the need for follow up purification [31]. Nevertheless, the RNA switch has never been used in such a setting and the protocol would need to be adapted and established.

Furthermore, it has been shown that the injection of iCMs improves cardiac function but can also lead to arrhythmias. Interestingly, studies in small animal models did not report arrhythmia [32, 33] in contrast to injection of cardiomyocytes after MI in non-human primate hearts [7, 34, 35]. To overcome this problem one strategy is to maturate or further enrich iCMs before transplantation. A relatively simple approach is the long-term cultivation of the cells, which leads to structural and functional maturation of the cells [36]. However, physical (e.g. electrical, mechanical) and biochemical (e.g. fatty acid) stimuli have also been tested and have shown effects such as an increase in maturation-related genes or sarcomere-like structures after treatment [37,38,39]. In order to further expand the areas of application, in particular pharmacological research or MI treatment of iCMs, it is probably necessary to apply various approaches to see a more mature phenotype. Another aspect is the use of mixed cardiomyocyte populations. Many studies are based on mixed populations consisting of ventricular, atrial and nodal cells, which can influence the reproducibility, but also the outcome in vitro and in vivo [40]. Therefore, our future research will focus on further purification of cardiac subgroups using the RNA switch and testing in vivo. Purification technologies based on exogenous gene expression is unlimited in contrast to magnetic activated cell sorting (MACS) or FACS relying on cell surface markers [41]. DNA-based method is associated with the risk of genomic integration; hence, mRNA is a new and safe alternative. So far, endothelial cells, hepatocytes, iCMs and INSULIN-producing cells have been purified by using cell-type specific miRNA-responsive switches [14]. The RNA switch is a very versatile purification tool, as it is possible to either target the desired cells directly or eliminate the unwanted population [12]. In addition, RNA switch technology has been further developed by combing it with MACS or utilizing multiple miRNA-OFF switches [42, 43]. Regardless of the technique, purification of cells should be an integral part of the workflow and a release criterion for follow-up experiments. Moreover, further in-depth characterization (transciptomic, proteomic, metabolic etc.) of iCMs after replating and freezing is still lacking, but this also exceeds the available resources and routine lab work. Therefore, uniform standards and guidelines for the production of iPSC and iPSC-derived cells would be essential to ensure quality and safety [44].

Conclusion

Many protocols have already been established but differ considerably [45]. Good manufacturing Practice (GMP) guidelines have been defined by the EMA and FDA to regulate the manufacturing and quality control of a product [46, 47]. For example, animal derived/based components in cell culture may influence the quality as well as safety. Martin et al. showed a xenogeneic immune reaction associated with animal components [48]. Over the last years animal-based cell culture products have been stepwise replaced by xeno-free culture systems of human or synthetic origin (Suppl Table 5). We have established a workflow for the generation of hiPSC and purified iCMs under xeno- and transgene-free conditions. PBMCs were reprogrammed by integration-free Sendai virus-based technique. Synthetic iMatrix-511 was used as a coating and is a chemically defined and xeno-free cell-culture substrate. We provide a platform with consistent operation of the whole process starting with blood samples, reprogramming, differentiation, purifying, freezing and replating iCMs of the same quality.

Data availability

Data and material that support the findings of this study are available on request from the corresponding author [M.G.].

Abbreviations

CIM:

Cardiac induction media

CMM:

Cardiomyocyte maintenance media

CMMII:

Cardiac maintenance medium II

CVD:

Cardiovascular disease

EMA:

European Medicines Agency

ESC:

Embryonic stem cells

FACS:

Fluorescence activated cell sorting

FDA:

Food and Drug Administration

GMP:

Good manufacturing practice

iCMs:

iPSC-derived cardiomyocytes

iPSCs:

Induced pluripotent stem cells

MACS:

Magnetic activated cell sorting

MI:

Myocardial infarction

MIM:

Mesoderm induction media

miRNA:

microRNA

PBMCs:

Peripheral blood mononuclear cells

PSC:

Pluripotent stem cell

References

  1. Tsao CW, et al. Heart disease and stroke statistics—2023 update: a report from the American heart association. Circulation. 2023;147(8):e93–621.

    Article  PubMed  Google Scholar 

  2. Coulombe KL, et al. Heart regeneration with engineered myocardial tissue. Annu Rev Biomed Eng. 2014;16(1):1–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zghebi SS, et al. Comorbidity clusters and in-hospital outcomes in patients admitted with acute myocardial infarction in the USA: A National population-based study. PLoS ONE. 2023;18(10):e0293314.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Parrotta EI, et al. Modeling cardiac disease mechanisms using induced pluripotent stem cell-derived cardiomyocytes: progress, promises and challenges. Int J Mol Sci. 2020;21(12):4354.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Matsa E, Ahrens JH, Wu JC. Human induced pluripotent stem cells as a platform for personalized and precision cardiovascular medicine. Physiol Rev. 2016;96(3):1093–126.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kawamura M, et al. Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a Porcine ischemic cardiomyopathy model. Circulation. 2012;126(11suppl1):S29–37.

    CAS  PubMed  Google Scholar 

  7. Shiba Y, et al. Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts. Nature. 2016;538(7625):388–91.

    Article  CAS  PubMed  Google Scholar 

  8. Leng Z, et al. Transplantation of ACE2-mesenchymal stem cells improves the outcome of patients with COVID-19 pneumonia. Aging Disease. 2020;11(2):216.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Ishida M, et al. Transplantation of human-induced pluripotent stem cell-derived cardiomyocytes is superior to somatic stem cell therapy for restoring cardiac function and oxygen consumption in a Porcine model of myocardial infarction. Transplantation. 2019;103(2):291–8.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Jha BS, Farnoodian M, Bharti K. Regulatory considerations for developing a phase I investigational new drug application for autologous induced pluripotent stem cells-based therapy product. Stem Cells Transl Med. 2021;10(2):198–208.

    Article  PubMed  Google Scholar 

  11. Fusaki N, et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Japan Acad Ser B. 2009;85(8):348–62.

    Article  CAS  Google Scholar 

  12. Fujita Y, et al. A versatile and robust cell purification system with an RNA-only circuit composed of microRNA-responsive ON and OFF switches. Sci Adv. 2022;8(1):eabj1793.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Parr CJ, et al. MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells. Sci Rep. 2016;6(1):32532.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Miki K, et al. Efficient detection and purification of cell populations using synthetic MicroRNA switches. Cell Stem Cell. 2015;16(6):699–711.

    Article  CAS  PubMed  Google Scholar 

  15. Fujikawa T, et al. Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. Am J Pathol. 2005;166(6):1781–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lee AS, et al. Effects of cell number on teratoma formation by human embryonic stem cells. Cell Cycle. 2009;8(16):2608–12.

    Article  CAS  PubMed  Google Scholar 

  17. Jones LS et al. Multidirectional filamented light biofabrication creates aligned and contractile cardiac tissues. Adv Sci, 2024: p. 2404509.

  18. Russo M, et al. Light in a heartbeat: bond scission by a single photon above 800 Nm. J Am Chem Soc. 2024;146(12):8417–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol. 2005;23(7):845–56.

    Article  CAS  PubMed  Google Scholar 

  20. Eschenhagen T, et al. Cardiomyocyte regeneration: a consensus statement. Circulation. 2017;136(7):680–6.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Lyra-Leite DM, et al. A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming. STAR Protoc. 2022;3(3):101560.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kehat I, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest. 2001;108(3):407–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Burridge PW, et al. Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells. 2007;25(4):929–38.

    Article  CAS  PubMed  Google Scholar 

  24. Laflamme MA, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol. 2007;25(9):1015–24.

    Article  CAS  PubMed  Google Scholar 

  25. Lian X, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via Temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A. 2012;109(27):E1848–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Burridge PW, et al. Chemically defined generation of human cardiomyocytes. Nat Methods. 2014;11(8):855–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lin Y, Zou J. Differentiation of cardiomyocytes from human pluripotent stem cells in fully chemically defined conditions. STAR Protoc, 2020. 1(1).

  28. Kleinsorge M, Cyganek L. Subtype-Directed differentiation of human iPSCs into atrial and ventricular cardiomyocytes. STAR Protoc. 2020;1(1):100026.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Kempf H, et al. Cardiac differentiation of human pluripotent stem cells in scalable suspension culture. Nat Protoc. 2015;10(9):1345–61.

    Article  CAS  PubMed  Google Scholar 

  30. Prondzynski M, et al. Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiac organoids in stirred suspension systems. Nat Commun. 2024;15(1):5929.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kriedemann N et al. Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks. Nat Protoc. 2024:1–29.

  32. Shiba Y, et al. Electrical integration of human embryonic stem cell-derived cardiomyocytes in a Guinea pig chronic infarct model. J Cardiovasc Pharmacol Therap. 2014;19(4):368–81.

    Article  Google Scholar 

  33. Shiba Y, et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature. 2012;489(7415):322–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chong JJ, et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature. 2014;510(7504):273–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Liu Y-W, et al. Human embryonic stem cell–derived cardiomyocytes restore function in infarcted hearts of non-human primates. Nat Biotechnol. 2018;36(7):597–605.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lundy SD, et al. Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells Dev. 2013;22(14):1991–2002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ma R, et al. Electrical stimulation enhances cardiac differentiation of human induced pluripotent stem cells for myocardial infarction therapy. Antioxid Redox Signal. 2018;28(5):371–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yoshida S, et al. Hydrogel microchambers integrated with organic electrodes for efficient electrical stimulation of human iPSC-derived cardiomyocytes. Macromol Biosci. 2019;19(6):1900060.

    Article  Google Scholar 

  39. Horikoshi Y, et al. Fatty acid-treated induced pluripotent stem cell-derived human cardiomyocytes exhibit adult cardiomyocyte-like energy metabolism phenotypes. Cells. 2019;8(9):1095.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lee JH, et al. Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm populations. Cell Stem Cell. 2017;21(2):179–94. e4.

    Article  CAS  PubMed  Google Scholar 

  41. Generali M et al. Purification technologies for induced pluripotent stem cell therapies. Nat Reviews Bioeng. 2024:1–14.

  42. Tsujisaka Y, et al. Purification of human iPSC-derived cells at large scale using MicroRNA switch and magnetic-activated cell sorting. Stem Cell Rep. 2022;17(7):1772–85.

    Article  CAS  Google Scholar 

  43. Endo K, Hayashi K, Saito H. High-resolution identification and separation of living cell types by multiple microRNA-responsive synthetic mRNAs. Sci Rep. 2016;6(1):21991.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang J, et al. Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions. Stem Cell Res Ther. 2015;6:1–11.

    Article  Google Scholar 

  45. Lyra-Leite DM, et al. A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming. STAR Protocols. 2022;3(3):101560.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Volume E. Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use. Part IV Guidelines on Good Manufacturing Practice Specific to Advanced Therapy Medicinal Products. Available online: https://health.ec.europa.eu/document/download/ad33d9dd-03f0-4bef-af53-21308ce2187d_en

  47. Commission E. Guidelines on good clinical practice specific to advanced therapy medicinal products. Belgium: European Commission Brussel; 2019.

    Google Scholar 

  48. Martin MJ, et al. Human embryonic stem cells express an Immunogenic nonhuman Sialic acid. Nat Med. 2005;11(2):228–32.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The work of the authors is supported by the the Mäxi Foundation and CiRA Foundation. The authors declare that they have not use AI-generated work in this manuscript” in this section.

Author information

Authors and Affiliations

  1. Institute for Regenerative Medicine, University of Zurich, Zurich, Switzerland

    M. Generali, D. Kehl, D. Meier, D. Zorndt, MY. Emmert & SP. Hoerstrup

  2. Center for Surgical Research, University of Zurich, University Hospital Zurich, Zurich, Switzerland

    K. Atrott

  3. Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan

    H. Saito

  4. Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, 113-0032, Japan

    H. Saito

  5. Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum der Charité (DHZC), Berlin, Germany

    MY. Emmert

  6. Charité Universitätsmedizin Berlin, Berlin, Germany

    MY. Emmert

  7. Wyss Zurich, University of Zurich and ETH Zurich, Zurich, Switzerland

    SP. Hoerstrup

Authors
  1. M. Generali
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. D. Kehl
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. D. Meier
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. D. Zorndt
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. K. Atrott
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. H. Saito
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. MY. Emmert
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. SP. Hoerstrup
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

MG: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing. DK: Collection and/or assembly of data, Data analysis and interpretation. DM: Collection and/or assembly of data, Data analysis and interpretation. DZ Collection and/or assembly of data, Data analysis and interpretation. KA: Collection and/or assembly of data. HS: Provision of material. MYE: Conception and design, Financial support. SPH: Conception and design, Financial support. All authors read and approved the final manuscript.

Corresponding author

Correspondence to M. Generali.

Ethics declarations

Ethical approval and consent to participate

Human peripheral blood was collected with written informed consent according to the permission from the cantonal ethics commission of Zurich, Switzerland [KEK-ZH-2014-0430] entitled “Periphere mononukleäre Blutzellen als Quelle für Tissue Engineering in der Regenerativen Medizin” (Amendmend 05.01.2015).

The veterinary office of the Canton Zurich, Switzerland approved all animal experiments (ZH174/2020) entitled “Safety assessment of induced pluripotent stem cell-derived cardiomyocytes” (approved 19.02.2021).

Consent for publication

All authors agreed to publication.

Competing interests

S.P.H. is a shareholder at Xeltis BV and LifeMatrix Technologies AG. M.Y.E. is a shareholder at LifeMatrix Technologies AG. H.S. is the investigator of a record listed on a patent application (PCT/JP2017/023513, filed by Kyoto University on 27 June 2017) related to the design of the RNA-ON switch. H.S. is listed on a patent application (Japanese patent application no. 2021-177971) related to the cell purification. H.S. own shares of aceRNA Technologies Ltd, where H.S. is an outside director. The authors declare that they have no other competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Generali, M., Kehl, D., Meier, D. et al. Generation and purification of iPSC-derived cardiomyocytes for clinical applications. Stem Cell Res Ther 16, 189 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04319-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04319-0

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512