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Secretome analysis during human induced pluripotent stem cell-derived cardiomyocyte differentiation

ABSTRACT: Introduction: Cell transplantation therapy using human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) is a promising treatment for ischemic heart disease. While paracrine effects mediated by the secretome are recognized as a key therapeutic mechanism, the dynamic changes in the secretome profile during the manufacturing process remain poorly understood. This study aimed to comprehensively elucidate the secretome dynamics of clinical-grade hiPSC-CM to identify their "Mode of Action" (MoA) and candidate quality attributes (CQAs). Methods: We integrated multi-omics profiling-including total RNA-seq, proteome analysis, and extracellular vesicle (EV)-derived miRNA-seq-with functional assays (proliferation, cell migration, and tube formation) using samples obtained across time points of the hiPSC-CM manufacturing process (Day 4-25). Results: High-purity hiPSC-CM (>95% cTnT positive) exhibited a distinct transcriptomic maturation between Day 16 and Day 25, characterized by the upregulation of cardiac maker genes. Proteomic clustering revealed four distinct stages, showing a functional transition from proliferation-centric signaling to tissue-repair signaling. Notably, the purified Day 25 secretome showed a qualitative shift toward a PDGF and SDF-1 rich profile. Simultaneously, EVs from Day 25 were enriched with "myomiRs" (miR-133b, -208b, -499b) and multiple anti-proliferative miRNAs (let-7e-5p, miR-145-5p). Functionally, the Day 25 secretome significantly enhanced mesenchymal stem/stromal cell (MSC) migration and promoted mature, highly branched endothelial tube formation compared to earlier stages. Conclusions: The hiPSC-CM secretome undergoes a programmed evolution during manufacturing, shifting from promoting undifferentiated growth to orchestrating complex tissue repair through stable angiogenesis and MSC recruitment. These findings establish a molecular foundation for the MoA of hiPSC-CM therapy and provide critical CQAs for ensuring the potency and consistency of clinical-grade cardiac products.

ORGANISM(S): Homo sapiens

PROVIDER: GSE319631 | GEO | 2026/07/24

REPOSITORIES: GEO

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Project description:Introduction: Cell transplantation therapy using human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) is a promising treatment for ischemic heart disease. While paracrine effects mediated by the secretome are recognized as a key therapeutic mechanism, the dynamic changes in the secretome profile during the manufacturing process remain poorly understood. This study aimed to comprehensively elucidate the secretome dynamics of clinical-grade hiPSC-CM to identify their "Mode of Action" (MoA) and candidate quality attributes (CQAs). Methods: We integrated multi-omics profiling-including total RNA-seq, proteome analysis, and extracellular vesicle (EV)-derived miRNA-seq-with functional assays (proliferation, cell migration, and tube formation) using samples obtained across time points of the hiPSC-CM manufacturing process (Day 4-25). Results: High-purity hiPSC-CM (>95% cTnT positive) exhibited a distinct transcriptomic maturation between Day 16 and Day 25, characterized by the upregulation of cardiac maker genes. Proteomic clustering revealed four distinct stages, showing a functional transition from proliferation-centric signaling to tissue-repair signaling. Notably, the purified Day 25 secretome showed a qualitative shift toward a PDGF and SDF-1 rich profile. Simultaneously, EVs from Day 25 were enriched with "myomiRs" (miR-133b, -208b, -499b) and multiple anti-proliferative miRNAs (let-7e-5p, miR-145-5p). Functionally, the Day 25 secretome significantly enhanced mesenchymal stem/stromal cell (MSC) migration and promoted mature, highly branched endothelial tube formation compared to earlier stages. Conclusions: The hiPSC-CM secretome undergoes a programmed evolution during manufacturing, shifting from promoting undifferentiated growth to orchestrating complex tissue repair through stable angiogenesis and MSC recruitment. These findings establish a molecular foundation for the MoA of hiPSC-CM therapy and provide critical CQAs for ensuring the potency and consistency of clinical-grade cardiac products.

Project description:BACKGROUND: Ischemia/reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI). The current standard of care focuses on supporting kidney function, stating the need for more efficient and targeted therapies to enhance repair. Mesenchymal Stromal Cells (MSCs) and their secretome, either as conditioned medium (CM) or extracellular vesicles (EVs), have emerged as promising options for regenerative therapy, however, their full potential in treating AKI remains unknown.METHODS: In this study, we employed an in vitro model of chemically-induced ischemia using antimycin A combined with 2-deoxy-D-glucose to induce ischemic injury in proximal tubule epithelial cells. Afterwards, we evaluated the effects of MSC secretome, CM or EVs obtained from adipose tissue, bone marrow and umbilical cord, on ameliorating the detrimental effects of ischemia. To assess the damage and treatment outcomes, we analyzed cell morphology, mitochondrial health parameters (mitochondrial activity, ATP production, mass and membrane potential) and overall cell metabolism by metabolomics.RESULTS: Our findings show that ischemic injury caused cytoskeletal changes confirmed by disruption of the F-actin network, energetic imbalance as revealed by a 50% decrease in the oxygen consumption rate, increased oxidative stress, mitochondrial dysfunction and reduced cell metabolism. Upon treatment with MSC secretome, the morphological derangements were partly restored and ATP production increased by 40-50%, with umbilical cord-derived EVs being most effective. Furthermore, MSC treatment led to phenotype restoration as indicated by an increase in cell bioenergetics, including increased levels of glycolysis intermediates, as well as an accumulation of antioxidant metabolites.CONCLUSION: Our in vitro model effectively replicated the in vivo-like morphological and molecular changes observed during ischemic injury. Additionally, treatment with MSC secretome ameliorated proximal tubule damage, highlighting its potential as a viable therapeutic option for targeting AKI.

Project description:Adipose-derived mesenchymal stromal cells (MSC(AT)) display immunomodulatory and angiogenic properties, but an improved understanding of quantitative critical quality attributes (CQAs) that inform basal MSC(AT) fitness ranges for immunomodulatory and/or angiogenic applications is urgently needed for effective clinical translation. We constructed an in vitro matrix of multivariate readouts to identify putative CQAs that were sensitive enough to discriminate between specific critical processing parameters (CPPs) chosen for their ability to enhance MSC immunomodulatory and angiogenic potencies, with consideration for donor heterogeneity. We compared 3D aggregate culture conditions (3D normoxic, 3D-N) and 2D hypoxic (2D-H) culture as non-genetic CPP conditions that augment immunomodulatory and angiogenic fitness of MSC(AT). We measured multivariate panels of curated genes, soluble factors, and morphometric features for MSC(AT) cultured under varying CPP and licensing conditions, and we benchmarked these against two functional and therapeutically relevant anchor assays – in vitro monocyte/macrophage (MΦ) polarization and in vitro angiogenesis. Our results showed that varying CPP conditions was the primary driver of MSC(AT) immunomodulatory fitness; 3D-N conditions induced greater MSC(AT)-mediated MΦ polarization toward inflammation-resolving subtypes. In contrast, donor heterogeneity was the primary driver of MSC(AT) angiogenic fitness. Our analysis further revealed panels of putative CQAs with minimum and maximum values that consisted of twenty MSC(AT) characteristics that informed immunomodulatory fitness ranges, and ten MSC(AT) characteristics that informed angiogenic fitness ranges. Interestingly, many of the putative CQAs consisted of angiogenic genes or soluble factors that were inversely correlated with immunomodulatory functions (THBS1, CCN2, EDN1, PDGFA, VEGFA, EDIL3, ANGPT1, and ANG genes), and positively correlated to angiogenic functions (VEGF protein), respectively. We applied desirability analysis to empirically rank the putative CQAs for MSC(AT) under varying CPP conditions and donors to numerically identify the desirable CPP conditions or donors with maximal MSC(AT) immunomodulatory and/or angiogenic fitness. Taken together, our approach enabled combinatorial analysis of the matrix of multivariate readouts to provide putative quantitative CQAs that were sensitive to variations in select CPPs that enhance MSC immunomodulatory/angiogenic potency, and donor heterogeneity. These putative CQAs may be used to prospectively screen potent MSC(AT) donors or cell culture conditions to optimize for desired basal MSC(AT) immunomodulatory or angiogenic fitness.

Project description:Background: Radiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass, irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications, neurodegeneration, andpremature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity, homeostasis, and repair. Here, we investigated the potential of the iPSC-derived MSC(iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines. Methods: We generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome, their modulation, and the molecular pathways they elicit. Results: Increasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ≤ 5 Gy) were treated with iMSC CM, endothelial cell viability, adherence, spheroid compactness, and proangiogenic sprout formation were significantly ameliorated, and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently, iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore, iMSC CM suppressed IR-induced NFκB activation, TNF-α release, and ROS production in THP1 cells. Additionally, iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3, iMSCs, or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2, IL6, IL8/CXCL8, ANG (Angiogenin), GROα/CXCL1, and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors, the PI3K/AKT/mTOR modulator PRAS40 and β-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism, translation, mitochondrial respiration, DNA damage repair, and neurodevelopment. Conclusions: The iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.

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Secretome analysis during human induced pluripotent stem cell-derived cardiomyocyte differentiation

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