Using Gene Editing to Boost Energy Production in Stem Cell-Derived Heart Cells for Better Development

A comprehensive literature search was performed in PubMed, Google Scholar, Scopus, Web of Science, bioRxiv/medRxiv, and the MDPI database covering 2005–2025. Search terms included: “iPSC-cardiomyocytes,” “mitochondrial biogenesis,” “PGC-1α NRF1 TFAM,” “CRISPRa,” “mitochondrial DNA editing,” “DdCBE,” “mitoTALEN,” “cardiomyocyte maturation,” “extracellular vesicle mitochondrial transfer,” and “engineered heart tissue maturation”.

We included primary research and reviews involving:

Exclusion Criteria

We excluded:

Two reviewers independently screened titles/abstracts and evaluated full texts. Disagreements were resolved by consensus. A PRISMA-style approach was followed to document: records identified, screened, assessed, and included.

To capture both nuclear and mitochondrial determinants of iPSC-CM maturation, we extracted parameters across editing modalities, environmental interventions, and functional readouts. For each eligible study, the following parameters were extracted:

For each eligible study, we recorded not only experimental parameters but also whether functional and metabolic readouts were reported. Metrics included mtDNA copy number, OCR (basal and maximal), spare respiratory capacity, ROS production, calcium transient fidelity, and sarcomere ultrastructure. This allowed a comparative assessment of the comprehensiveness and reproducibility of maturation studies and identification of gaps that CRISPR-guided interventions could address. In summary, the extracted parameters capture the full spectrum of determinants shaping iPSC-CM maturation, spanning nuclear, mitochondrial, and environmental influences. By linking genetic interventions with functional readouts, this dataset enables comparative evaluation of strategies and highlights translational gaps. The categories encompass both upstream regulators (nuclear and mtDNA) and downstream outcomes (metabolic and electrophysiological), providing an integrated framework to assess combinatorial approaches for achieving adult-like cardiomyocyte function.

To ensure rigorous evaluation of mitochondrial genome editing outcomes, we extracted detailed sequencing-related parameters from all included studies. Extracted variables included: (1) sequencing platform (short-read NGS, long-read, or duplex sequencing), (2) average and per-base coverage depth, (3) error-correction strategies, and (4) variant-calling thresholds relevant to heteroplasmy quantification.

Short-read NGS datasets were assessed for read quality (Q ≥ 30) and adapter trimming. Alignment parameters were optimized for mtDNA circularity. Minimum accepted mean coverage was ≥500×, with ≥1000× used as the preferred threshold for detecting low-frequency heteroplasmy (<1%).

Long-read sequencing (PacBio HiFi, ONT) was extracted when studies evaluated large mtDNA deletions, structural variants, or mtDNA-nuclear DNA chimeric reads. For long-read platforms, high-fidelity error filtering or consensus-building algorithms (e.g., R10.4.1 Q20+, circular consensus sequencing) were documented.

Duplex sequencing datasets were evaluated separately due to their ultra-low error rate (≈10⁻⁷–10⁻⁸). Studies using duplex consensus sequencing (DCS) were annotated for strand-tagging methodology, unique molecular identifiers (UMIs), and the required ≥500× DCS depth to confirm low-frequency off-target edits.

Variant interpretation parameters included: (1) heteroplasmy detection limits (0.1%, 0.5%, or 1%), (2) allele-fraction thresholds for calling on-target edits (≥5% unless justified), and (3) filters for strand bias, read position bias, and background error models. Studies lacking sufficient coverage or appropriate mitochondrial-specific variant filters were flagged during quality assessment.

Because mtDNA is present in high copy number, we extracted reporting on copy-number normalization strategies (e.g., nuclear DNA ratios, TFAM-corrected quantification) to ensure accurate interpretation of variant allele fractions in edited iPSC-CMs.

CRISPRa activation of PGC-1α is prioritized for its established role in increasing mitochondrial mass and OXPHOS activity. DdCBE was selected due to its precise cytosine editing and low off-target potential in the mitochondrial genome. PGC-1α/NRF1 transcriptional activation is linked to increased mtDNA copy number, cristae density, and OXPHOS activity, demonstrating functional maturation. EV-mediated mitochondrial transfer complements these edits by improving ROS homeostasis and calcium handling. Together, these parameters allowed cross-study normalization of mtDNA editing efficiency, off-target assessment, and heteroplasmy dynamics in iPSC-CM maturation paradigms.

A narrative synthesis approach was used to integrate nuclear–mitochondrial pathways with mtDNA maintenance mechanisms, group CRISPR-based strategies by transcriptional activation, epigenetic remodeling or mtDNA editing, evaluate synergy between genetic editing and environmental maturation, identify translational gaps using prior publications, and compare findings qualitatively due to protocol heterogeneity across studies. This qualitative synthesis framework builds upon the comparative mitochondrial maturation analysis previously established in [4], with updated inclusion of CRISPR-based nuclear and mitochondrial genome editing strategies.

Following qualitative cross-tabulation, integrative workflows were prioritized using the following criteria: (i) frequency of functional improvement across independent iPSC-CM studies; (ii) magnitude of reported metabolic and electrophysiological enhancement when available (e.g., OCR, ATP production, ROS handling, calcium kinetics); (iii) mechanistic complementarity across nuclear, mitochondrial, and environmental interventions; and (iv) feasibility and translational relevance, including delivery modality, reproducibility, and off-target risk. The three workflows presented represent the most frequently supported and mechanistically synergistic strategies identified in the literature.

Because of substantial heterogeneity in differentiation protocols, genetic tools, culture duration, and outcome reporting, formal quantitative meta-analysis was not feasible. Instead, extracted data were synthesized qualitatively by cross-tabulation of interventions against reported functional outcomes (e.g., OCR, ATP production, ROS handling, calcium dynamics, mitochondrial ultrastructure). Interventions were prioritized based on (i) frequency of reported functional improvement across independent studies, (ii) breadth of mitochondrial domains affected, and (iii) mechanistic complementarity across nuclear, mitochondrial, and environmental layers. These criteria informed the selection of the three integrative workflows presented in. Section 7

While formal risk-of-bias scoring was not required, we evaluated:

As a review article, no new data involving humans or animals were collected. All included research adhered to the ethical standards of their originating institutions.

The transcriptional coactivator PGC-1α functions as the dominant upstream regulator of mitochondrial biogenesis in cardiomyocytes [17]. By coactivating NRF1/2 and ERRα, PGC-1α coordinates nuclear expression of respiratory chain subunits, fatty-acid oxidation enzymes, mitochondrial ribosomal proteins, and protein import machinery. Critically, this nuclear program is coupled to mitochondrial genome replication through induction of TFAM, enabling synchronized expansion of mitochondrial mass and function [18,19,20,21,22,23,24,25,26].

In postnatal cardiomyocytes, upregulation of PGC-1α coincides with a surge in mitochondrial proliferation, cristae maturation, and the metabolic switch toward fatty-acid oxidation [27,28]. In iPSC-CMs, this axis remains transcriptionally blunted [1,4,29,30,31], resulting in reduced mtDNA copy number, impaired cristae architecture, diminished OXPHOS flux, and insufficient ATP supply for mature excitation–contraction coupling.

Recent work further establishes the PGC-1α–NRF1–TFAM axis as a dominant control node, governing not only mitochondrial expansion but also redox buffering, stress resilience, and metabolic adaptability [32,33,34]. In contrast, mitochondrial dynamics and mitophagy primarily refine the quality, distribution, and turnover of mitochondria generated downstream. This hierarchical organization positions PGC-1α–NRF1–TFAM signaling as the highest-leverage target for restoring adult-like mitochondrial competency in iPSC-CMs.

Mitochondrial DNA encodes essential subunits of the electron transport chain, along with rRNAs and tRNAs required for intramitochondrial translation [35]. Its replication depends on POLG, TWINKLE, and TFAM, with TFAM abundance tightly correlated with mtDNA copy number [36,37,38,39,40]. In iPSC-CMs, reduced expression of TFAM and POLG contributes to low mtDNA content, disorganized nucleoids, and aberrant cristae morphology [4,41,42,43,44,45,46,47].

Incomplete maturation of mitochondrial import machinery further restricts incorporation of nuclear-encoded OXPHOS proteins, compounding energetic insufficiency. These defects underscore mtDNA maintenance as a critical downstream bottleneck, which must be addressed in concert with upstream nuclear activation to achieve functional mitochondrial maturation.

Mitochondrial biogenesis is tightly coupled to mitochondrial dynamics and mitophagy, which together ensure network integrity and quality control. Fusion proteins (MFN1/2, OPA1) promote cristae continuity and mtDNA distribution, while fission mediated by DRP1 facilitates organelle turnover and redistribution [48,49,50,51,52,53,54,55]. Mitophagy, primarily via PINK1–Parkin signaling, selectively removes damaged mitochondria, preventing accumulation of dysfunctional organelles [56,57,58].

During cardiomyocyte maturation, fusion predominates, supporting elongated networks and enhanced metabolic efficiency. In iPSC-CMs, dysregulated dynamics and excessive fragmentation disrupt this balance, yielding bioenergetically inefficient networks [1,4,8]. Importantly, these pathways optimize rather than initiate mitochondrial maturation; their maximal benefit emerges when upstream biogenesis programs are restored. Accordingly, combinatorial strategies activating PGC-1α–NRF1–TFAM signaling while promoting fusion and controlled mitophagy are predicted to synergistically restore cristae architecture, OXPHOS efficiency, and excitation–contraction coupling [58,59,60] (Figure 3).

During cardiac development, mitochondrial morphology evolves in parallel with metabolic transitions [1,4]. Fetal cardiomyocytes rely predominantly on anaerobic glycolysis [1,61], possessing sparse, spherical mitochondria with few cristae [1,4]. Correspondingly, these cells exhibit low basal and maximal oxygen consumption rates (OCR), reduced ATP production, and limited ROS buffering capacity, reflecting immature bioenergetic function.

After birth, exposure to oxygen and lipid-rich nutrients triggers a surge in oxidative metabolism [1,4,62], accompanied by increased mitochondrial density, elongated morphology [63], and intricate cristae organization [4]. This metabolic reprogramming engages the transcriptional regulators outlined in Section 3.1, along with enhanced fatty acid oxidation enzymes (CPT1B, ACADs) [64,65,66] and improved calcium–mitochondria crosstalk [8,67]. Adult cardiomyocytes display extensive sarcomere-anchored mitochondrial arrays optimized for ATP delivery, efficient OCR, and dynamic ROS signaling to support continuous contractile activity [4,8].

In contrast, iPSC-CMs largely recapitulate the fetal state, retaining low OXPHOS flux, reduced ATP reserves, and blunted redox buffering. This developmental mismatch defines the metabolic immaturity that engineering strategies seek to overcome. This developmental perspective establishes a framework for subsequent strategies employing nuclear genome modulation, mtDNA editing, and environmental maturation cues, which are discussed in. Section 4

Collectively, mitochondrial biogenesis, dynamics, and mitophagy form an interdependent regulatory triad governing cardiomyocyte energetic capacity [1,2,4,8]. In iPSC-CMs, incomplete coordination of these processes sustains a fetal-like mitochondrial phenotype. Among these layers, nuclear-driven mitochondrial biogenesis via the PGC-1α–NRF1–TFAM axis is the primary determinant, while dynamics and mitophagy refine network quality and performance [68,69,70,71]. This hierarchy provides the biological framework for genome-guided and environmental maturation strategies discussed below.

iPSC-CMs consistently display an immature mitochondrial phenotype characterized by perinuclear localization, fragmented networks, and poorly developed cristae [4,72]. Dysregulated dynamics—excessive fission and insufficient fusion—further impair network integration with sarcomeres, limiting efficient ATP delivery [8,49,50,51]. These deficits are not subtle: failing to account for mitochondrial immaturity systematically compromises metabolic performance, electrophysiological fidelity, and stress adaptability.

Metabolically, iPSC-CMs retain a glycolytic bias, with reduced basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and impaired fatty-acid oxidation [2,4,41,73,74,75]. Altered NAD⁺/NADH ratios and limited ROS buffering exacerbate vulnerability to energetic and calcium-handling stress [4,13,76,77,78,79]. Gene expression analyses consistently demonstrate reduced transcription of PGC-1α, NRF1/2, TFAM, and OXPHOS subunits, corroborating these functional deficits [4,8,29,41,70].

Standardized metrics enable rigorous comparison across studies, including mtDNA copy number, citrate synthase activity, OCR/SRC, mitochondrial membrane potential, ROS production, and respiratory complex abundance [17,22,29,37,41,64,80,81,82,83,84]. Recent studies suggest transcriptional or genome-editing strategies, including CRISPRa and mitochondrial base editors (DdCBE, mitoTALENs), can partially overcome these deficits, producing measurable improvements in mitochondrial mass, respiration, and calcium handling [85].

Comparative studies demonstrate that metabolic conditioning partially improves OXPHOS and mtDNA content, electromechanical stimulation enhances network connectivity and SRC, and CRISPRa-mediated nuclear activation produces the largest gains in mtDNA copy number, cristae density, OCR, and calcium handling. For example, CRISPRa activation of PGC-1α and NRF1 yields approximately two-fold increases in mtDNA content and maximal OCR, exceeding improvements achieved by metabolic conditioning alone [70,86,87,88]. Collectively, these findings indicate mitochondrial immaturity is a major barrier to achieving adult-like functional performance in iPSC-CMs, highlighting the need for integrated genetic, metabolic, and structural strategies.

Mitochondrial immaturity manifests functionally as impaired excitation–contraction coupling, reduced calcium transient amplitude, increased arrhythmogenic susceptibility, and limited stress adaptability. Integrating mitochondrial metrics with electrophysiological and contractile readouts reveals that restoring mitochondrial performance is prerequisite for achieving adult-like functional fidelity in iPSC-CMs.

Genetic and epigenetic interventions target the regulatory hierarchy defined in Section 3 [1,2,3,4]. In immature iPSC-CMs, this regulatory axis is suppressed, resulting in reduced mitochondrial biogenesis, underdeveloped cristae architecture, and persistent metabolic immaturity [4].

CRISPR activation (CRISPRa) recruits transcriptional activators (VP64, p65, Rta) to promoters of PGC-1α, NRF1/2, TFAM, and GATA4, inducing coordinated upregulation of nuclear-encoded mitochondrial genes. Combinatorial activation produces synergistic effects, enhancing OXPHOS subunit expression and fatty-acid oxidation enzymes (CPT1B, ACAD family) [6].

CRISPR interference (CRISPRi) complements activation by silencing negative regulators such as HDAC3, RIP140, and PPARγ corepressors, enabling fine-tuned bidirectional control. Inducible and temporally staged CRISPR systems further allow maturation protocols that better recapitulate postnatal metabolic transitions [7].

Among nuclear interventions, CRISPRa-mediated activation of PGC-1α consistently emerges as the most effective strategy for establishing a global transcriptional and metabolic framework for mitochondrial expansion, particularly when integrated with CRISPRi and downstream maturation cues.

CRISPRa-driven nuclear modulation induces convergent structural, metabolic, and electrophysiological improvements in iPSC-CMs. These include increased mtDNA copy number, enhanced cristae density, and redistribution of mitochondria from perinuclear clusters toward sarcomeric regions, reflecting improved organelle integration.

Functionally, these structural changes correlate with increased maximal oxygen consumption, elevated ATP production, reduced oxidative stress, and improved calcium transient amplitude and kinetics, collectively enhancing excitation–contraction coupling and contractile fidelity [6,10].

CRISPRi-mediated repression of inhibitory regulators further amplifies these effects, demonstrating that precise tuning of nuclear transcriptional programs can drive durable mitochondrial maturation. Compared with metabolic or electromechanical conditioning alone, nuclear genome modulation offers broader transcriptional coverage and programmability, albeit with dependence on sustained or staged activation.

Delivery of nuclear genome modulators remains a critical translational bottleneck. AAV vectors offer cardiomyocyte tropism but are constrained by payload size, whereas lentiviral systems provide higher capacity at the cost of insertional risk. Non-viral platforms—including synthetic mRNA and lipid nanoparticles—enable transient, high-efficiency expression without genomic integration, reducing cytotoxicity and improving clinical relevance [8,9].

Emerging strategies using compact Cas variants (e.g., SaCas9, KKH-SaCas9) and peptide- or microfluidic-assisted delivery improve multiplexing efficiency while minimizing off-target effects [10,11]. Optimization of vector choice, promoter specificity, and timing is essential for uniform activation across heterogeneous iPSC-CM populations.

While nuclear modulation expands mitochondrial capacity and metabolic potential, it cannot directly correct mtDNA-encoded defects, motivating complementary mitochondrial genome editing approaches.

Direct mtDNA editing complements nuclear genome modulation by addressing intrinsic mitochondrial defects that nuclear interventions alone cannot resolve. Canonical CRISPR–Cas9 systems are ineffective in mitochondria due to gRNA import limitations [12], necessitating alternative platforms.

DdCBE (derived from bacterial DddA deaminase) enables precise C•G → T•A base conversions without inducing double-strand breaks, preserving organelle integrity. In contrast, mitoTALENs and mitoZFNs induce targeted cleavage to promote heteroplasmy shifts, selectively eliminating pathogenic mtDNA variants [13,14].

Each platform carries trade-offs: DdCBEs are constrained by narrow editing windows and sequence context, whereas mitoTALENs and mitoZFNs require target-specific protein engineering. Nonetheless, these tools directly correct mtDNA-encoded ETC defects, improving electron transport efficiency, stabilizing membrane potential, and reducing ROS burden [15].

Thus, mtDNA editing resolves intrinsic mitochondrial constraints, while nuclear activation establishes global biogenic and metabolic programming—together enabling comprehensive mitochondrial maturation.

Edited mtDNA-edited iPSC-CMs exhibit restored complex I and IV activity, improved ATP synthesis, stabilized membrane potential, and enhanced calcium buffering capacity. These improvements are indispensable for durable excitation–contraction coupling and stress resilience.

Nuclear editing primarily expands mitochondrial quantity and metabolic programming, whereas mtDNA editing restores intrinsic mitochondrial quality. Their combination produces complementary and synergistic effects, particularly relevant in patient-derived iPSC-CMs harboring pathogenic mtDNA variants.

Delivery and heteroplasmy management remain key challenges, as high proportions of edited mtDNA are required for functional rescue [16]. Functional assessment integrates ATP generation, ROS balance, calcium handling, and OXPHOS flux, providing a comprehensive evaluation of organelle competence.

A comparative summary of nuclear versus mitochondrial editing tools, their targets, strengths, and limitations is presented in Table 2.

Mitochondrial delivery remains technically challenging. Mitochondrial-targeted AAVs, adenoviruses, and lipid nanoparticles are under active optimization to improve organelle import while minimizing cytotoxicity.

Patient-derived iPSC-CM studies demonstrate that even modest mtDNA defects can destabilize ETC function and calcium homeostasis, underscoring the translational relevance of precise mitochondrial editing [68]. mtDNA editing is most impactful when intrinsic mitochondrial defects limit function, whereas nuclear activation is better suited for global mitochondrial expansion.

Epigenetic modulation provides a stabilizing regulatory layer, ensuring sustained transcriptional accessibility and long-term fidelity of mitochondrial biogenesis programs. dCas9-fused epigenetic effectors (e.g., dCas9-p300, dCas9-KRAB) remodel chromatin at promoters of PGC-1α, NRF1, and TFAM, enhancing activation without permanent genomic alteration [17].

Non-coding RNAs—including miRNAs (e.g., miR-181c, miR-494) and lncRNAs (e.g., Lnc-MD1)—coordinate mitochondrial dynamics with metabolic gene networks. CRISPR-mediated modulation of these RNAs harmonizes transcriptional and post-transcriptional control [18,19].

Epigenetic interventions sustain long-term activation of mitochondrial regulators, improve network organization, enhance OXPHOS capacity, and stabilize redox homeostasis. Regulatory RNA modulation further supports ETC stability and mitochondrial turnover, reinforcing maturation consistency across iPSC-CM populations.

Epigenetic tools can be delivered via viral or non-viral platforms, with dosage and temporal control critical for reproducibility. Synthetic RNA mimics and inhibitors enable scalable modulation. Integration of nuclear, mitochondrial, and epigenetic strategies establishes a multidimensional framework for overcoming mitochondrial immaturity (Figure 4).

Together with nuclear and mtDNA interventions, epigenetic strategies set the stage for an integrated paradigm of durable mitochondrial maturation, summarized in the next section.

Durable mitochondrial maturation in iPSC-CMs arises from coordinated, multi-layered engineering rather than isolated interventions. CRISPRa-driven nuclear activation establishes the transcriptional and metabolic framework, mtDNA editing corrects intrinsic organelle constraints, and epigenetic modulation stabilizes regulatory accessibility.

When combined with appropriate delivery systems and environmental conditioning, these strategies restore adult-like mitochondrial competency—characterized by stable mtDNA maintenance, organized cristae, balanced redox homeostasis, and mature excitation–contraction coupling—providing the mechanistic foundation for subsequent translational maturation platforms.

Mature cardiomyocytes predominantly rely on fatty-acid oxidation (FAO) for 60–90% of ATP production, contrasting with the glycolytic bias of fetal or iPSC-CMs [1,4,64]. Metabolic conditioning mimics this postnatal energy transition through:

Although substrate switching alone partially enhances FAO and mitochondrial morphology, full bioenergetic maturation is only realized when metabolic cues are coordinated with transcriptional activation (e.g., CRISPRa of PGC-1α) and electromechanical stimulation. Integration of substrate, hormone, and oxygen cues produces synergistic gains, bridging metabolic gaps that genetic editing alone may not fully address.

While substrate, hormonal, and oxygen cues partially enhance mitochondrial morphology and FAO, their full potential is realized when integrated with transcriptional activation (e.g., CRISPRa of PGC-1α) and electromechanical stimulation, setting the stage for the structural cues discussed in the next section.

Electromechanical cues emulate the postnatal heart’s mechanical and electrical microenvironment. They align mitochondrial maturation with contractile development and metabolic output:

Consistent with systematic analyses showing that electrical stimulation drives enhanced mitochondrial development and metabolic maturation relative to non-stimulated controls, pulsatile pacing (e.g., 1–2 Hz) in combination with ECM/nanopatterning significantly enhances metabolic and electrophysiological maturity markers in iPSC-CMs. Mechanical and electrical stimulation improves mitochondrial network connectivity; however, these benefits are most pronounced when paired with metabolic conditioning and nuclear biogenesis activation, underscoring the necessity of multi-modal integration.

These interventions highlight the interplay between biophysical cues and transcriptional programs, allowing mitochondria to expand in concert with contractile architecture. In isolation, electromechanical and structural cues improve mitochondrial distribution and functional coupling but do not fully reset mitochondrial identity; their principal impact emerges when paired with genome-level biogenesis programs that lock in oxidative capacity.

These electromechanical and structural interventions improve mitochondrial connectivity and contractile alignment; however, their maximal benefits occur in combination with metabolic conditioning and upstream genome editing, highlighting the need for interdependent multi-modal strategies before moving to intercellular approaches.

Intercellular communication via extracellular vesicles (EVs) offers a natural route to enhance mitochondrial function:

Preclinical models demonstrate that mitochondria-enriched EVs can restore ATP production and enhance oxidative metabolism in recipient cardiomyocytes relative to untreated cells, supporting their potential to improve metabolic phenotypes. A recent study demonstrated that EV-mediated mitochondrial transfer enhances OXPHOS capacity and restores contractile bioenergetics in metabolically impaired cardiomyocytes, providing mechanistic support for EVs as a potent non-genetic augmentation strategy [90].

This strategy mimics the ideal: leveraging cooperative cellular intelligence to achieve systemic bioenergetic enhancement without direct genetic manipulation. EV-mediated mitochondrial transfer enhances functional integration of recipient mitochondria, but maximal restoration of OXPHOS and calcium handling occurs when EV delivery is combined with upstream nuclear and mitochondrial editing, highlighting interdependence between top-down and bottom-up interventions.

While EV-mediated mitochondrial transfer can transiently enhance OXPHOS and stress resilience, durable maturation depends on concurrent nuclear and mitochondrial genome engineering to ensure long-term integration, turnover, and identity maintenance. Consistent with the framework outlined in Table 1, these approaches primarily reinforce mitochondrial network integration and functional output, but require upstream genetic programming to achieve stable adult-like identity.

EV-mediated mitochondrial transfer enhances network integration and OXPHOS but achieves durable effects only when paired with nuclear and mitochondrial genome programming, bridging the environmental and genetic strategies described above and informing pharmacologic enhancement in the next section.

Chemical activators provide precise temporal control over mitochondrial biogenesis pathways:

Small-molecule activation of PGC-1α-related pathways, such as with asiatic acid (AA) or GW501516, has been shown to double basal mitochondrial respiration and ATP production in treated iPSC-CMs compared with control cells in metabolic stress tests. Pharmacologic activators of PGC-1α and AMPK accelerate maturation, but their efficacy is contingent upon concurrent structural and metabolic cues, such as 3D tissue architecture and substrate switching, demonstrating the conditional nature of combinatorial strategies.

Thus, small-molecule activation of PGC-1α and AMPK accelerates mitochondrial maturation kinetics but remains insufficient as a standalone intervention, achieving maximal impact only when embedded within coordinated genetic, metabolic, and structural maturation frameworks. Fatty acid-enhanced metabolic conditioning consistently increases basal and maximal oxygen consumption rates, ATP-linked respiration, and spare respiratory capacity relative to glucose-conditioned controls, reflecting a shift toward β-oxidation-dependent energetics. Small-molecule activation of PPAR/PGC-1α pathways produces moderate but reproducible increases in basal respiration and ATP-linked OCR (typically ~1.5–2-fold across studies), enhancing substrate flexibility without fully recapitulating adult mitochondrial capacity. Electromechanical stimulation, particularly physiological pacing (~1–2 Hz), robustly improves sarcomere organization and calcium handling, indirectly supporting mitochondrial efficiency despite less frequent reporting of direct metabolic metrics. Three-dimensional engineered tissues combined with pacing consistently outperform 2D systems, yielding superior mitochondrial network organization and tissue-level functional integration. However, no single non-genetic strategy alone achieves adult-like mitochondrial maturity, reinforcing the necessity of multimodal integration with genome-guided interventions. To contextualize these non-genetic interventions, Table 3 summarizes key strategies, their primary functional readouts, typical effects, and complementary notes, highlighting how each approach reinforces mitochondrial maturation established by upstream nuclear and mitochondrial genome programming.

Functional and structural outcomes of metabolic, electromechanical, 3D, and small-molecule interventions are summarized. Metrics include oxygen consumption rate (OCR), ATP production, spare respiratory capacity (SRC), sarcomere alignment, and calcium handling. Effects are reported relative to baseline glucose-conditioned controls unless otherwise noted. This table emphasizes the complementary role of environmental and pharmacologic cues in amplifying genetic and epigenetic maturation programs.

Comparative evidence from multiple studies indicates that non-genetic maturation strategies produce quantifiable and strategy-specific improvements in iPSC-CM mitochondrial and functional metrics. Fatty acid-enhanced metabolic media significantly increases basal and maximal oxygen consumption rates, ATP-linked respiration, and spare respiratory capacity relative to glucose-conditioned controls, demonstrating a shift toward adult-like oxidative metabolism. Small molecule activators of PPAR/PGC-1α pathways similarly enhance mitochondrial respiratory capacity and substrate flexibility, yielding higher basal respiration and ATP production relative to untreated cells. Electromechanical stimulation protocols, especially pacing at physiological frequencies (~1–2 Hz), consistently improve structural features (sarcomere alignment) and calcium handling metrics, contributing indirectly to enhanced energetic competence. Engineered 3D tissues combined with pacing further elevate tissue-level functional markers and mitochondrial network density beyond 2D approaches. Quantitative comparisons from metabolic profiling reviews confirm that none of these non-genetic cues alone fully recapitulate adult mitochondrial maturity, underscoring the rationale for multimodal integration with genetic and transcriptional programming.

A review of prior studies employing nuclear activation (e.g., PGC-1α overexpression or CRISPRa), fatty-acid-enriched metabolic media, or electrical pacing reveals partial but incomplete evidence for adult-like mitochondrial maturation [4,5,12,56]. Across these studies, mtDNA copy number was increased in approximately 60% of reports following PGC-1α activation or metabolic substrate switching [11,22,53]. However, ultrastructural maturation metrics—particularly cristae density and inner membrane organization—were infrequently quantified, limiting conclusions regarding mitochondrial architectural maturity [5,22,49].

Functional bioenergetic assessments were similarly heterogeneous. While oxygen consumption rate (OCR) was commonly reported, many studies restricted analysis to basal OCR, with maximal respiration and spare respiratory capacity (SRC) measured inconsistently [5,22,49]. Measurements of mitochondrial ROS handling and calcium transient fidelity—key indicators of redox balance and excitation–metabolism coupling—were reported in fewer than 30% of studies. Moreover, multi-omics validation (proteomics and metabolomics) linking transcriptional activation to downstream metabolic remodeling was largely absent.

These methodological gaps constrain interpretation of whether observed changes reflect true adult-like mitochondrial competency or partial metabolic adaptation. CRISPRa-mediated nuclear activation, when combined with controlled metabolic conditioning and electromechanical pacing, directly addresses these limitations by standardizing transcriptional input and enabling reproducible downstream functional and multi-omics validation. Accordingly, Workflow 1 is recommended based on moderate-to-strong evidence for transcriptional and metabolic effects, coupled with high synergistic potential when nuclear activation is sequentially integrated with environmental maturation cues.

Workflow 2: CRISPRa (GATA4 + NRF1) + Extracellular Vesicle-Mediated Mitochondrial Augmentation → 3D Engineered Heart Tissue.

Building on nuclear activation, combinatorial strategies incorporate additional transcriptional targets to enhance structural–metabolic integration (e.g., sarcomere assembly, network alignment). Extracellular vesicles (EVs) containing functional mitochondria or modulatory RNAs are administered to supplement endogenous organelles and signaling. GATA4 is included to complement NRF1 because it primarily drives sarcomere assembly and supports mitochondrial network alignment, making it critical for integrated structural–metabolic maturation; while NRF1 alone enhances biogenesis, the combination ensures both organelle and contractile maturation. Embedding iPSC-CMs in 3D engineered heart tissue provides cell–cell and matrix interactions that further stabilize mitochondrial organization. Electromechanical conditioning within these constructs consolidates alignment, with functional readouts including ATP production, ROS handling, and calcium dynamics.

Studies utilizing EV-mediated mitochondrial transfer or signaling augmentation demonstrate promising but variable effects on mitochondrial content, ATP production, and oxidative metabolism in iPSC-derived cardiomyocytes [7,8,14]. EV treatment has been reported to increase mitochondrial mass and improve ATP availability [5,7,8,11,14]; however, the durability of these effects and their integration with host mitochondrial networks are rarely assessed beyond short-term time points. Furthermore, heterogeneity in EV source, cargo composition, and dosing strategies complicates cross-study comparison.

Separately, 3D EHT models consistently improve sarcomere alignment, force generation, and metabolic maturation relative to 2D cultures [10,53,56,63]. Nonetheless, many EHT studies focus on contractile outputs without comprehensive mitochondrial phenotyping, and few directly interrogate mitochondrial network organization, ROS buffering capacity, or calcium–mitochondria coupling [22].

Importantly, studies combining EV delivery with 3D tissue context and electromechanical conditioning remain scarce, despite strong mechanistic rationale. The integration of CRISPRa-driven nuclear activation with EV-mediated mitochondrial augmentation and 3D EHT culture addresses these gaps by synchronizing transcriptional programming, organelle supplementation, and biomechanical stabilization. Workflow 2 is therefore recommended on the basis of emerging but consistent evidence for structural–metabolic enhancement, with high anticipated synergy when EV-based strategies are deployed within mechanically and electrically conditioned 3D tissues. Workflows 2 and 3 address limitations not resolved by nuclear activation alone, including structural–metabolic integration and intrinsic mtDNA defects (Table 4).

Workflow 3: Mitochondrial DNA Correction (DdCBE) in Patient iPSC Lines → Maturation Regimen → Functional Validation.

In lines harboring pathogenic mtDNA variants, mitochondrial genome integrity is restored using DdCBE or mitoTALEN-mediated base editing, leading to direct improvements in ETC performance and oxidative capacity. Following correction, iPSC-derived cardiomyocytes (iPSC-CMs) are subjected to staged maturation incorporating metabolic, electrical, and mechanical cues. Functional validation parallels that of Workflows 1–2, with the addition of heteroplasmy quantification to confirm adequate mtDNA correction.

mtDNA editing is prioritized because nuclear interventions alone cannot resolve intrinsic mitochondrial genome defects that directly constrain ETC function and OXPHOS efficiency. Among currently available tools, DdCBE provides superior precision and reduced off-target activity relative to earlier nuclease-based approaches, making it the most effective strategy for restoring adult-like mitochondrial competency.

Correction of pathogenic mtDNA variants using DdCBE or mitoTALENs primarily addresses disease modeling applications, rather than constitutive mitochondrial maturation in healthy iPSC-CMs. While mtDNA editing ensures functional ETC integrity in patient-derived lines, it may not be necessary for general adult-like mitochondrial development in non-diseased iPSC-CMs and is therefore treated here as a secondary, context-specific strategy.

Within this framework, CRISPR-guided nuclear activation establishes the primary transcriptional and metabolic program, while mtDNA correction and environmental conditioning serve as essential secondary modifiers that stabilize and consolidate mitochondrial maturation. Upon achieving ≥80% edited mtDNA, corrected iPSC-CMs undergo a defined maturation regimen involving fatty-acid substrate switching, thyroid hormone supplementation, and electromechanical pacing. Post-maturation analyses assess ATP production, redox balance, calcium handling, and integrated OXPHOS flux. Translational validation may then be performed using small-animal transplantation or engineered heart tissue (EHT) implantation to confirm engraftment, metabolic fidelity, and contractile competence. Table 3 operationalizes the evidence from Table 1 and Table 2 into actionable workflows with recommended timing, dosing, and assays, facilitating reproducible experimental design.

Mitochondrial genome editing using DdCBE or mitoTALENs has demonstrated clear proof-of-concept for correcting pathogenic mtDNA variants and improving electron transport chain (ETC) function in patient-derived iPSC lines [15,32]. Reported outcomes include enhanced oxidative phosphorylation efficiency, increased maximal respiration, and partial restoration of ATP production. However, delivery efficiency, heteroplasmy control, and long-term stability of edited mtDNA populations remain incompletely characterized across studies.

Notably, mtDNA editing studies are predominantly conducted in disease-modeling contexts, and few directly examine whether mtDNA correction alone is sufficient to drive adult-like mitochondrial maturation without concurrent nuclear or environmental interventions. Multi-omics validation and integrated functional readouts—particularly calcium handling and redox balance—are inconsistently reported, limiting assessment of systems-level recovery.

Despite these gaps, mtDNA editing uniquely addresses intrinsic mitochondrial genome defects that cannot be resolved by nuclear activation alone. When paired with post-editing maturation regimens incorporating metabolic substrate switching and electromechanical conditioning, mtDNA correction enables stabilization of ETC function within a supportive transcriptional and biomechanical environment. Accordingly, Workflow 3 is recommended as a context-specific, high-evidence strategy for disease modeling, with conditional synergy dependent on integration with nuclear and environmental maturation cues. Table 5 provides recommended controls, timing, dosing, and assays for implementing the combinatorial interventions summarized in Table 4, highlighting practical considerations for reproducible maturation experiments.

These integrative workflows exemplify synergistic maturation, in which CRISPR-guided nuclear and mitochondrial interventions are coupled with environmental conditioning to recapitulate adult cardiomyocyte energetics. Critical parameters—including timing, dosage, and delivery vector choice—must be empirically optimized per iPSC line and cardiomyocyte subtype, reflecting line-specific transcriptional plasticity and metabolic responsiveness. Combinatorial pipelines allow for sequential or parallel modulation, enabling temporal orchestration of gene activation, mtDNA correction, substrate adaptation, and mechanical conditioning. The recommended controls, timing, dosing, and assays in this table reflect current best practices for integrating nuclear and mitochondrial interventions with environmental conditioning. However, significant literature gaps remain as identified in Table 1 and Table 2, including: incomplete optimization of combinatorial activation strategies, limited long-term stability and reproducibility of mtDNA editing, variability across iPSC lines and cardiomyocyte subtypes, and insufficient standardized, multi-omic functional validation. These gaps underscore the need for empirical, cell-line-specific optimization and further preclinical validation to achieve robust, adult-like cardiomyocyte maturation.

While CRISPRa/i strategies targeting PGC-1α, NRF1/2, GATA4, and ERRα are well-established for promoting mitochondrial biogenesis in other cardiac or non-cardiac systems, their direct application in iPSC-CM specification and energetic maturation remains largely untested. Quantitative activation efficiency, temporal dynamics, and functional outcomes specifically in iPSC-CMs require systematic validation. As such, CRISPRa-based transcriptional modulation currently represents a hypothesis-driven scaffold rather than a fully validated method for adult-like mitochondrial maturation.

Although environmental maturation strategies have been reviewed extensively, including in our previous work [1,2], the integration of these approaches with CRISPR-guided transcriptional activation and mtDNA editing in temporally sequenced, multi-modal pipelines represents a novel framework. By combining metabolic conditioning, electromechanical stimulation, extracellular vesicle supplementation, and nuclear/mitochondrial genome modulation, this manuscript proposes synergistic workflows that quantitatively reinforce mitochondrial identity in iPSC-CMs.

In summary, iPSC-CM maturation requires integrated control across nuclear, mitochondrial, and environmental levels. CRISPR-guided genome editing synergizes with metabolic, electromechanical, and EV-mediated interventions to achieve adult-like mitochondrial structure and function. These workflows establish the mechanistic foundation for rigorous safety, off-target, and translational assessment described in the following section.

The regulatory landscape for mitochondrial genome editing and stem cell-derived cardiomyocyte therapies remains in rapid evolution. Unlike nuclear gene editing, mtDNA editing lacks a defined FDA/EMA pathway, partly due to its non-Mendelian inheritance, heteroplasmy dynamics, and absence of endogenous mitochondrial DNA repair systems. The FDA currently evaluates mtDNA editing technologies under advanced therapy medicinal products (ATMP) classification, requiring:

For nuclear CRISPRa/i therapies, regulators emphasize transcriptional specificity, absence of nuclease activity, and reversibility. Synthetic mRNA- and LNP-based CRISPRa vectors are viewed favorably due to transient expression and lower genotoxicity risks.

Translational success will require biomanufacturing platforms capable of producing GMP-grade edited iPSC-CMs, standardized mitochondrial maturation assays, and harmonized multi-omics release criteria. Integration of CRISPR editing with 3D engineered heart tissues or patch grafts must also meet structural, electrophysiological, and biodistribution standards before progressing to first-in-human trials.

Safety in mitochondrial biogenesis engineering is not merely the absence of toxicity—it is the controlled establishment of mitonuclear harmony. The dual-genome nature of cardiomyocyte energetics demands that editing strategies be precise, transient, and quantitatively validated. High-fidelity sequencing, immune-safe delivery systems, metabolic buffering, and regulatory-aligned manufacturing pipelines form the cornerstone of safe translation. Approached through this rigorously analytical yet forward-looking framework, CRISPR-guided mitochondrial biogenesis can advance toward clinical-grade maturation of iPSC-CMs, opening new trajectories for disease modeling, cardiotoxicity screening, and regenerative cardiology.

Taken together, the safety and translational landscape of mitochondrial biogenesis engineering underscores the need for dual-genome precision, quantitative validation, and immune-compatible delivery systems. Robust off-target profiling, metabolic buffering, and regulatory-aligned workflows are essential to ensure clinical-grade iPSC-CM maturation. These considerations inform the standardized reporting and dataset structures proposed into harmonize methodology across laboratories. Section 9

At minimum, all studies should report:

These items are proposed as recommended reporting standards for future experimental studies; no supplementary datasets were generated as part of this review

No new data were created or analyzed in this study. Data sharing is not applicable.