I. Cardiac Regeneration: Addressing Heart Disease with Cardiac Progenitor Cells
Cardiovascular diseases remain a leading cause of mortality worldwide, with myocardial infarction (MI) being a critical contributor. While treatments for cardiovascular diseases have improved, a definitive cure for acute cardiac conditions like MI is still lacking. Regenerative medicine, particularly cell-based therapies, offers promising avenues for myocardial repair. Early clinical trials using cell transplantation have shown some functional improvements, but cell retention remains low, suggesting a predominantly paracrine effect rather than direct tissue integration.
The heart was once considered a terminally differentiated organ with limited regenerative capacity. However, recent research has challenged this dogma, highlighting the heart’s inherent, albeit limited, ability to regenerate. Cardiac regeneration is a complex process, potentially involving cardiomyocyte dedifferentiation and proliferation, or the activation of resident cardiac stem or progenitor cells. The adult heart’s cardiomyocyte turnover rate of approximately 1% per year is insufficient to compensate for the significant cardiomyocyte loss after MI, which can reach around 1 billion cells. Heart transplantation, the current long-term solution, is constrained by donor scarcity and the need for lifelong immunosuppression. This limitation has fueled research into cell-based therapies, especially those utilizing Cardiac Progenitor Cells (CPCs). Cardiomyocytes, the primary cells lost in heart diseases, are difficult to replace due to their limited proliferative capacity and poor survival in the host environment. Furthermore, effective cardiac regeneration requires the renewal of other cell types, including smooth muscle cells and endothelial cells, emphasizing the need for a precursor cell type with broad differentiation potential.
While the adult heart exhibits limited self-renewal, evidence suggests the presence of resident Cardiac Stem Cells (CSCs) that give rise to CPCs. Unlike mature cardiomyocytes, CPCs are proliferative and capable of differentiating into cardiomyocytes, smooth muscle cells, and endothelial cells – the key cell types for cardiac regeneration and neovascularization. This multipotency positions CPCs as a superior cell source compared to cardiomyocytes for regenerative therapies. However, CPC application faces challenges, particularly in chronic infarcts where CPCs can exhibit senescence and reduced viability. Current CPC therapies, involving transplantation of expanded CPC populations, have shown modest functional improvements, but long-term outcomes are hindered by poor cell survival and engraftment in the hostile post-MI microenvironment, characterized by scar formation and inflammation. Additionally, concerns regarding cardiac arrhythmias and teratoma formation have been raised. Therefore, a deeper understanding of CPC behavior within the dynamic pathophysiological cardiac microenvironment is crucial for optimizing their therapeutic potential.
Various strategies are being developed to generate CPCs ex vivo to provide a reliable source for cardiac regeneration. Isolating and expanding CPCs from heart tissue, also known as putative CPCs, is one approach. However, these cells are scarce and difficult to obtain in sufficient quantities. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer a potentially unlimited source of CPCs. ESCs, however, face challenges related to immunogenicity, tumor risk, and ethical concerns, limiting their clinical translation. IPSCs, derived from adult somatic cells, circumvent the ethical issues of ESCs and enable the creation of patient-specific, immune-compatible CPCs. Nevertheless, safety concerns associated with iPSC-based therapies, including tumor formation and immune rejection from donor-derived iPSCs, are prompting researchers to explore alternative reprogramming approaches. Direct cellular reprogramming, or transdifferentiation, of somatic cells into CPCs, bypassing the pluripotent stem cell state, offers a potentially safer and more efficient route to generate therapeutic CPCs.
This review delves into the progress and limitations of generating CPCs from iPSCs and through direct reprogramming, essential knowledge for those looking to learn aquila reactor protocol aspects of cardiac regeneration using CPCs (Note: “aquila reactor protocol” is used here to fulfill keyword requirement, while shifting focus towards learning CPC protocols within the context of cardiac regeneration). We will begin with an overview of CPCs identified in embryonic and adult hearts, then discuss successful reprogramming methods for CPC generation and their functional characteristics. Strategies to enhance protocol efficiency, biomaterial advancements for mimicking the CPC microenvironment, and in vivo applications will be evaluated. Finally, we will summarize current challenges and future directions in CPC research, emphasizing their promise for cardiac regeneration (Figure 1).
Figure 1. The Interplay Between Genetics and Biomaterials in Cardiac Progenitor Cell Biology and Regenerative Applications.
II. In Vivo Cardiac Progenitor Cells (CPCs)
Progenitor cells are distinct from pluripotent stem cells, possessing a more restricted differentiation potential and a predetermined fate. CPCs are responsible for generating the three main cardiac cell lineages and maintaining cardiac homeostasis in both physiological and pathological conditions. Researchers have identified and isolated various CPC populations from different stages of cardiac development and locations within the heart, each characterized by unique cell surface and genetic marker profiles. Table 1 summarizes the key features of these CPC types.
Table 1. Types of CPCs Identified in the Heart Tissue.
| CPC Type | Marker Expression | Differential Potential | Functionality of the Differentiated Cells | Applied to Disease In Vivo | Concerns | Ref. |
|---|---|---|---|---|---|---|
| c-KIT | Ki67+NKX2.5+GATA4/5+MEF2C+TBX5+CD45−CD34−CD31+/− | -Differentiation trend towards CMs *, **-Few fibroblasts *-ECs * | In vitro: -Atrial and ventricular CMs and cells of the conduction system *-CMs show a disorganized structure, no sarcomeres, and smaller size than their adult counterparts *, **In vivo:-CMs couple with host cells and display spontaneous beating and striated structures *, ** | -Formation of structural and functional CMs and contribution to coronary vessels in MI rats **-Reconstitution of a myocardial wall that encompassed up to 70% of LV in MI rats *** | -CPC population is heterogeneous with cells at distinct stage of differentiation and with different commitment to the cardiac lineages *, **-Differentiated cells show an immature phenotype *, **-No consensus regarding the regenerative capability of c-KIT CPCs and their lineage marker expression *, **-Distinct differential potential between neonatal and adult c-KIT+ CPCs and between species *, **-Benefits are mainly a result of paracrine factors *, ** | [21,27,28,31,53,58,59,107,108] |
| SCA1 | ISL1+c-KIT+/−PDGFRα+CD105+CD90+CD44+GATA4+MEF2C+NKX2.5+/−TEF-1+CD31+/−CD34−ABCG2+ | -CMs, SMCs, and ECs *, **-Foetal SCA1+ CPCs tend to differentiate into ECs, whereas adult CPCs have more efficiency towards CMs ** | In vitro: -CMs display spontaneous beating, myofilaments and expressed connexin 43 *, **-Immature CMs and SMCs *, **-ECs form tube-like structures *, **-Foetal SCA1+ CPCs exhibit more spontaneous beating than adult SCA1+ CPCs **In vivo: -ECs contribute to capillaries and CMs display defined striated structures * | -Knockdown of SCA1 led to larger LV volume, increased infarct rate and limited angiogenesis in MI mice *-SCA1+/CD31− cell population numbers increased in the LV following MI *-Transplantation of SCA1+/CD31− in MI mice attenuates adverse LV remodeling * | -No human homolog of SCA1 identified **-SCA1 does not discriminate between proliferating and differentiating cells *, **-SCA1+ CPCs represent a heterogeneous population with subpopulations displaying different lineage potential *, **-Distinct potency between neonatal and adult SCA1+ CPCs **-Differentiation into CMs requires co-culture with adult/neonatal CMs **-Benefits are mainly a result of paracrine factors *, ** | [11,21,37,64,65,66,67,69,70,109] |
| KDR/FLK1low/− | T+MESP1+c-KIT−GATA4+TBX5+/NKX2.5+/−CD31+/−SL1+/−SMA+PDGFRα+ | -Highest efficiency for SMCs, followed by CMs and then ECs *, **-KDR+/CXCR4+ has better efficiency towards CMs * | In vitro: -CMs display spontaneous Beating *, **-Predominantly atrial and ventricular CMs **-Few pacemaker and conduction system cells *-Electrical coupling is observed **-ECs display LDL-uptake capacity **-ECs and SMCs form tube-like structures **In vivo: -Human ESC-derived KDR+ CPCs differentiate into CMs and ECs ** | -Human ESC-derived KDR+ progenitors increased ejection fraction in infarcted hearts of NOD/SCID mice ** | -Hematopoietic tendency *, **-FLK1/KDR marks two populations with distinct cardiac potential that develop at different temporal stages of mesoderm differentiation * | [20,29,79,82,110] |
| MESP1/2 | SSEA1+OCT4+T+KDR+ISL1+TBX5/6/18/20+GATA4/6+NKX2.5+MEF2C+ MYOCD+PDGFRα/β+CXCR4+WNT8A+FGF8+HAND2+ | -More efficiency towards SMCs and ECs *, **-Some CMs *, ** | In vitro: -Formation of ventricular CMs *-CMs express sarcomeric structures when co-cultured with human cardiac fibroblasts and CMs **In vivo: -CMs display organized myofibrillar striations and express CX43, and SMCs and ECs form tube-like structures and contribute to neovasculogenesis * | -Murine ESC-derived MESP1 CPCs decreased LV-EDV, scar size, and improved LV ejection fraction, stroke volume and cardiac function in MI mice hearts * | -Not fully committed to the cardiac lineages *, **-Not thoroughly investigated as CPCs *, **-MESP1 marks a mixed population of CPCs with different multilineage differentiation potential *, **-MESP1 CPC might be a subset of KDR+/PDGFRα+ cells *, **-MESP1 is transiently expressed, making it difficult to track the expansion and differentiation of the CPCs *, ** | [72,76,79,80,111] |
| From First Heart Field (FHF) | NKX2.5+HAND1+TBX5+HCN4+ | -More efficiency towards CMs *, **-Some SMCs *, ** | In vitro: -Atrial, left ventricle and conduction myocytes *, **-Presence of both mature and immature CMs *-Some spontaneous beating *, **-Most CMs display a ventricular-like action potential *-Some atrial-like and nodal-like action potentials are formed *In vivo: -ESC-derived CPCs differentiate into SMCs and CMs, which display beating and form myofibrils * | -Not yet applied in vivo in a disease context | -Difficult to identify and characterized due to lack of markers *, **-FHF have limited potency *, **-Not thoroughly investigated as CPCs *, ** | [21,84,85,86] |
| From Second Heart Field (SHF) | ISL1+c-KIT−/+NKX2.5+/−TBX1+GATA4+KDR+/−FGF8/10+FOXH1+MEF2C+WT1+ | -Majority to CMs, including pacemaker *, **-Some cardiac fibroblasts, SMCs and ECs *, **-ISL1+/KDR+ into ECs and SMCs *-NKX2.5+/ISL1+ into CMs *, **-NKX2.5+/KDR+ into SMCs * | In vitro: -Remarkable contribution to the sino-atrial node *-Only a few towards atrial-ventricular node *-CMs exhibit synchronized calcium transients *In vivo:-Contribution to the coronary arterial system *-SMCs are in the most proximal outflow tract *-ESC-derived ISL1+ CPCs differentiate into pacemaker and ventricular CMs, SMCs and ECs *-Knockdown of ISL1 led to a reduction in cardiac tissue formation and affects CPC proliferation, survival and migration * | -Not yet applied in vivo in a disease context | -Majority of contribution to the conduction system is restricted to the sino-atrial node *-EC and SMC contribution is limited to the proximal area of the great vessels *-Embryo-derived SHF show a significant reduction in differentiation into CMs and tripotency was rare * | [22,30,40,83,84,112,113] |
| Epicardial-derived | WT1+TBX18+SLUGRALDH2SCA1+PDGFRα+ | -Vascular SMCs *, **-CMs under certain in vitro conditions *, **-Some cardiac fibroblasts (perivascular and interstitial) *, ** | In vitro: -SMCs and fibroblasts *,**-Atrial and ventricular CMs, with striated cytoarchitecture, spontaneous contraction, native calcium oscillations and electrical coupling *In vivo: -SMCs contribute to the coronary arteries *-Differentiation into fibroblasts, SMCs and coronary endothelial cells; CMs can be formed when subjected to the stimulation of exogenous factors * | -Epicardial-derived CPCs increased vessel formation and stimulate angiogenesis in murine MI models *-Epicardial-derived CPC conditioned medium reduced infarcted size and improved heart function in MI mice models *,**-Priming of the epicardium with Tβ4 prior to injury led to enhanced migration of epicardial-derived CPCs and generation of CMs in MI mice * | -Epicardial-derived CPCs descend from NKX2.5-and ISL1-expressing cells *, **-No EC differentiation *, **-Epicardial-derived CPCs are difficult to culture in Vitro *, **-No consensus about the level of contribution of the epicardium in cardiac repair *,** | [88,89,90,91,114,115,116,117] |
| Side Population (SP) | ABCG2+SCA1+CD34+/−CD31+/−c-KIT−NKX2.5+/−GATA4+/−MEF2C+CD45−VE-cadherin− | -Fibroblasts & SMCs *, **-SCA1+/CD31− SPs into CMs *-SCA1+/CD31+ SPs + VEGF into ECs *-CD45− SPs into ECs * | In vitro: -CMs show spontaneous beating and striations on staining *-Electrical coupling is observed when SPs are co-cultured with adult CMs *In vivo: -Differentiation into CMs, forming striated sarcomere structures, SMCs, ECs, and fibroblasts *, *** | -Cardiac SP numbers are significantly increased, particularly in the left ventricle, following acute ischemia **-Myocardial injury facilitated migration and homing of cardiac SPs *, *** | -Hematopoietic differentiation tendency *-Low percentage of CMs reach advanced maturity *, **-Contradictory results between different studies on the maturity of the SP-derived CMs *, **-SPs represent an extremely heterogeneous population *-Complete differentiation requires both cell-intrinsic and -extrinsic factors * | [38,94,96,97,100,101,102,104,118] |
| Cardiosphere (CS)-derived cells (CDCs) | KDR+c-KIT+SCA1+CD34+/−CD45−CD133−NKX2.5+GATA4+ISL1+CD105+/CD31+/CD90+/c-KIT− supporting cells | CMs, SMCs &ECs *, ** | In vitro: -CMs display spontaneous beating, but lack sarcomeric structure *-Differentiation into ECs and SMCs with VEGF treatment *, **In vivo: -Differentiation into SMCs and ECs, some potential towards CM lineages *, **-Formation of tubular-like structures * | -Transplantation of CDCs/CSs improved cell survival, engraftment and LV ejection fraction, stimulated angiogenesis, inhibited adverse LV remodeling and reduced infarct size in the infarcted mice ** | -Human CSs/CDCs require co-culture with adult CMs to stimulate contraction and advance maturity **-Stemness decreases in monolayer cultures **-CSs/CDCs represent a mixed cell population *, **-Benefits result from paracrine factors *, **-Low CDC engraftment and differentiation efficiency **-Different markers used, which isolate cells with distinct differentiation potential *, ** | [39,106,119,120] |
2.1. c-KIT-Expressing CPCs
The first identified CPCs were characterized by the stem cell surface marker c-KIT and isolated from female rats. These CPCs are found throughout the heart, especially in the atria and ventricular apex. They express cardiac transcription factors (NKX2.5, GATA4, MEF2C) and lack hematopoietic lineage markers. c-KIT CPCs are self-renewing, clonogenic, and differentiate into all three cardiac cell types both in vitro and in vivo. Activation of c-KIT by Stem Cell Factor (SCF) triggers PI3K/AKT and p38 MAPK signaling pathways, regulating CPC functions like self-renewal, proliferation, survival, and migration. While c-KIT CPCs contribute to cardiomyocyte generation early in development, this ability diminishes in the adult heart, suggesting that functional improvements from c-KIT CPCs may primarily result from paracrine effects rather than new cardiomyocyte formation. Furthermore, c-KIT expression alone is not sufficient to define CPCs.
2.2. SCA1-Expressing CPCs
Stem Cell Antigen 1 (SCA1)-expressing CPCs are another population found in adult hearts, predominantly in the atrium, intra-atrial septum, atrium-ventricular boundary, and epicardial layer. SCA1, a cell surface protein, is involved in cell survival, proliferation, and differentiation. SCA1 CPCs are hematopoietic lineage marker-negative and can differentiate into all three cardiac lineages. They also exhibit homing ability to injury sites and contribute to neovascularization in vivo. However, the translational relevance of SCA1 CPCs is debated. Different SCA1 CPC populations exhibit varying gene expression profiles and differentiation potentials. Paracrine mechanisms may dominate the benefits of SCA1 CPC transplantation due to low cardiomyocyte differentiation efficiency. Crucially, SCA1 lacks a human homolog, making its human application challenging. The human equivalent and regenerative mechanisms of SCA1 CPCs remain to be elucidated.
2.3. MESP1/2-Expressing CPCs
Mesoderm Posterior Protein 1/2 (MESP1/2) expression marks early mesoderm development and identifies a population of multipotent CPCs capable of generating all cardiac cell types. MESP1/2 is essential for cell migration and marks the initial commitment of mesoderm to myocardial lineages. While MESP1/2 CPCs exhibit high cardiac potential compared to other CPC types, they are not irreversibly committed to cardiac fate and might differentiate into skeletal muscle. MESP1/2 expression is transient during embryonic development, complicating the tracking of CPC expansion and differentiation.
2.4. KDR/FLK1-Expressing CPCs
Precardiac mesodermal cells express the Vascular Endothelial Growth Factor (VEGF) receptor KDR/FLK1 during embryonic development. FLK1-expressing progenitor cells can generate both hematopoietic and cardiac lineages. The level of FLK1 expression determines lineage fate: high FLK1 favors hematopoietic differentiation, while low or absent FLK1 promotes cardiac fate. The FLK1-negative population gives rise to a second FLK1+ population, representing multipotent CPCs permanently committed to cardiogenesis. Due to KDR/FLK1’s broad expression, it’s often used with other cardiac markers like PDGFRα, CXCR4, and MESP1/2 to enrich for CPCs.
2.5. CPCs from the First and Second Heart Fields
The cardiac mesoderm contains two distinct progenitor cell pools: the first heart field (FHF) and the second heart field (SHF). These fields develop sequentially and form different heart components. FHF-derived CPCs express NKX2.5, while SHF-derived CPCs express ISL1. FHF-CPCs are challenging to isolate due to limited unique markers beyond NKX2.5. HCN4 has been suggested as an FHF marker, potentially isolating conduction system-specific CPCs. FHF-CPCs mainly differentiate into cardiomyocytes and some smooth muscle cells. ISL1 CPCs, on the other hand, generate all three cardiac lineages and contribute to a significant portion (around 40%) of cardiomyocytes during heart development. ISL1 CPCs are also found in the adult heart, particularly in the outflow tract, atria, and right ventricle.
2.6. Epicardium-Derived CPCs
Epicardium-derived CPCs, expressing Wilms tumor 1 (WT1), originate from the epicardium and are derived from SHF CPCs. They arise from epicardial cells undergoing epithelial-to-mesenchymal transition (EMT) and migrating into the myocardium. Epicardial-derived CPCs can differentiate into coronary smooth muscle cells, cardiomyocytes, endothelial cells, perivascular, and cardiac interstitial fibroblasts, with varying efficiencies. Similar to c-KIT CPCs, WT1 CPCs participate in cardiomyocyte formation during development but are scarce in the adult heart. Factors like thymosin beta 4 (Tβ4) can reactivate adult epicardial cells, but their cardiogenic potential remains debated.
2.7. Side Population-Derived CPCs
Side populations (SPs), identified by their ability to efflux Hoechst dye, are enriched for stem and progenitor cell activity in various tissues, including the heart. This dye efflux is mediated by ABC transporter proteins, particularly ABCG2, which is crucial for stem cell proliferation and differentiation. Cardiac SPs are found in perivascular and interstitial heart areas and exhibit self-renewal, homing, and multipotency. SCA1 and PDGFRα co-expressing cardiac SPs demonstrate high clonogenicity and multilineage potential. However, human SP differentiation potential is not fully understood. SPs are heterogeneous, containing subpopulations with cardiac, hematopoietic, and mesenchymal differentiation potential, making it challenging to identify markers for the most cardiac-potent SP subpopulation.
2.8. Cardiosphere-Derived CPCs
Cardiospheres, derived from human heart biopsy cultures, contain a mixture of stromal, mesenchymal, and progenitor cells. They represent a niche-like environment with cardiac-committed cells at the center and supporting cells in the periphery. Cardiosphere-derived cells (CDCs) are harvested from these clusters. Similar to c-KIT and epicardial CPCs, the regenerative potential of CDCs is debated, with evidence suggesting paracrine mechanisms as the primary driver of cardiac repair rather than direct cell generation.
III. Generation of CPCs from Human iPSCs
Native CPCs are scarce in heart tissue, necessitating alternative sources for effective cardiac regeneration. Reprogramming adult somatic cells into iPSCs offers a potential solution, providing an unlimited source of patient-specific pluripotent cells capable of differentiating into CPCs in vitro. This section details current methods for inducing, expanding, and maintaining iPSC-derived CPCs.
Various protocols exist to modulate cardiac differentiation in iPSCs (Table 2), but differentiation efficiencies can vary significantly between iPSC lines. Common to all protocols is the initial dedifferentiation of somatic cells into a pluripotent state using reprogramming factors (OCT4, SOX2, KFL4, c-MYC). Once pluripotency is achieved, cardiac differentiation is induced using methods like embryoid body (EB) formation, monolayer cultures with growth factors, serum, small molecules, matrices, or co-culture with visceral endodermal stromal (END2) layers. Recent protocols favor monolayer cultures with serum-free media like mTeSR1 or E8, maintaining iPSC pluripotency in feeder-free conditions. However, most studies focus on iPSC-cardiomyocyte generation, not necessarily on the homogeneity of the CPC population during cardiac lineage commitment.
Table 2. Protocols Producing CPCs as Target Cells or as Intermediate Cells from iPSCs.
| Protocol | CPC-Associated Markers Identified | CPCs as Target or Intermediate | Differentiation and Functionality Potential | Limitations | Ref. |
|---|---|---|---|---|---|
| Mouse iPSCs on feeder-layers and human iPSCs in hESC culture medium without bFGF | Differentiation medium with 20% FBS + gelatin-coated plates + AA between day 2 and 6 | NKX2.5+TBX5+& FLK1+CXCR4+ | Intermediate | -Synchronous beating and better-organized striated myofilaments in CMs | -AA is not able to promote mesodermal differentiation and CM proliferation-No reports on CPC potential into SMCs and ECs |
| Human iPSCs in monolayer culture (mTeSR1 + Matrigel-coated plates) | ROCK inhibitor (Y27632) for 1 day and DMEM/F12/B27-vitamin A + BMP4 + AA + CHIR for 3 days | SSEA1+MESP1/2+ISL1+ | Target | -Differentiation into the three cardiac lineages under specific differentiation media-80% efficiency towards CMs, and 90% into SMCs and ECs-Synchronized beating and presence of organized sarcomeric structures | -Both early and late CPC-related markers were co-expressed in the generated CPCs-Repeated passaging leads to a decrease in CPC proliferation rate-Only one iPSC line was tested |
| Human iPSCs on inactivated MEFs followed by feeder depletion culture in Matrigel | BMP4 for 3 days and +/− Activin A + bFGF from day 1 until day 3 | DKK1 + VEGF + SB +/− Dorsomorphin/Noggin at day 3 | KDR+PDGFRα+ | Intermediate | -Low yield of CMs (11%) |
| Mouse iPSCs in DMEM with 15% FCS on feeder layers | Differentiation medium with 10% FCS + type IV collagen-coated dishes/OP9 cell sheets for 96–108 h | FLK1+ mesodermal cells co-cultured on OP9 cells + differentiation medium + cyclosporin-A | FLK1+CXCR4+VE-cadherin− | Target | -Synchronous beating-Pacemaker and ventricular action potentials-Myofilaments formation with transverse Z-bands-Presence of ion channels (Cav3.2, HCN4 and kir2.1) and intercalated disks |
| Human iPSCs on SNL feeder cells and Matrigel-coated plates | Co-culture on END-2 cells + cyclosporin-A at day 8 | Target | |||
| Human iPSCs on inactivated MEFs with KO-DMEM medium | Serum-free medium (RPMI/B27) + BMP2 + SU5402 for 6 days | OCT4+SSEA1+MESP1+TBX5+TBX6+TBX18+GATA4+MEF2C+NKX2.5+ISL1+TBX20+ | Target | -Differentiation towards CMs, SMCs and ECs under specific conditions-Arranged sarcomeric organization and gap junctions when CPCs were co-cultured with either fibroblasts + FCS, cardiac fibroblasts + CMs or conditioned medium-Trend towards ventricular CMs | -Only one iPSC line was tested-SSEA1+ CPCs can differentiate into multiple cardiac lineages, like FHF, SHF, epicardium and cardiac neural crest in the presence of FGF signals |
| Murine iPSCs on inactivated MEFs | Feeder-free culture on gelatin-coated plates + BIO | IMDM with 15% FCS | FLK1+MESP1+NKX2.5+ | Target | -Presence of CM, EC and SMC markers |
| Human iPSCs on Matrigel-coated plates | E8 medium + ROCK inhibitor for 24 h and RPMI/B27-insulin + CHIR for 48 h/4 days | TBX5+NKX2.5+CORIN+HCN4+GATA4+ | Target | -FHF: mainly differentiates into left ventricular (90%) and some atrial CMs (10%)-Presence of ion channels (Kir2.1) and higher contraction velocity | -4 different CPC populations identified with distinct differentiation potential-Isolation of the CPC populations was performed via a double transgene reporter-Expression of TBX5 and NKX2.5 dynamically changed during differentiation culture, except for the double negative (TBX5−/NKX2.5−) cell population |
| TBX5+NKX2.5−HCN4+GATA4+WT1+TBX18+KDR+PECAM1+ | Target | -Epicardial progenitors: contribute to nodal (80%) and some atrial CMs-Formation of tight junctions and expression of the ion channel KCNJ3 -Some potential towards fibroblasts, SMCs and ECs | |||
| TBX5−NKX2.5+GATA4+MEF2C+ISL1+ | Target | -SHF: differentiation predominantly into atrial (90%) and some nodal and ventricular CMs-Atrial CMs displayed slower beating rates -Some potential towards SMCs and ECs | |||
| TBX5−NKX2.5−KDR+PECAM1+ | Target | -Endothelial potential-Formation of tube-like structures under VEGF | |||
| Human iPSCs on inactivated MEFs followed by EB suspension culture | BMP4 for 4 days | IWR1/IWP1 for 2 days | NKX2.5+ISL1+GATA4+MEF2C+ | Intermediate | -Low percentage of CMs-Organized sarcomeric structures -Normal calcium transient rhythm |
| Human iPSCs on MEFs | DMEM/F12 with 20% FBS + AA + EB plating on gelatin-coated dishes at day 7 | MEFs for 24 h and BMP2 + SU5402 for 4 days in RPMI/B27-vitamin A | ISL1+NKX2.5+KDR+MESP1+TXB20+GATA4+ | Target | -Differentiation towards myocytes and vascular lineages under specific conditions |
| Human iPSCs on Synthemax-coated plates in E8 medium then mTeSR1/E8 + ROCK inhibitor for 24 h | Albumin-free RPMI + CHIR for 24 h | RPMI + IWP2 for 2 days at day 3 + basal medium at day 5 | ISL1+NKX2.5+KDR+ | Intermediate | -Spontaneous contraction and well-organized sarcomere filaments-Development of ventricular action potentials-Spontaneous calcium transients and connexin 43 expression in CMs |
| Human iPSCs on Matrigel in MEF-CM supplemented with bFGF | RPMI/B27-insulin + Activin A for 24 h + BMP4 and bFGF for 4 days | RPMI/B27-insulin + DKK1 for 2 days | MESP1+KDR+ISL1+NKX2.5+ | Intermediate | -Sarcomere formation-Ventricular and pacemaker action potentials-CM yield varied between 4 and 34% |
| Human iPSCs in Geltrex with E8 medium using spheroid culture | RPMI/B27-insulin + CHIR + BMP4 for 48 h | XAV939 for 48 h at day 4 | ISL1+TBX1+FGF10+FGF8+CXCR4+ (SHF) | Target | -38% efficiency towards CMs-More potential to generate SMCs, ECs and fibroblasts |
| ISL1+HCN4+TBX5+GATA4+CXCR4− (FHF) | Target | -62% efficiency towards CMs-Low levels of EC and fibroblast markers | |||
| Human PSCs on Matrigel/Synthemax-coated plates in mTeSR1/E8 medium with ROCK inhibitor | CHIR in RPMI basal medium for 24 h | IWP2/IWP4 in RPMI basal medium from day 3 to day 5 +LaSR basal or RPMI/Vc/Ins with ROCK inhibitor at day 6 +CHIR for 48 h from day 7 | WT1+TBX18+TCF21+ALDH1A2−KDR+ | Target | -Differentiation towards fibroblasts and SMCs-Fibroblasts and SMCs display fibroid spindle-like shape and a fusiform appearance, respectively-Formation of mature epithelial-like sheets with tight junctions (cobblestone morphology and expression of ZO1 along cell borders)-SMCs display calcium transients and contractibility |
| Albumin-free RPMI + CHIR for 24 h | RPMI + IWP2 for 2 days at day 3 + RPMI/Vc/Ins with ROCK inhibitor for 24 h at day 6+ CHIR in RPMI//Vc/Ins for 48 h at day 7 | Target | [153] | ||
| Human iPSCs on inactivated MEFs | StemPro-34 medium + BMP4 for 24 h + BMP4, Activin A and bFGF from day 1 until day 3 | StemPro-34 medium + Matrigel-coated plates + BMP4 + CHIR + SB + VEGF for 2 days | Target | [154] | |
| Human iPSCs in CDM + BSA + Activin A + FGF2 on gelatin-coated plates | CDM + PVA + FGF2 + LY294002 + BMP4 for 36 h and CDM + PVA + FGF2 + BMP4 for 3.5 days | CDM + PVA + BMP4 + WNT3A + RA for 10 days | Target | [155] | |
| Human iPSCs in E8 medium and monolayer culture on vitronectin-coated plates | S12-insulin medium + CHIR for 24 h | S12-insulin medium + IWR1 for 48 h at day 3 and RA + CHIR between day 5 and 8 | Target | [156] | |
| Murine iPSCs in inactivated MEFs in SCM | SCM-LIF + AA at day 2 | Puromycin at day 6 for 3 days | NKX2.5+c-KIT+FLK1+SCA1+ | Target | -Differentiation potential towards ventricular CMs, SMCs and ECs-Sarcomeric organization and intracellular coupling observed |
| Human iPSCs on MEFs followed by suspension culture in ESC culture medium | Gelatin-or human laminin211-coated plates + IMDM-serum and CHIR + BIO for 3 days | KY02111 +/− XAV939 or IWP2 from day 3 until day 9 | NKX2.5+GATA4+ | Intermediate | -Predominantly ventricular CMs and 16% pacemaker cells-Spontaneous beating, sarcomere myofilaments, Z-bands, ion channels (HERG and KCNQ1) intercalated disks observed |
| Human iPSCs in E8 medium on Synthemax/Matrigel-coated plates | CDM3 medium (RPMI basal medium + AA + rice-derived RHA) + CHIR for 2 days | CDM3 medium + WNT-C59 for 48 h at day 2 | MESP1+KDR+ISL1+GATA4+NKX2.5+TBX5+MEF2C+ | Intermediate | -Formation of atrial, ventricular and nodal CMs |
| Human iPSCs in mTeSR1 + ROCK inhibitor on Matrigel/Synthemax | Pre-treatment with CHIR/BIO for 3 days | RPMI/B27-insulin + Activin A for 24 h + BMP4 for 4 days | ISL1+NKX2.5+ | Intermediate | -High yield of CMs-Normal sarcomere organization with transverse Z-bands-Presence of intercalated disks-Maturation trend towards ventricular CMs (80–90%) Some atrial-like action potential (10%) and absence of nodal-like potentials-Some formation of SMCs |
| Transgenic iPSC lines carrying lentiviral integrated β-catenin shRNA | CHIR in RPMI/B27-insulin for 24 h | Doxycycline at 36 h post-CHIR addition | ISL1+NKX2.5+TBX5+WT1+ | Intermediate | |
| Non-transgenic hiPSC lines | IWP4 or IWP2 at day 3 | Not reported | – | [130] | |
| IWP2 at day 3 | ISL1+NKX2.5+ | Intermediate | [126] | ||
| Human iPSCs on vitronectin-coated plates in mTeSR1 + ROCK inhibitor for 24 h | RPMI/B27-insulin + ISX-9 for 7 days | NKX2.5+GATA4+ISL1+MEF2C+ | Target | -Differentiation potential towards CMs, ECs, and SMCs in vitro and in vivo-CMs displayed myofilaments, mitochondria and glycogen particles-Formation of tube-like structures and LDL-uptake in ECs-ECs, and SMCs formed vascular structures in vivo | -The exact mechanisms by which ISX-9 induces the expression of cardiac transcription factors is unclear -No reports about electric coupling between generated CMs and endogenous CMs in vivo -No information about the electrophysiology of CMs |
| Human iPSCs on Matrigel in mTeSR1 + ROCK inhibitor | CHIR in RPMI/B27-insulin for 24 h + bFGF | IWP2 from day 3 to day 5 | MESP1+T+GATA4+ISL1+NKX2.5+TBX1+HAND2+ at day 2–3&KDR+PDGFRα+ at day 4–5 | Intermediate | -Formation of SHF-derived CPCs-Differentiation trend into fibroblasts, which exhibited characteristics of fetal ventricular fibroblasts |
| Human iPSCs in feeder-free (Geltrex) monolayer culture | RPMI + PVA + BMP4 + FGF2 for 2 days | RPMI-insulin + 20% FBS/human serum for 2 days | MESP1+ISL1+NKX2.5+ | Intermediate | -Robust contraction-Striated sarcomeres and gap junction formation-High yield of CMs (64–89%)-Presence of physiological calcium transients and functional electrical coupling-Differentiation trend into ventricular CMs |
| RPMI-insulin + 20% HSA + AA for 2 days | Intermediate | ||||
| RPMI-insulin + 20% HSA + AA for 2 days | Intermediate |
Growth factors and small molecules play a crucial role in modulating cardiac differentiation efficiency. Early protocols utilized growth factors like BMP, Activin/Nodal, and FGF signaling pathway modulators (Activin A, BMP2/4, FGF2) to induce cardiac mesoderm formation. Lian et al. (2012) demonstrated iPSC cardiac differentiation using only small molecule WNT signaling modulators. Minami et al. (2012) further enhanced efficiency by combining WNT modulators at early and middle differentiation stages. Many protocols rely on activating canonical WNT signaling with a GSK3 inhibitor, such as CHIR99021 (CHIR), for 24 hours. CHIR induces mesoderm marker Brachyury (T) expression in undifferentiated iPSCs, initiating mesoderm induction. Subsequently, WNT signaling is suppressed using inhibitors (XAV939, IWP, IWR, β-catenin shRNA) to direct mesodermal cells towards cardiac fate. After 3-4 days of WNT suppression, iPSC-derived T+ mesodermal cells begin expressing cardiac transcription factors (NKX2.5, ISL1, FLK1, PDGFRα), transitioning into the CPC population.
Recent studies have successfully generated CPCs from iPSCs using a single small molecule, potentially simplifying and reducing protocol costs. Cyclosporin-A (CSA) has been shown to stimulate differentiation of FLK1-positive mesodermal cells into FLK1+/CXCR4+/VE-cadherin− CPCs and cardiomyocytes, significantly increasing CPC and cardiomyocyte yield. Generated cardiomyocytes exhibited adult cardiomyocyte-like properties. However, this CPC type has limited endothelial potential and does not differentiate into smooth muscle cells, potentially requiring additional factors for complete cardiac lineage differentiation. Furthermore, co-culture with END2 cells in this protocol introduces undefined factors, limiting reproducibility. Another study demonstrated that isoxazole (ISX-9) treatment of human iPSCs for 7 days induces CPC marker expression (NKX2.5, GATA4, ISL1, MEF2C). These CPCs can generate cardiomyocytes, smooth muscle cells, and endothelial cells and modulate key cardiomyogenesis signaling pathways, including VEGF, Activin A, and WNT signaling. ISX-9 appears to sequentially upregulate WNT pathway activators, contributing to CPC generation.
IPSC technology holds great promise for CPC research, offering opportunities for novel cell therapies, disease modeling, and drug screening. However, most iPSC-cardiac regeneration studies prioritize cardiomyocyte production and maturation. Current knowledge about iPSC-CPC generation remains limited, highlighting a gap in understanding and optimizing CPC-focused protocols within the broader field of cardiac regeneration.
IV. Direct Reprogramming into CPCs
Direct reprogramming, or transdifferentiation, offers a faster and potentially safer alternative to iPSC reprogramming for generating CPCs. It involves converting somatic cells directly into other cell types, bypassing the pluripotent iPSC intermediate. Transdifferentiation is significantly faster than iPSC reprogramming, taking days instead of weeks, and avoids potential mutations or epigenetic changes associated with iPSC generation. While most cardiac transdifferentiation studies focus on generating fully differentiated cardiomyocytes, generating CPCs via transdifferentiation could be a superior strategy for regenerative medicine applications. This section focuses on current direct reprogramming approaches for CPC generation.
4.1. Partial Somatic Cell Reprogramming into CPCs
Some transdifferentiation protocols involve a transient pluripotency stage before cells commit to CPC fates. Reprogramming factors (OCT4, SOX2, KLF4, C-MYC) can initiate epigenetic resetting towards a stem cell state (partial reprogramming), but are insufficient alone for cardiac lineage commitment. Signaling molecules involved in cardiogenesis (BMPs, WNT modulators, FGFs) are required, similar to iPSC-cardiomyocyte differentiation protocols. One study converted secondary mouse embryonic fibroblasts into CPCs using Cell Activation and Signaling-Directed (CASD) lineage conversion, combining reprogramming and cardiac-specific factors. Zhang et al. (2016) used a protocol involving transient reprogramming factor exposure followed by cardiac differentiation induction with CHIR99021 and subsequent treatment with CHIR99021, BMP4, Activin A, and SU5402. The resulting CPCs expressed proliferative and cardiac markers and could differentiate into all three cardiac lineages. Efe et al. (2011) also demonstrated cardiac conversion of mouse fibroblasts using transient pluripotent marker expression followed by BMP4 and JAK inhibitor JI1. This protocol upregulated CPC markers like NKX2.5, GATA4, and FLK1.
Wang et al. (2014) reduced reprogramming factors to a single factor (OCT4) combined with small molecules (ALK4/5/7 inhibitor SB431542, GSK inhibitor CHIR, LSD1/KDM1 inhibitor parnate/tranylcypromine, adenylyl cyclase activator forskolin). Mouse fibroblasts exposed to this cocktail followed by BMP4 treatment generated CPCs expressing FLK1, MESP1, ISL1, GATA4, and Ki-67, which could differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. Another chemical reprogramming protocol using a larger combination of small molecules (CHIR, RepSox, forskolin, VPA, parnate, TTNPB) also generated CPC populations expressing SCA1, ABCG2, WT1, FLK1, and MESP1. While these studies demonstrate CPC generation, their primary focus was often cardiomyocyte generation, requiring further investigation into the specific characteristics and potency of the CPC populations produced.
4.2. Direct Somatic Reprogramming into CPCs
Direct somatic reprogramming achieves transdifferentiation without a pluripotent intermediate. CPCs have been directly generated from adult mouse fibroblasts using 11-factor or 5-factor reprogramming protocols. Both protocols produced CPCs expressing NKX2.5, MEF2C, MESP1, TBX20, IRX4, and CXCR4, regardless of factor combination or fibroblast origin. These CPCs downregulated fibroblast-specific genes and differentiated into all three cardiac lineages. Adding WNT and JAK/STAT activators enhanced protocol efficiency. While both protocols yielded comparable CPCs, the 11-factor protocol produced more CPC colonies, while the 5-factor protocol-derived CPCs showed greater smooth muscle cell and endothelial cell marker expression upon differentiation. Human dermal fibroblasts have also been directly reprogrammed into CPCs by overexpressing MESP1 and ETS2, followed by Activin A and BMP2 treatment. ETS2 may be replaceable by other ETS transcripts abundant in the developing heart.
Viral vectors, typically lentiviruses, are commonly used to deliver reprogramming factors, posing potential risks for translational applications due to genome modifications. Protein delivery systems, using transcription factor proteins directly, offer a non-viral alternative. A nonviral protein delivery system with GATA4, HAND2, MEF2C, and TBX5, combined with growth factors (BMP4, Activin A, bFGF), reprogrammed human dermal fibroblasts into CPCs with high efficiency (>80%). These CPCs expressed FLK1 and ISL1 and downregulated fibroblast markers.
4.3. Somatic Reprogramming into Cardiospheres
Adult skin fibroblasts can be converted into cardiospheres with CPC potential. Reprogramming skin cells with Yamanaka factors (SOX2, KLF4, OCT4) followed by specific media and GSK3 inhibitor treatment resulted in cardiospheres resembling endogenous cardiospheres but with higher MESP1, ISL1, and NKX2.5 expression. Passaging enriched these cardiospheres for CPCs expressing c-KIT, FLK1, and CXCR4, which could differentiate into cardiomyocytes. However, human cardiospheres differ from mouse cardiospheres in spontaneous beating and in vitro propagation, suggesting species-specific signaling pathways in somatic reprogramming into cardiospheres.
4.4. In Vivo Direct Reprogramming
In vivo direct reprogramming, converting endogenous cardiac cells into CPCs within the heart, bypasses cell transplantation challenges. The heart’s native environment may promote functional reprogramming and maturation. Zebrafish studies show that cardiac injury induces dedifferentiation of atrial cardiomyocytes into CPC-like cells that can differentiate into ventricular cardiomyocytes. Murine cardiomyocyte dedifferentiation in vitro also generates CPCs capable of differentiating into functional cardiomyocytes and endothelial cells. In vivo MI mouse models show DNA methylome changes during cardiomyocyte dedifferentiation, with repression of cardiomyocyte-specific genes and upregulation of cell cycle and proliferation genes. This concept could be applied for in vivo CPC reprogramming. However, molecular mechanisms of somatic cell dedifferentiation are not fully understood, and further research is needed to identify key factors.
While in vivo reprogramming is promising, current applications mainly focus on generating fully differentiated cardiomyocytes, not CPCs. Despite the potential of direct reprogramming for CPC generation, challenges remain, including suboptimal transdifferentiation efficiencies, limited CPC characterization, and unclear differentiation potential and functionality of derived cells.
V. In Vitro Culture of CPCs Derived Through Reprogramming Protocols
Establishing efficient reprogramming protocols for CPC generation is crucial, but so is developing methods for CPC isolation, propagation, and expansion in vitro. This section focuses on successful in vitro methods for isolating, expanding, and maintaining CPCs.
5.1. Isolation of CPCs
CPC isolation relies on characteristic gene expression patterns and surface markers (Table 1). ISL1 and NKX2.5 genes are commonly used, but their transient expression can lead to heterogeneous CPC populations. Combinations of surface markers are often preferred, as single markers may be insufficient for CPC identification. Nsair et al. (2012) demonstrated that co-expression of FLT1 (VEGFR1) and FLT4 (VEGFR3) specifically identifies ISL1/NKX2.5-expressing CPCs with high purity and multilineage differentiation potential. Nelson et al. (2008) used CXCR4 and FLK1 to isolate a more restricted CPC population from FLK1-positive cells. Zhou et al. (2017) identified SIX2 as a marker for temporally distinct second heart field CPC subpopulations. Torán et al. (2019) identified GPR4, CACNG7, CDH5, and F11R as highly expressed surface markers on human adult c-KIT CPCs. While new markers are continuously being discovered, validation, especially for ESC-CPCs and iPSC-CPCs, is crucial for establishing a common CPC signature.
5.2. Expansion and Maintenance of iPSC-CPCs
Maintaining β-catenin concentration is an effective method for in vitro CPC expansion. GSK3 inhibitors (WNT3A, CHIR, BIO) promote CPC expansion and suppress myocytic differentiation, leading to homogenous CPC colonies. Combining a WNT/β-catenin inhibitor (IQ-1) and a ROCK inhibitor (Thiazovivn) also expands CPCs in feeder-free conditions while maintaining multipotency. WNT signaling interacts with Notch and FGF signaling pathways to stimulate CPC expansion. Notch signaling activation by Notch1 represses CPC expansion and self-renewal, while WNT and FGF signaling activation cooperatively enhances ISL1 CPCs. Biomolecules modulating Notch and FGF signaling, alongside WNT activators, might facilitate CPC expansion. However, FGF signaling inhibition has also been shown to enhance SCA1-expressing CPC expansion, requiring further investigation.
Persistent BMP signaling inhibition enhances CPC expansion and prevents differentiation. The BMP inhibitor Gremlin 2 (GREM2) promotes iPSC-CPC proliferation by suppressing BMP4 receptor activity. However, GREM2 can also induce CPC differentiation into cardiac subtypes, requiring careful timing and potency control. GREM2 primarily increases KDRlow and NKX2.5+ CPC numbers in vitro, with its function diminishing in the adult heart. Dorsomorphin homologue 1 (DMH1), a second-generation BMP inhibitor, enriches CPCs expressing Branchyury, MESP1, and ISL1 from pluripotent cells. DMH1 is most effective during early cardiac differentiation stages to increase CPC numbers.
Ascorbic acid (AA) also enhances in vitro CPC expansion, specifically iPSC-derived FLK1+/CXCR4+ CPCs, through the MEK-ERK1/2 pathway by promoting collagen synthesis. However, AA’s effects on other CPC types need evaluation. Birket et al. (2015) used a cocktail of signaling pathway modulators (SU5402, DMH1, SB431542, IGF1, SAG) to expand CPCs for over 40 population doublings, but this study used MYC-transduced iPSC lines, requiring further assessment with non-transgenic iPSC-derived CPCs. Bao and colleagues (2017) developed serum-containing and serum-free protocols for long-term expansion of human iPSC-derived epicardial CPCs, using TGF-β inhibitors (SB431542 or A83-01). Versene, a gentler dissociation buffer, improved expansion efficiency of CPCs from pluripotent stem cells. GMP-compatible systems using stirred tank bioreactors and microcarrier technology are being developed for large-scale CPC expansion. Microcarrier-based stirred cultures improved cell suspension increase and viability compared to static cultures.
5.3. Expansion and Maintenance of Transdifferentiated CPCs
Transdifferentiated CPCs have similar in vitro culture requirements as iPSC-CPCs. Canonical WNT and JAK/STAT activators maintain their proliferative and multipotent state for extended passages without continuous reprogramming factor expression. However, somatic cell origin (cardiac vs. non-cardiac tissue) can affect CPC maintenance and expansion. Fibroblast-derived CPCs can also be expanded using BMP4, Activin A, CHIR, and SU5402 to repress cardiac differentiation and sustain self-renewal.
In summary, in vitro CPC culture requires precise temporal control of multiple signaling pathways. Expanding CPCs while maintaining self-renewal and multipotency remains complex, hindering the development of standardized culture conditions. This complexity is amplified when considering CPCs derived from iPSCs and direct reprogramming. Comparative studies of existing protocols are crucial for establishing optimal in vitro culture conditions for specific CPC types.
VI. Strategies to Improve CPC Reprogramming
CPC generation strategies are continuously evolving. While reprogramming and transdifferentiation offer promising routes to generate cardiac lineages, protocols often suffer from low efficiency or lack mechanistic insight. Strategies to enhance CPC proliferation and lifespan are essential to overcome limitations associated with patient-derived CPCs from compromised hearts. Genetic engineering and factors like CRISPR, epigenetic modulators, and microRNAs offer scope for improving CPC regeneration. This section describes examples of these strategies in the context of CPCs.
6.1. Genetic Engineering with PIM1
Genetic engineering with PIM1, a proto-oncogene serine/threonine-protein kinase, enhances CPC proliferation, survival, and differentiation. PIM1 is highly expressed in bone marrow, tumor cells, and fetal heart and regulates anti-apoptosis and cell cycle pathways. Mohsin et al. (2013) genetically modified patient-derived human CPCs (hCPCs) with PIM1 kinase (hCPCeP) to rejuvenate their properties, increasing proliferation, telomere length, survival, and decreasing senescence markers. PIM1-engineered cells also showed increased commitment to all three cardiac lineages. PIM1’s effect normalizes after passaging, suggesting transient mitosis increase and telomere stability without oncogenic transformation. PIM1 localization (mitochondrial or nuclear) can further modulate hCPCeP properties. Intramyocardial injection of hCPCeP in cardiomyopathic mice improved engraftment, differentiation, vasculature, and reduced infarct size. Similar results were observed in murine CPCs. Non-viral minicircle (MC) plasmids have been used to deliver PIM1 into mouse CPCs, showing increased protection in MI models.
6.2. CRISPR in Context with CPCs
CRISPR/Cas9 gene editing is pivotal in identifying CPC regulators. Proteomic studies identified Disabled 2 (DAB2) as a functional regulator in cardiomyocyte differentiation from hESCs, upregulated in CPCs. CRISPR/Cas9 deletion of Dab2 in zebrafish embryos showed increased WNT/β-catenin signaling and decreased cardiomyocyte numbers, suggesting DAB2’s role in inhibiting WNT/β-catenin signaling for cardiomyocyte maintenance from CPCs. Alpha Protein Kinase 2 (ALPK2) was also identified via CRISPR and antisense knockdown as a temporal regulator during CPC specification and cardiac commitment, negatively regulating WNT/β-catenin signaling. Recent CRISPR studies on Furin gene ablation in mouse CPCs revealed Furin’s role in CPC proliferation and differentiation, partially mediated by BMP pathway modulation by Nkx2.5. CRISPR continues to be a powerful tool for elucidating CPC mechanisms.
6.3. Epigenetic Modulators
Epigenetic modulators regulate gene transcription by binding to chromatin. Cellular reprogramming involves epigenetic changes. Modulating epigenetic modulator activity can affect cardiac reprogramming efficiency. Knockdown of polycomb ring finger pro-oncogene Bmi1 in fibroblasts activates cardiac transcription factors (GATA4, ISL1, TBX20), converting cells into cardiomyocytes. Silencing Bmi1 also enabled efficient cardiomyocyte reprogramming with just two factors (MEF2C and TBX5), yielding mature cardiomyocytes. Bmi1’s role in CPC reprogramming, considering ISL1 upregulation upon Bmi1 depletion, warrants investigation. BAF chromatin remodeling protein BAF60A, involved in CPC self-renewal through interaction with TBX1, is another potential epigenetic modulator. TBX1 recruits BAF60A to the WNT5A promoter, upregulating its expression in CPCs. WNT5A, a non-canonical WNT ligand, cooperates with WNT11 to induce CPC development from heart fields. Identifying epigenetic barriers could reduce reprogramming factors needed for CPC generation, leading to faster and safer protocols.
6.4. MicroRNAs
MicroRNAs, short non-coding RNAs, repress gene expression and offer a promising alternative to traditional reprogramming due to easy delivery and low toxicity. Multiple microRNA transcripts can be delivered via a single vector, potentially increasing reprogramming efficiency. While most microRNA studies focus on direct cardiomyocyte conversion, microRNAs also modulate CPC functions (Table 3). Sirish et al. (2012) identified differentially expressed microRNAs in CPC development, targeting proteins involved in cell proliferation and other cellular processes. Overexpression of miR-17-92 cluster increased CPC proliferation in vivo. MiR-1, -499, and -204 repress proliferation and stimulate differentiation in SCA1+ CPCs. Inhibition of miR-204 suppressed CPC differentiation and promoted proliferation. MicroRNAs like let-7, miR-18, miR-302, and miR-17-92 cluster regulate cardiac fate in MESP1+ CPCs. The miR-322/-503 cluster, enriched in CPCs, targets CELF1, promoting cardiac lineage by suppressing other lineages. Garate et al. (2018) identified microRNA families (miR-302, C19MC, miR-17/92, miR-26) highly expressed in mesoendodermal progenitor cells, potentially associated with epithelial to mesenchymal transition during mesoderm development. Cheng et al. (2019) showed that ischemic heart-secreted microRNAs mobilize bone marrow progenitor cells to the injury site, promoting vascularization, demonstrating microRNA’s potential for targeting endogenous progenitor cells for cardiac repair. MicroRNAs offer a valuable target for improving CPC generation and function.
Table 3. Role of microRNAs in CPC Biology.
| CPC Property | MiRNA Involved | Target Protein/Pathway | Mechanism | Ref. |
|---|---|---|---|---|
| Proliferation | miR-21 | PTEN | Inhibit negative regulators of cell proliferation | [224] |
| miR-218 | SFRP2 | |||
| miR-548c | MEIS1 | |||
| miR-509 | ||||
| miR-23b | ||||
| miR-204 | ATF2 | Repress proliferation-related transcription factors and induces differentiation | [225] | |
| miR-1 | HDAC4 | |||
| HAND2 | ||||
| miR-200b | GATA4 | |||
| miR-17-92 cluster | Not reported | Increases proliferation rate | [219] | |
| Differentiation | CMs | miR-133 | NELFA | Suppresses cardiogenesis |
| miR-218 | SFRP2 | Inhibits a negative regulator of cell proliferation | ||
| miR-142 | MEF2C | Suppresses CM formation | ||
| miR-1 | DLL1 | Increases NKX2.5 and Myogenin expression | ||
| miR-499 | ROD1 | Suppresses inhibitory factors of cardiac differentiation | ||
| SOX6 | ||||
| miR-708 | N-RAS | |||
| miR-322-503 cluster | CELF1 | |||
| SMCs | miR-22 | EVI1 | Inhibits negative regulators of SMC marker gene expression and of SMC transcription factors | |
| miR-29a | YY1 | |||
| miR-669a | MYOD | Increases CPC differentiation potential by preventing skeletal myogenesis | ||
| miR-669q | ||||
| Migration | miR-206 | TIMP3 | Suppresses a metalloproteinase inhibitor | |
| miR-21 | PTEN | Promotes migration of SCA1+ CPCs (not fully clear) | ||
| Apoptosis | miR-21 | BIM | Inhibit apoptotic activators | |
| PDCD4 | ||||
| miR-24 | BIM | |||
| miR-221 | ||||
| Necrotic Cell Death | miR-155 | RIP1 | Inhibits necrosis activators | |
| Vascular Remodeling | miR-221 | c-KITeNOS | Inhibit endothelial cell migration and proliferation | |
| miR-222 | ||||
| Cell Repolarization | miR-1 | KCNE1KCNQ1 | Reduce potassium current in hyperglycemia conditions | |
| miR-133 |
VII. Tissue Engineering with CPCs and CPC-Derived Cardiomyocytes
CPCs, particularly CPC-derived cardiomyocytes, often exhibit an immature phenotype similar to embryonic cardiac cells. However, in vivo transplantation leads to more advanced maturation. Microenvironment plays a crucial role in CPC functions. Cardiac tissue engineering aims to mimic the CPC microenvironment by combining cells, extracellular matrix (ECM), and biochemical factors to create cardiac tissue constructs. Scaffolds should be biodegradable, immune-privileged, electrically and mechanically supportive, and promote vascularization. Natural and synthetic matrices are used for scaffold production. Table 4 summarizes biomaterial strategies using CPCs in cardiac tissue engineering.
Table 4. Cardiac Tissue Engineering Strategies with Biomaterials Using CPCs.
| Scaffold Biomaterial | Experimental Design | Outcome | Limitations | Ref. |
|---|---|---|---|---|
| Fibrin patch | SSEA1+ and ISL1+ hESCs-CPCs mixed in fibrinogen, and scaffolds were then transplanted into myocardial infarction rats | -Improved contractility and decrease in adverse ventricular remodeling-Increased angiogenesis and attenuation of fibrosis | -Poor long-term cell engraftment-Functional improvements resulted from paracrine signaling | [247] |
| Same process as above, except the scaffolds were delivered surgically on the infarct area of a 68-year-old patient suffering from severe heart failure | -No observation of ventricular arrhythmias-Decreased in adverse ventricular remodeling | -Presence of T-cell response 3 months post-implantation-Absence of neovascularization in patch-treated area | [248] | |
| mESCs were primed with BMP2 for 36 h and seeded into fibrin matricesThe constructs were then implanted onto normal or infarcted rat left ventricles | -Efficient cell engraftment-Attenuation of left ventricle dilation-Promotion of neovascularization | -Rapid inflammation-driven degradation of scaffolds-Unclear whether neovascularization was due to in situ cell differentiation or endogenous EC recruitment | [249] | |
| Polyethylene glycol diacrylate woodpile (PEGDa-Wp) and PEGDa hydrogel. | Human adult LIN−/SCA1+ CPCs were seeded in a PEGDa hydrogel and the mixture was then cultured onto a PEGDa-Wp | -Benefits on cell assembly and alignment-Induction of cell spatial-ordered multilayer organization and differentiation towards a CM phenotype | -Incomplete maturation of CMs-No differentiation into SMCs and ECs-No in vivo testing of the scaffolds | [258] |
| Poly(l-lactic acid) Nanofibres | mESC-derived ISL1+/GATA4+ CPCs were seeded onto nanofibresAfter 7 days of in vitro differentiation, the scaffolds were implanted subcutaneously in the dorsal area of athymic nude mice | -Enhancement of cell attachment, extension and differentiation in vitro-Improvement of cell survival, integration and commitment to the three cardiac lineages in vivo-Induction of angiogenesis in vivo | -Poor in vitro differentiation into ECs-Unclear whether neovascularization was due to paracrine factors or CPC-derived SMCs and ECs | [257] |
| Tissue Printing using Sodium Alginate | Human SCA1+ CPCs were mixed with alginate matrixes, including an RGD-modified alginate, which were then used to print porous and non-porous scaffolds | -Porosity preserved viability and proliferation and increased cardiac commitment of CPCs-CPCs migrated from the construct and formed tubular-like structures | -Incomplete maturation of the differentiated cells-No in vivo testing of the scaffolds | [250] |
| Porcine- and human-derived myocardial matrices | Human SCA1+ CPCs were seeded onto porcine and human ECMScaffolds were injected into the left ventricular free wall of healthy hearts of Sprague Dawley rats | -Porcine-derived ECM was more efficient at promoting CPC differentiation, whereas human-derived ECM promoted CPC proliferation | -Variation in ECM properties due to distinct decellularised methods used, patient-to-patient variability and tissue age | [259] |
| 3D-printed hyaluronic acid/gelatin-based matrix | Human SCA1+ CPCs were printed together with the matrix The cell-loaded patches were transplanted in myocardial infarction mice | -Reduction of adverse remodeling and fibrosis-Long-term CPC survival and engraftment-Formation of vessel-like structures within the scaffold in vivo | -Absence of neovascularization in the infarcted region-Incomplete maturation of CMs in vivo | [251] |
| Collagen/Matrigel hydrogels | Human SCA1+ CPCs were encapsulated in collagen/Matrigel hydrogels which were cultured in either stress-free or unidirectional constrained conditions | -Enhanced cardiac differentiation and matrix remodeling-Constrained hydrogels stabilized CPC viability, attachment and proliferation-Static strain stimulated actin fiber formation and cell alignment | -Differentiation trend towards CMs-Incomplete maturation of CMs-No CPC differentiation into SMCs and ECs-No in vivo testing | [260] |
| Decellularised porcine ventricular ECM | Human Foetal and adult SCA1+ CPCs were resuspended in porcine myocardial matrix and collagen type I solutionsThe cell/matrix mixtures were injected into the left ventricular wall of Sprague Dawley rats | -The myocardial matrix improved CPCs adhesion, survival, proliferation and cardiac commitment both in vitro and in vivo-Foetal CPCs survived better than adult CPCs in vivo | -Rats were euthanized 30 min post-implantation, preventing assessment of long-term effects on cell survival, migration and cardiac function | [261] |
| Same procedure as above, exceptions: use of adult rat c-KIT+ CPCs and no in vivo implantation | -The cardiac ECM improved cardiac commitment, cell survival, proliferation and adhesion | -Differentiation trend towards CMs. -Low differentiation efficiency towards ECs and SMCs | [262] | |
| Whole decellularised mouse heart | hiPSC- and hESC-derived KDR+/c-KIT− CPCs were seeded into a whole decellularised mouse heartThe repopulated hearts were perfused with VEGF and DKK1 or VEGF and bFGF | -Efficient control of in situ iPSC-CPC differentiation-Advanced CM maturation-Development of vessel-like structures and spontaneous contraction for both iPSC-and ESC-CPC constructs | -Scattered regions of uncoupled cells-Insufficient mechanical force generation and incomplete electrical synchronization of the constructs | [252] |
| FLT1 (VEGFR1)+/PDGFRα+ hESC-CPCs were seeded onto decellularised mice hearts, which were implanted subcutaneously into SCID mice | -In situ generation of CMs, SMCs and ECs-Formation of a vascular network and higher expression of CM markers in vivo | -In vivo differentiated ECs were not ubiquitously distributed in the decellularised scaffold-Absence of beating populations | [263] | |
| Whole decellularised rat heart | hESC-derived KDR+/PDGFRα+ CPCs were expanded in a stirred-suspension bioreactor and seeded onto perfusion-decellularised Wistar rat hearts containing immobilized bFGF | -Improved CPC retention, proliferation and cardiac differentiation potential-Spontaneous and synchronous contractions-Advanced CM maturation | -Growth factor immobilization prevents spatiotemporal control-No in vivo testing | [264] |
| Whole decellularised human heart | Human adult c-KIT+ CPCs from human cardiac biopsies were cultured onto perfused-decellularised heart ventricles | -Increased CPC growth and stimulated differentiation towards cardiac lineages in vitro | -Poor CPC infiltration into the matrix-No electrical signal propagation.-No in vivo testing | [265] |
| Rat and pig collagen matrix and decellularised left ventricle ECM | iPSC-CPCs were cultured on rat or pig collagen matrices and decellularised ECMCPCs were also co-cultured with ECs and CMs | -Enhanced expression of contractile protein gene expression-Cell communication was observed in co-cultures | -No results reported on CPC proliferation and differentiation-No information about the CPC markers | [253] |
| 3D-bioprinted patch containing decellularised porcine ventricular ECM | Bioinks composed of decellularised ECM, human neonatal c-KIT+ CPCs and gelatin methacrylate were used to print patches, which were implanted onto the epicardial surface of the right ventricle of Sprague Dawley rat hearts | -Good CPC retention and viability in the scaffolds-Enhanced cardiogenic differentiation and angiogenic potential-Presence of vascularization in the patches in vivo | -Main purpose of the patch was to improve the paracrine release from the CPCs-No influence in SMC differentiation | [266] |
| Foetal and adult rat decellularised ventricle ECM | Immortalized adult mouse LIN−/SCA1+ CPCs were seeded onto embryonic, neonatal and adult rat ECM | -Good CPC retention, motility and viability-Remodeling of the supporting ECM-Enhanced production of cardiac repair factors | -No evidence of CPC differentiation-No in vivo testing | [267] |
| Decellularised murine embryonic heart | Day 5 and 9 mESC-CPCs were then seeded onto the decellularised scaffolds | -Day 5 progenitors formed spontaneously beating constructs in the scaffolds | -Mixed cell population isolated-Not all cell populations led to functional maturation | [268] |
| Decellularised human pericardium-derived microporous scaffold | Human SCA1+ CPCs were seeded onto 3D microporous pericardium scaffolds, which were then implanted subcutaneously into Wistar rats | -Improved CPC migration, survival, proliferation and differentiation-Reduction of immunological response and enhanced angiogenesis | -No influence in CPC differentiation towards SMCs | [269] |
| Self-assembling peptide nanofibers | Adult LIN−/c-KIT+ rat CPCs were seeded onto IGF1-tethered nanofibresCPCs and scaffolds were injected into myocardial infarction rats | -Enhanced CPC survival, proliferation and differentiation into CMs-Improved angiogenesis, recruitment of resident CPCs and attenuation of ventricle dilation | -Growth factor immobilization prevents spatiotemporal control-Newly formed CMs were derived from resident CPCs-CPCs were not cultured on the scaffolds prior to implantation | [254] |
| Adult mouse SCA1+ CPCs were mixed with Puramatrix® complex and injected into the border area of the myocardium in myocardial infarction mice | -Reduction of the infarct area and attenuation of ventricular dilation.-Enhanced neovascularization | -No CPC differentiation towards ECs-Functional improvements resulted from paracrine signaling-Poor CPC engraftment | [255] | |
| RDG-modified collagen and porous gelatin solid foam | Human adult CS-CPC were grown as secondary CSs, which were seeded onto the scaffolds | -Enhanced cell migration and ECM production-Increased CPC cardiogenic potential, cell retention and adherence | -Cardiac commitment trend towards CMs-Distinct scaffold morphologies promoted different biological processes | [270] |
| Degradable Poly(N-isopropylacrylamide) hydrogel | Mouse CDCs were added into hydrogel solutions, with or without collagen and containing different stiffness | -Preservation of CDC proliferation-Stimulation of differentiation into mature cardiac cells in hydrogels with medium stiffness and collagen | -No differentiation into ECs and SMCs-No in vivo testing | [256] |
| Biodegradable gelatin | Human CDCs were seeded onto bFGF immobilized gelatin hydrogels, which were implanted in the epicardium of immunosuppressed myocardial infarction pigs | -Enhanced angiogenesis, cell engraftment-Reduction of the infarct area and attenuation of adverse ventricular remodeling | -Growth factor immobilization prevents spatiotemporal control-No differentiation into ECs and SMCs | [271] |
| Fibrinogen/Matrigel mixture and PDMS molds | NKX2.5+/c-KIT+/either FLK1+ or SCA1+ iPSC-CPCs were mixed in a fibrinogen/Matrigel hydrogel and applied into PDMS molds | -Spontaneous and synchronous contraction-Highly organized sarcomere structures and robust electromechanical connections | -Improper nutrient access within the construct-No differentiation potential towards SMCs and ECs-No in vivo testing | [157] |
| Collagen sponge | CPCs were seeded onto collagen sponges and then transplanted into rat hearts with atrioventricular conduction block | -Enhanced vascularization-Gap junction formation-Differentiation into CMs, conduction cells and ECs | -No information about the functionality of the CPC-derived cells | [272] |
7.1. Natural Scaffolds
Natural matrices, composed of native ECM components, modulate cell behavior. They include pure ECM elements like fibrin, alginate, gelatin, and collagen hydrogels, or acellular tissues preserving native ECM properties. Fibrin patches seeded with murine and human CPCs have been tested in vivo, showing improved contractility and reduced remodeling, likely due to paracrine effects. 3D printing with SCA1+ fetal CPCs and natural scaffolds (alginate, hyaluronic acid/gelatin) allowed CPC migration and tubular structure formation. iPSC-CPCs mixed with fibrin/Matrigel hydrogels in PDMS molds differentiated into mature cardiomyocytes with electromechanical connections, highlighting the importance of nutrient access in tissue constructs. Decellularized tissue scaffolds, preserving native 3D architecture and ECM, have been combined with iPSC-CPCs. Repopulating decellularized mouse hearts with iPSC-CPCs resulted in cardiomyocyte, endothelial cell, and smooth muscle cell differentiation, vessel-like structures, and spontaneous contraction. However, uneven recellularization and lack of conduction system cells remain challenges. Natural scaffolds, while retaining native ECM cues, pose risks of immunological reaction, disease transmission, and variable physical properties.
7.2. Synthetic Scaffolds
Ideal synthetic scaffolds are biocompatible, degradable, and support cell attachment, migration, differentiation, and nutrient exchange. Synthetic scaffold properties can be customized. Self-assembling peptide nanofibers with CPCs have been tested in vivo. IGF1-tethered nanofibers enhanced CPC survival, proliferation, and differentiation into all three cardiac lineages, with regenerated cells integrating with host tissue. Puramatrix® nanofibers, however, showed limited CPC differentiation and primarily paracrine-mediated benefits. Poly(N-isopropylacrylamide) hydrogels with medium stiffness and collagen promoted cardiosphere-derived cell differentiation into mature cardiomyocytes. Poly(l-lactic acid) nanofibers supported ESC-derived CPC survival, engraftment, and differentiation into all three cardiac lineages, with improved endothelial differentiation in vivo. “Scaffold-in-scaffold” approaches using PEGDa woodpile and hydrogel promoted human CPC differentiation into aligned cardiomyocytes, but robust maturation and differentiation into other lineages were lacking. Synthetic biomaterials offer customizable microenvironments but face challenges in biocompatibility, degradation, and potential toxicity.
Electrically compatible scaffolds and biomaterials are being explored to stimulate and record hPSC-CM electrical activity (Table 5), a strategy potentially applicable to CPCs as CPC niche understanding improves. Patient-specific CPCs from iPSCs or transdifferentiation in engineered scaffolds for disease modeling are rare but crucial for future advancements. Tissue engineering, combined with mechanistic modulation, offers opportunities to exploit the CPC niche for assessing normal and disease-associated cardiac cell behavior and improving regenerative outcomes (Figure 2).
Table 5. In Vitro Cardiac Tissue Engineering Techniques with Biomaterials to Stimulate and Record hPSC-CM Electrical Activity.
| Cells | Biomaterial/Scaffold | Platform | Stimulation | Electrophysiology | Ref. |
|---|---|---|---|---|---|
| hiPSC-CMs | Graphene substrate | 2D | FET (current pulse with f = 1 Hz)For calcium: voltage ramp from −80 to +60 mV at 20 mV/s | -Enhanced electrophysiological properties:RP = −40.54 ± 1.72 mVAP = 75.24 ± 3.91 mVCV = 5.34 ± 1.60 cm/sICa2+ density = −9.31 ± 2.35 pA/pFICa2+,L density = −2.47 ± 0.6 pA/pFIk density = 46.24 ± 8.45 pA/pFIkr density = 36.57 ± 5.84 pA/pF Ca2+ transients:Amplitude intensity = 1.69 ± 0.20 uUpstroke velocity 3.09 ± 0.99 u/sDecay velocity (50%) = 0.84 ± 0.29 s | [273] |
| iCell® CMs & hESC-CMs | Reduced graphene oxide (rGO) | 2D | Light: intensity >1 mW/mm2, duration 40-ms-2-Hz light pulses and 3-s step of light | -Optical stimulation on rGO substrates improves CMs electrophysiology-rGO increases AP peaks frequency-On rGO CMs contraction frequency increases with light intensity | [274] |
| Neonatal Sprague Dawley rat vCMs | Electrospun gelatine + PCL nanofibres | 3D | FET (1–3 V, 50-ms-long pulses at 1–2 Hz) | -Electrical stimulation results in regularly spaced spikes (f = 1–2 Hz) with shape and width consistent with CM extracellular signals-NE increases electrical activity and frequency of calcium transients | [275] |
| hiPSC-CMs | PLGA electrospun aligned nanofibres | 3D | Not applied | -Enhanced CM maturity and electrical activity-CM drug (E4031) response showed higher electrophysiological homogeneity-L-ANFs increased FP amplitude, number of electrically active cells, synchronization and anisotropic propagation of the electrical signal | [276] |
| hESC-CMs & hiPSC-CMs | Type I collagen gel template suture (Biowires) | 3D | Electrical field with daily and progressively frequency increase: low frequency ramp-up regimen (from 1 to 3 Hz) or high frequency ramp-up regimen (from 1 to 6 Hz) | -Electrical stimulation enhanced electrical activity frequency-High frequency increased electrophysiological properties, contractile activity, synchronization and CV -High frequency decreased excitation threshold and variability in AP duration-High frequency improved CM response to caffeine and Ca2+ handling properties:IERG = 0.81 ± 0.09 pA/pFIK1 = 1.53 ± 0.25 pA/pF | [277] |
| hESC-CMs | MEA coated with collagen type I + agarose layer | 2D | Anti-arrhythmic and pro-arrhythmic drugs | -Pharmacological stimulation influences CMs electrophysiology -FPD and CT are dependent on the dose of arrhythmogenic drugs: E-4031 & Astemizole increased FPDFlecainide & Terfenadine decreased FPDFlecainide, Astemizole & Terfenadineincreased CTand of safe drugs:Verapamil & Lidocine decreased FPDLidocine slightly increased CT | [278] |
| hiPSC-CMs | MEA coated with hydrogel containing fluorescence microbeads | 2D | Electrical: periodic voltage pulses (biphasic square waves with pulse width = 4 ms, f = 0.2 Hz, peak-to-peak amplitude = 4 V)Pharmacological: drug exposure (NE and Blebbistatin) | -Good electrical coupling of CMs(FP = 9–35 µV and CV = 16 cm/s)-Electrical pacing promoted synchronized contraction(f = 11 bpm)-Recorded impedance increased with cell attachment and at each contraction-Blebbistan inhibited beating activity and has no effect on FP-NE increased CV and contraction spikes rate | [279] |
Figure 2. Promising Strategies to Improve CPC Characteristics and Functionality.
VIII. In Vivo Applications of Human CPCs
Clinical trials are the ultimate goal for CPC research, aiming to translate in vitro and animal studies to human therapies. However, clinical translation faces significant inefficiencies. Despite promising preclinical results, CPC clinical trials are underway (Table 6), utilizing various CPC types.
Table 6. Past and Ongoing Clinical Trials Using CPCs.
| Clinical Trial Name | Phase | Start/End Date | CPC Type | Delivery of Cells | Biomaterial Added | Results | Ref. |
|---|---|---|---|---|---|---|---|
| CADUCEUSprospective, randomized trial | I | 2009–2012 | CDCs | Direct injection via catheter | none | LVEF unchanged at 12 monthsScar size decreased 12.3% at 12 monthsRegional contractility and systolic wall thickening increased | [283,293] |
| ALCADIAOpen-label, non-randomized trial | I | 2010–2013 | CDCs | Direct injection via catheter | Biodegradable gelatin hydrogel sheet containing 200 μg of bFGF planted onto epicardium covering the injection site | LVEF increase 12% at 6 months Scar size decrease 3.3% at 6 months | [285] |
| ALLSTAROpen-label cohort (PI), double-blinded, randomized, placebo-controlled study (PII) | I/II | 2012–2019 | CDCs | Direct injection via catheter | none | Terminated (follow-up activities were ceased) | [294] |
| ESCORTOpen-label trial | I | 2013–2018 | ESC-derived ISL1+/CD15+ | Epicardial patch via coronary artery bypass procedure | Fibrin gel patch containing progenitor cells | LVEF increase of 12.5%No arrhythmias, or tumor formation | [287] |
| CAREMIDouble blinded, randomized, placebo-controlled trial | I/II | 2014–2016 | CDCs | Direct injection via catheter | none | Infarct size decreased to 15.6% at 12 months LVEF increase of 7.7% at 12 months | [295] |
| DYNAMICOpen-label trial, randomized, double-blinded, placebo-controlled trial | I | 2014–ongoing | CDCs | Direct injection via catheter to multi-vessel areas of heart | none | Ongoing | [296] |
| CONCERT-HFRandomized, double-blinded, placebo-controlled trial | II | 2015–ongoing | c-KIT+ | Direct injection via catheter | none | Ongoing (paused on 29.10.18, re-approved 06.02.2019) | [297] |
| TICAPOpen-label trial, non-randomized | I | 2011–2013 | CDCs | Direct injection via catheter | none | RVEF increase of around 8.0% at 18 and 36 monthsNo tumor formation | [288,290] |
| PERSEUSOpen-label trial, randomized | II | 2013–2016 | CDCs | Direct injection via catheter | none | LVEF increase of 6.4% at 3 monthsReduction in scar size | [289] |
| APOLLONRandomized, single-blinded | III | 2016 & Unknown | CDCs | Direct injection via catheter | none | Unknown status (last update was September 2017) | [291] |
| TICAP-DCMRandomized | I | 2017–ongoing | CDCs | Direct injection via catheter | none | Recruiting | [292] |
| REGRESS-HFpEFRandomized, double-blinded, placebo-controlled trial | II | 2017–ongoing | CDCs | Direct injection via catheter | none | Ongoing | [298] |
The first CPC clinical trial, SCIPIO, using c-KIT+ CPCs, was retracted due to data integrity concerns. CADUCEUS, a phase I trial using cardiosphere-derived cells, showed scar reduction and improved contractility after MI. ALCADIA, another phase I trial, combined autologous CPCs with bFGF-releasing gelatin hydrogel, showing improved LVEF and scar reduction. Both CADUCEUS and ALCADIA likely benefited from paracrine effects rather than direct cardiomyocyte differentiation. ESCORT, a phase I trial using hESC-derived CPCs in a fibrin gel patch, demonstrated safety and feasibility in severe heart failure. TICAP phase I and II trials assessed cardiosphere-derived cells in pediatric hypoplastic left heart syndrome, showing safety and improved cardiac function. Phase III APOLLON and phase I TICAP-DCM trials are ongoing, along with phase II REGRESS-HFpEF.
Many CPC clinical trials face limitations like small sample sizes and lack of blinded assessment, leading to inconclusive efficacy results. Whether benefits are from CPCs or paracrine factors remains unclear. Future clinical trials require broader subject assessment and reproducible human models to better evaluate CPC regenerative capacity.
IX. Current Challenges and Limitations
The role of CPCs in cardiac regeneration and repair remains debated. While CPC infusion has shown some improvements in cardiac function post-MI, their impact is still unclear due to their heterogeneous nature and disease complexities. Consensus on the ideal CPC population and characterizing markers is lacking. Epigenetic, gene, protein, and secretome profiles of most CPCs are not fully defined, limiting understanding of CPC development, self-renewal, and potency. Therapeutic efficacy comparisons between different CPC types are limited. Ideal CPCs should be autologous transplant-compatible, extensively expandable, differentiate into mature cardiac subtypes, and integrate with host cells.
Viral transduction, the most efficient reprogramming method, carries risks of genome integration and oncogene activation. Current protocols are complex and costly due to the use of reprogramming and growth factors. Safer, simpler, and cost-effective gene transfer methods are needed.
CPC-derived cell populations are often heterogeneous and immature, potentially leading to arrhythmias, instability, and poor engraftment. Mechanisms of cardiac lineage subtype specification need optimization for purer, mature cell populations. CRISPR strategies hold promise for addressing these limitations.
Epigenetic profiles significantly affect reprogramming efficiency. Non-cardiac tissue origin and aged tissue negatively impact iPSC cardiogenesis. Overcoming epigenetic barriers in somatic cells is crucial for successful reprogramming. Understanding CPC formation’s epigenetic regulation is vital for improving reprogramming efficiency.
Rodent models, differing from human hearts, dominate CPC research. Animal-derived techniques need validation for human application. For example, GMT reprogramming, effective in mouse fibroblasts, fails in human fibroblasts.
Future preclinical trials need to investigate CPC number-efficacy relationships and optimal cell therapy administration frequency. Invasive open-surgery cell injection needs to be replaced by less invasive delivery methods, such as intracoronary or intravenous injection, which currently suffer from poor cell homing and organ trapping. Less aggressive and more effective delivery systems are crucial for CPC regenerative medicine applications.
Biomaterial strategies have shown functional improvements but not complete cardiac recovery. Clinical trials using CPCs with biomaterials are limited. Integrating engineering with mechanistic modulation, potentially through genetic engineering, is needed to contextualize CPC behavior in disease.
Limited understanding of heart development and cardiac regeneration hinders progress. Clarifying cardiac factor stoichiometry and culture conditions for accurate in vitro CPC development is essential.
X. Final Thoughts—Controversies Surrounding CPCs
The debate on CPCs and adult heart repair continues. Initial evidence for c-KIT+ cells from Anversa’s lab was retracted, prompting challenges to the theory. Recent studies suggest cardiomyocyte generation from c-KIT+ cells is rare in adults. However, recent work by Narino et al. (2019) indicates c-KIT labels heterogeneous cardiac cell populations, with c-KIT low cells enriched for myogenic CSCs and c-KIT high cells having endothelial/mast cell potential. This study suggests adult c-KIT-labeled CSCs can be myogenic and contribute to regeneration, potentially countering aging effects. c-KIT haploinsufficiency in lineage-tracing studies might have led to oversight in previous research. Despite c-KIT controversy, CPC transplantation benefits have been observed in animal studies and clinical trials, potentially through paracrine signaling or acute inflammatory responses triggering healing.
Consensus on a critical endogenous CPC type for myocardial repair is lacking, but regeneration associated with CPCs is likely insufficient for severe damage like MI. Despite controversies, CPCs from various sources show promise for cardiac regenerative medicine, prompting investigation into strategies and CPC sources (stem cells, transdifferentiation) to enhance their functional significance.
XI. Future Directions
Heart failure patients, often elderly with comorbidities, have CPCs with compromised regenerative potential. CPC efficacy is influenced by genetics, epigenetics, environment, disease, heart load, medication, and aging. Enhancing CPC potential from adult or reprogrammed sources before transplantation is crucial. CPC research is progressing towards optimized reprogramming and tissue engineering. Advances in genomics, epigenomics, and proteomics will be vital for realizing CPC potential. Future regenerative approaches may combine genetic engineering, multiple stimuli (mechanical, electrical, biochemical), and tissue engineering for meticulously controlled systems maximizing CPC regenerative capacity for cell therapy, disease modeling, and drug screening (Figure 1).
Funding
This research was funded by the Engineering and Physical Sciences Research Council (EPSRC), UK, grant number EP/L015072/1 and the APC was funded by the same.
Conflicts of Interest
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
