Stable Phenotype and Function of Immortalized Lin − Sca-1 + Cardiac Progenitor Cells in Long-Term Culture: A Step Closer to Standardization

Abstract

Putative cardiac progenitor cells (CPCs) have been identified in the myocardium and are regarded as promising candidates for cardiac cell-based therapies. Although two distinct populations of CPCs reached the clinical setting, more detailed studies are required to portray the optimal cell type and therapeutic setting to drive robust cell engraftment and cardiomyogenesis after injury. Owing to the scarcity of the CPCs and the need for reproducibility, the generation of faithful cellular models would facilitate this scrutiny. Here, we evaluate whether immortalized Lin⁻Sca-1⁺ CPCs (iCPC^Sca-1) represent their native-cell counterpart, thereby constituting a robust in vitro model system for standardized investigation in the cardiac field. iCPC^Sca-1 were established in vitro as plastic adherent cells endowed with robust self-renewal capacity while preserving a stable phenotype in long-term culture. iCPC^Sca-1 differentiated into cardiomyocytic-, endothelial-, and smooth muscle-like cells when subjected to appropriate stimuli. The cell line consistently displayed features of Lin⁻Sca-1⁺ CPCs in vitro, as well as in vivo after intramyocardial delivery in the onset of myocardial infarction (MI). Transplanted iCPC^Sca-1 significantly attenuated the functional and anatomical alterations caused by MI while promoting neovascularization. iCPC^Sca-1 are further shown to engraft, establish functional connections, and differentiate in loco into cardiomyocyte- and vasculature-like cells. These data validate iCPC^Sca-1 as an in vitro model system for Lin⁻Sca-1⁺ progenitors and for systematic dissection of mechanisms underlying CPC subsets engraftment/differentiation in vivo. Moreover, iCPC^Sca-1 can be regarded as a ready-to-use CPCs source for pre-clinical bioengineering studies toward the development of novel strategies for restoration of the damaged myocardium.

Introduction

Cardiac cell therapy was shown to be a very complex endeavor for which a completely innovative vision and novel technologies are necessary. Indeed, in spite of intensive scientific and economic efforts, the technological exploitation of the stem cell potential has generated frustrating results owing to the unsystematic and simplistic approaches considered so far. Myocardial cell delivery in experimental animals and patients results in a very low number (∼3–10%) of cell persistence in the myocardium due to significant cell death and/or loss to the bloodstream with subsequent entrapment into the lungs [1,2]. Although some beneficial effects have been reported, the improvement of cardiac function has been greatly ascribed to the release of paracrine factors rather than to an increased presence of functional cardiomyocytes (CM) in the injured myocardium [3 –5]. Regardless, in the last decade, adult cardiac progenitor cells (CPCs) have gathered abundant attention [6 –18], paving the way to a quick transition from the bench to the bedside [19,20]. In fact, two independent clinical trials, each addressing a different subset of progenitor cells (c-Kit-expressing cells for SCIPIO [19] and cardiosphere-derived cells for CADUCEUS [20]), were carried out to assess their therapeutic potential in the myocardial infarction (MI) setting. Although the short-term results on a reduced patient number have shown a partial amelioration of the heart condition, most aspects of the CPCs biology are still undefined. Hence, only an indisputable demonstration of the beneficial effects of these strategies can suppress the innumerable doubts regarding protocols based on harsh manipulations of stem/progenitor cells [21]. Among many others, the factors governing the admirable equilibrium of the myocardial in vivo complexity, such as the interactions between progenitor cells and environmental components, remain largely undefined. Therefore, the only possibility to mimic the dynamics of myocardial homeostasis in order to establish safe and reliable repair procedures is a stringent “standardization” of the different aspects of myocardial complexity [22].

A clear understanding on CPCs biology is impaired by the scarcity of progenitors present within the myocardium as well as by time-consuming isolation procedures, and, importantly, by their phenotypic variability when isolated and expanded in different laboratories [23]. Consistently, progenitor cells that apparently partake in the same population can display non-comparable features. To circumvent these limitations, the aim of this study was to create a benchmark to be used as a reference for progenitor cell populations isolated from the myocardium in different laboratories on the basis of a plethora of non-specific cell-surface markers [6 –15,17,18]. For this purpose, lineage negative/Sca-1⁺ (Lin⁻Sca-1⁺) CPCs have been isolated from murine hearts and immortalized via telomerase catalytic subunit [murine telomerase catalytic subunit (mTERT)] overexpression. Recently, the role of telomerase activity in maintaining CPC viability and regenerative potential was demonstrated [24]. Aging leads to telomeric shortening in CPCs, thus leading to a senescent phenotype, as shown by the expression of p16INK4a. Such events have been correlated to cardiac function impairment, suggesting that CPC loss could be a main determinant in heart failure. Moreover, the overexpression of the human telomerase reverse transcriptase (TERT) catalytic subunit in various cell lines resulted in extended cellular lifespan [25 –27] without detectable changes characteristic of malignant transformation [28].

In the present study, a cell line of immortalized Sca-1⁺ cardiac progenitor cells (iCPC^Sca-1) has been specifically generated for the first time. iCPC^Sca-1 were extensively characterized to assess whether after immortalization these cells had preserved the hallmarks of their native-cell counterpart and the recognized capability to engraft and differentiate when transplanted in an MI murine model. Cells grew in vitro as adherent cells with a typical spindle-shape morphology and displayed robust self-renewal capacity, while preserving a CPC tri-lineage potential (ie, differentiated in CM-, endothelial-, and smooth muscle like-cells). iCPC^Sca-1 transplanted into the MI border zone significantly reduced the MI-induced left ventricle (LV) anatomical and functional alterations. Importantly, the cells engrafted, established functional connections, and differentiated in loco into cardiomyocyte- and vasculature-like cells, as previously shown for their native counterparts. Thus, the iCPC^Sca-1 line constitutes an unlimited source of Sca-1⁺ CPC replicates and can be used as a model system for in vitro high-throughput studies and for the dissection of the in vivo role of adult heart-resident progenitors.

Methods

Animals

C57BL/6 mice aged 6–12 weeks were used. Procedures were approved by Instituto de Biologia Molecular e Celular–Instituto de Engenharia Biomédica Animal Ethics Committee and National Direção Geral de Veterinária (permit no: 022793), and are in conformity with the European Parliament Directive 2010/63/EU. Humane endpoints were followed according to the Organization for Economic Co-operation and Development Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation (2000).

Mice were anesthetized by an intraperitoneal injection (ip) of medetomidine (1 mg/kg; Sededorm) and ketamine (75 mg/kg; Clorketam), and its adequacy was monitored by the pedal withdrawal reflex.

Isolation and culture of Sca-1⁺ CPCs

CPCs were isolated as previously described [29]: 6 week male C57BL/6 mice hearts were minced and digested using 0.25% Trypsin/ethylenediaminetetraaceticacid (EDTA) and collagenase II (1,500 U; Worthington) in phosphate-buffered saline (PBS). Tissue fragments were cultured in Dulbecco's Modified Eagle Medium (DMEM; Lonza) that was supplemented with 10% fetal calf serum (Lonza), 100 IU/mL penicillin and 100 μg/mL streptomycin, insulin-transferrin-selenium 1×, 300 ng/mL retinoic acid, 0.8 μg/mL linoleic acid, 2 mM l-glutamine, 0.1 ng/mL insulin-like growth factor 1, and 0.1 ng/mL endothelial growth factor (complete medium). After 12–15 days, adherent cells migrating out from fragments were harvested and magnetically sorted using anti-hematopoietic lineages antibody cocktail (Miltenyi Biotec). Sca-1⁺ cells were enriched from the Lin⁻ fraction by two sets of magnetic cell sorting protocols (Miltenyi Biotec) and expanded in culture in complete medium before transfection.

Establishment of iCPC^Sca-1

Murine TERT coding sequence, obtained from pGRN190 (kind gift from Geron Corporation), was inserted into EcoRI restriction site in pcINeo plasmid (Promega). Lin⁻Sca-1⁺ cells were transfected with pCINeo-TERT or control vector using the Calcium Phosphate Method (Promega), according to the manufacturer's instructions and incubated in G418-containing medium (Sigma-Aldrich) for at least 3 weeks. For clonal selection, immortalized cells were plated at single-cell density by limiting dilution and cultured in Sca-1⁺-conditioned medium. Clones derived from a single cell were expanded under G418 selection and characterized for Sca-1 expression by flow cytometry. Proliferative capacity of the established line was calculated using the formula log₁₀ (total no./start no.)/log₂.

In vivo tumorigenicity assay

The tumorigenicity of iCPC^Sca-1 was assayed by a heterotopic injection into syngeneic animals [30,31]. Eight-week-old C57BL/6 female mice were subjected to a subcutaneous injection of 1×10⁶ cells [in 0.2 mL of 2% fetal bovine serum (FBS) in α-minimum essential medium (α-MEM)] of iCPC^Sca-1 at each shoulder pad. Animals were monitored daily for the appearance of palpable tumors. Mice were necropsied after a 5-month surveillance and the injection region as well as the heart, spleen, lung, and liver were harvested, fixed in 10% formalin neutral buffer (VWR BDH and Prolabo), and paraffin embedded.

Flow cytometry

Before flow-cytometry analysis, single-cell suspensions were labeled with the following antibodies: APC—anti-PDGFRα (Biolegend), c-Kit and Flk-1 (eBiosciences); PE—anti-CD105 (Biolegend), CD106 (Biolegend), CD44 (Immunotools), CD45 (Immunotools), CD34 (Biolegend), CD31 (BD Pharmingen), CD62L (Immunotools), CD40L (BD Pharmingen), CD90.2 (BD Pharmingen), and Stro-1 (Santa Cruz Biotech); and FITC—anti Sca-1 (eBiosciences) and CD40 (BD Pharmingen). Samples were acquired on an FACS Canto II and analyzed using FlowJo software.

Gene expression

For the reverse transcription polymerase chain reaction (RT-PCR), total RNA was extracted using Trizol Reagent (Sigma-Aldrich), reverse transcribed according to the manufacturer's instructions (Bioline) and PCR was performed using BIOTAQ DNA polymerase (Bioline) and gene-specific primers. For quantitative RT-PCR (qRT-PCR) analysis, cDNAs were synthesized using PrimeScript^™ RT reagent kit following the manufacturer's instructions (Takara Bio, Inc.). qRT-PCR was performed using iQ^™ Sybr^® Green Supermix (Bio-Rad) and gene-specific primers. Reactions were carried out in triplicate on the iCycler iQ5 Real-Time PCR system (Bio-Rad). Values were normalized to glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. Primer sequences are available on request.

Immunocytochemistry

iCPC^Sca-1 were fixed with 4% paraformaldehyde (PFA) or cold methanol for GATA-4 detection, permeabilized with 0.2% (cytoplasmic) or 1% Triton X-100/PBS (nuclear epitopes), and blocked for 1 h with 1% goat serum, 1% bovine serum albumin (BSA), or M.O.M.™ Immunodetection Kit (Vector Lab). Incubation with anti-Nestin (Abcam), anti-GATA-4 (Santa Cruz Biotech.), anti-Sarcomeric α-actinin (α-actinin; Sigma-Aldrich), anti-α smooth muscle actin (α-SMA; Sigma-Aldrich), or anti-von Willebrand Factor (vWF; Sigma-Aldrich) was carried out for 1–2 h at room temperature (RT). Incubation with secondary antibodies was for 1 h at RT, with the exception of M.O.M.™ Biotinylated Anti-Mouse IgG Reagent (Vector Lab) by which the manufacturer's instructions were followed. Slides were mounted in Vectashield with 4,6-diamino-2-phenylindole (DAPI; Vector Lab) and observed on the inverted fluorescence microscope Axiovert 200 (Zeiss).

iCPC^Sca-1 differentiation

For cardiomyogenic differentiation, cells were (i) co-cultured with neonatal CM for 7 days, (ii) cultured in the presence of transforming growth factor-β (TGF-β) for 21 days [32], or (iii) maintained using a commercial cardiomyocyte differentiation medium (Millipore) for 21 days.

For the co-culture set-up, CM were isolated from neonatal (postnatal day 1–3) C57BL/6 as previously described [33]. iCPC^Sca-1 were pre-stained with viable red fluorescent dye Vybrant^™ DiI (Molecular Probes) before seeding onto CMs for 7 days.

For endothelial differentiation, iCPC^Sca-1 were pre-conditioned for 10 days in Endothelial Growth Medium with EGM BulletKit (EGM; Lonza) or in α-MEM (Gibco) with 10% FBS as control. After this period, cells were plated for immunocytochemistry (ICC), processed for CD31 flow-cytometry staining, or seeded onto Matrigel Growth Factor-Reduced Matrix (BD Biosciences). After overnight culture, cells were fixed and stained for vWF (PBS/0.05% Tween-20/1% BSA for 4 h at RT) or for calcein AM (BD Biosciences). Briefly, cells were incubated for 30 min at 37°C in calcein/PBS and imaged using the inverted fluorescence microscope Axiovert 200 (Zeiss). To evaluate tube formation, number of branch points and tube length were assessed using ImageJ1.42.

For smooth muscle cell differentiation, iCPC^Sca-1 were cultured in DMEM high glucose containing 2% FBS and 50 ng/mL of platelet-derived growth factor BB (PDGF-BB; R&D Systems) for 10 days, as described earlier [7].

MI, iCPC^Sca-1 delivery, and echocardiography

MI was induced by permanent ligation of left anterior descending (LAD) coronary artery as described elsewhere [34] with minor alterations. After anesthesia, female C57BL/6 were intubated and ventilated using a small-animal respirator (Minivent 845; Harvard Apparatus). The heart was exposed (Ø 5–7 mm) via left thoracotomy on the third intercostal space, and the pericardial sac was gently disrupted. A non-absorbable 7-0 suture (Silkam^®; B. Braun) was used for ligation. Immediately afterward, 5×10⁵ iCPC^Sca-1 (n=8) or vehicle (0.5% BSA/PBS) only (n=9) were delivered by four intramyocardial injections (5 μL each) with a 30-gauge syringe (Hamilton company). Thoracic incision was closed using an absorbable 6-0 suture (Safil^®; B. Braun), and surgical staples were used for skin closure. Anesthesia was reverted by atipamezole (ip, 5 mg/kg; Revertor). Analgesia and fluid therapy were performed by ip delivery of butorphanol (1 mg/kg; Butador) and a subcutaneous injection of 5% glucose physiological saline, respectively. This procedure was repeated every 12 h for approximately 72 h post-surgery or until full recovery.

Echocardiographic assessment

Transthoracic echocardiography was performed at 2 weeks after LAD coronary artery ligation using a portable ultrasound apparatus (GE Vivid I; General Electric) that was equipped with a 12 MHz linear probe (GE 12L-RS Linear Array Transducer; General Electric). To evaluate LV structural changes, several parameters from M-mode were measured, that is, the LV internal diameter at diastole (LVIDd) and at systole (LVIDs), the interventricular septum at diastole and at systole, the LV posterior wall at diastole and at systole (LVPWs), and heart rate. Left ventricular ejection fraction (EF) and fraction shortening (FS) were calculated as an index of systolic function: FS (%)=[(LVIDd−LVIDs)/LVIDd] ×100 and EF (%)=[(LVIDd3–LVIDs3)/LVIDd3] ×100.

Histological procedures

Hearts were harvested at 2 weeks post-surgery after an injection with potassium chloride and fixed in 10% Formalin neutral buffer (VWR BDH and Prolabo) for approximately 24 h before paraffin embedding. Representative LV sampling (∼12 sections at 3 μm) was obtained by transverse sectioning from apex to base with 300 μm intervals.

Masson's Trichrome (MT) staining was performed using the Trichrome (Masson) Stain kit (Sigma-Aldrich) with the following modifications: Nuclei were pre-stained with Celestine Blue solution following Gill's Hematoxylin staining, and tissue was incubated for 1 h in Bouin's solution before muscle staining with Biebrich Scarlet-Acid Fuchsin. Sections were photographed with a stereomicroscope (Leica) before morphometric analysis.

MI size calculation and morphometric analysis

Infarct size measurement was based on collagen deposition in the ischemic LV wall 2 weeks post-infarction and calculated by the area method using the semi-automated program MIQuant [35]. The thickness of LV-free wall was measured on the infarction region using ImageJ1.42 on MT-stained sections.

Fluorescence in situ hybridization

Sections were subjected to antigen retrieval using 10 mM sodium citrate Buffer (pH 6; Sigma-Aldrich) at 98°C for 10 min followed by 0.01% acid pepsin (Sigma-Aldrich) at 37°C for 10 min. For fluorescence in situ hybridization (FISH), samples were dehydrated and incubated with a specific probe to whole mouse Y-chromosome (Y-Chr; Cambio) for 5 min at 82°C and overnight at 37°C. Samples were incubated with Streptavidin (Invitrogen) for 30 min, mounted in Vectashield with DAPI (Vector Lab), and observed on the inverted fluorescence microscope Axiovert 200 (Zeiss).

For α-actinin and CD31 (Santa Cruz Biotech.) immunostaining combined with FISH (immuno-FISH), antigen retrieval was achieved by pronase treatment for 30 min at 37°C or with 10 mM Tris/1 mM EDTA (pH 9.0) at 98°C for 30 min. Samples were permeabilized with 0.2% Triton X-100 and blocked using 4% FBS and 1% BSA or M.O.M Immunodetection Kit (Vector Lab). Incubation with anti-Connexin43 (Cx43; Abcam), anti-α-SMA(Sigma-Aldrich) and anti-CD31 was performed at RT for 1–2 h and overnight at 4°C for anti-α-actinin (Sigma-Aldrich). After a 1 h incubation with the secondary antibody (Invitrogen), slides were rinsed in 50 mM MgCl₂/PBS for 5 min and post-fixed with 4% PFA and 50 mM MgCl₂/PBS for 10 minutes. Samples were dehydrated and incubated with Y-Chr probe as described earlier.

Blood vessel density quantification

After antigen retrieval using 10 mM Tris/1 mM EDTA (pH 9.0; Sigma-Aldrich) at 98°C for 30 min, samples were permeabilized with 0.2% Triton X-100 and blocked for 1 h with 4% FBS and 1% BSA. Slides were incubated with anti-CD31 for 1–2 h at RT, 1 h incubated with secondary antibody and mounted in Vectashield with DAPI. Images were captured using MosaiX (AxioVision modules; Carl Zeiss). CD31⁺ cells were counted in 30 fields per heart over 3–5 sections at infarcted and border zone regions along the heart's long axis (n=9 control group, n=8 iCPC^Sca-1) using ImageJ1.42. Density was calculated as CD31-positive cells per square millimeter (mm²).

Data and statistical analysis

Statistical significance was evaluated with SPSS v.19.0 using Mann–Whitney or one-way analysis of variance with post hoc Tukey's test. Values are presented as mean±standard error of the mean. P<0.05 was considered statistically significant. *P<0.05, **P<0.01, and ***P<0.001.

Results

Generation of an immortalized line representative of Lin⁻Sca-1⁺ adult CPCs

To immortalize Lin⁻Sca-1⁺ CPCs by inducing ectopic expression of mTERT, cells expressing Sca-1 were isolated from murine hearts and transfected with pCINeo vector encoding the mTERT.

Further analysis was restricted to one transfected clone of myocardium-resident Sca-1⁺ progenitors, that is, the iCPC^Sca-1. The remaining G418-resistent isolated clones were subjected to transcriptional profiling (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/scd) and cryopreserved for future investigation.

iCPC^Sca-1 grew to confluence as a monolayer of spindle-shaped adherent cells similar to their native counterparts (Fig. 1A) and presented a doubling time of ∼21.6 h. iCPC^Sca-1 were continuously sub-cultured for more than 24 months with no signs of differentiation and maintained high levels of Sca-1 expression. Conversely, primary (non-transfected) Lin⁻Sca-1⁺ CPC decreased Sca-1 expression after 1 month in culture and senesced after 15–20 passages (Supplementary Fig. S1).

FIG. 1.

Generation and characterization of immortalized Lin⁻Sca-1⁺ cardiac progenitors (iCPC^Sca-1) cell line. (A) Representative image of primary (CPC^Sca-1) and immortalized (iCPC^Sca-1) Sca-1⁺ CPC. (B) Successful integration of pCINeo-murine telomerase catalytic subunit into iCPC^Sca-1 genome was confirmed by Southern blotting for Neomycin. The empty plasmid was used as a positive control. (C) Chromosome counts on (▪) iCPC^Sca-1 metaphase spreads show aneuploidy (n=42) when compared with (▪) Sca-1⁺ counterparts (n=23). (D) Flow cytometry analysis of iCPC^Sca-1 displays high expression of Sca-1 and mesenchymal-associated antigens (PDGFRα, CD29, CD44, CD105, and CD106). iCPC^Sca-1 lack hematopoietic- (CD45, c-kit) and endothelial-affiliated (Flk-1, CD31, and CD34) markers. Bars are represented as mean±standard error of the mean (SEM). (E). Reverse transcription polymerase chain reaction (RT-PCR) profile of primary CPC^Sca-1 and iCPC^Sca-1 reveals the expression of stemness-related genes (Bcrp1, Bmi-1, and Nestin) and early cardiac transcriptional regulators (Gata-4, Isl-1, Mef2c, and Tef-1). Mature contractile myofilaments [α- and β-cardiac myosin heavy chain beta (Mhc)] and pluripotency (Sox2, Nanog, and Oct4) transcripts were not detected. Embryonic stem cells (ESC); bone marrow (BM), and heart (Hrt) were used as controls. (F) Flow cytometry displays the profile of Sca-1 expression in primary CPC^Sca-1 and iCPC^Sca-1, and immunocytochemistry (ICC) corroborates the expected sub-cellular location of progenitor-associated proteins GATA-4 and Nestin. Scale bar: 10 μm. Color images available online at www.liebertpub.com/scd

Genome integration of mTERT was confirmed by Southern blotting using a specific probe for the neomycin phosphotransferase gene (Fig. 1B). Chromosome number per metaphase plate of iCPC^Sca-1 was compared with Sca-1⁺ primary cells; the majority of the cells were diploid and displayed a median chromosome number of 44 and 40 in iCPC^Sca-1 and primary culture, respectively (Fig. 1C). In addition, tetraploid cells were present in both conditions. The tumorigenic potential of iCPC^Sca-1 was evaluated by means of a classical tumorigenesis assay in which syngeneic animals are used as recipients for heterotopic delivery of the cell inocula [30]. Hence, iCPC^Sca-1 were subcutaneously delivered to the shoulder pads of syngeneic animals that were daily monitored for tumor progression. No local mass formation was observed after a 5-month inspection period and normal histological appearance was verified for the heart, spleen, lungs, and liver (Supplementary Fig. S2).

iCPC^Sca-1 display a typical tri-lineage potential and a stable phenotype after long-term in vitro culture

Prospective identification of bona fide CPCs based on the unique expression of surface markers is at the present not possible. Hence, it is generally accepted that an accredited CPC will meet the following criteria: (i) derive from the adult heart; (ii) display either Sca-1 or c-Kit at their surface and lack markers of hematopoietic and endothelial commitment/differentiation; (iii) express cardiac-affiliated transcription factors while lacking mature proteins; and (iv) differentiate both in vitro and in vivo into CM, endothelial cells, and smooth muscle cells. iCPC^Sca-1 were, thus, characterized with regard to the cell surface phenotype, transcriptional profile, and functional properties in order to verify whether this cell line meets the premises mentioned earlier.

Flow cytometry analysis indicated a phenotype consistent with adult Lin⁻Sca-1⁺ CPCs, that is, a population constituted of cells with high Sca-1 levels (99.8%±0.21%), lacking hematopoietic (Stro-1, CD45, CD34) and endothelial (Flk-1, CD31 and CD34) markers, and expressing mesenchymal-associated proteins (eg, CD29, CD44, CD105, CD106, and PDGFRα), several of which are critical to cell migration, adhesion, and communication (Fig. 1D). Furthermore, the characterized clone (Clone No. 3, Supplementary Table S1) did not express c-Kit, a protein that singly or together with Sca-1 has been used to isolate CPCs. A detailed transcriptional profile was carried out in parallel with primary Sca-1⁺ CPC (CPC^Sca-1), embryonic stem cells, bone marrow, and heart as controls. The side-by-side transcriptional analysis demonstrated that iCPC^Sca-1 and primary Sca-1⁺ cells consistently expressed stemness-related genes (Bcrp1, Bmi-1, and Nestin) and early cardiac transcription factors (Gata-4, Isl-1, Mef2c, and Tef-1) while lacking transcripts for mature contractile myofilaments (α- and β-Mhc), fibroblasts [transcription factor 21 (Tcf21)], and regulators of pluripotency (Sox2, Nanog, Oct4), thus corroborating that iCPC^Sca-1 preserves the phenotype of cardiac-affiliated Sca-1⁺ multipotent progenitors (Fig. 1E). It should be noted that both primary and immortalized CPC^Sca-1 expressed c-Myc, a transcription factor known to block differentiation in distinct model systems [36] and particularly shown to synergize with Pim-1 to induce the proliferation and survival of CPCs [37]. Overall, the data demonstrated that the immortalization of the cells did not alter the transcriptional profile of Sca-1⁺ CPCs. The only obvious difference between the established line and the native counterparts is the lack of c-Kit in the immortalized cells, as a result of clone selection. Flow cytometry analysis clearly exposed the enrichment for the Sca-1-expressing fraction in CPC^Sca-1 (91.8%) and iCPC^Sca-1 (99.8%). Moreover, likely as a result of clonal selection, the iCPC^Sca-1 displayed a stronger and less disperse pattern of Sca-1 expression as compared with primary cells. Proper sub-cellular allocation of stemness-associated proteins GATA-4 and Nestin was further confirmed by ICC for both primary and immortalized cells (Fig. 1F).

To test iCPC^Sca-1 multipotency, distinct sub-passages were cultured under cell differentiation conditions. Cardiomyocytic differentiation potential was evaluated by co-culture with mouse neonatal CM. iCPC^Sca-1 were pre-stained with viable red fluorescent dye Vybrant DiI to enable detection in the co-culture system. After a week, iCPC^Sca-1 up-regulated GATA-4, displayed a functional pattern of Cx43 and a typical myofibrillar assembly of sarcomeric α-actinin (α-actinin) (Fig. 2A). Although the gold-standard protocol to trigger CPCs differentiation is the direct co-culture with neonatal CM [33], this assay is prone to misleading interpretations due to spontaneous cell fusion events [38,39]. Hence, we further addressed iCPC^Sca-1 differentiation in CM by culturing the cell line for 21 days in cardiomyocytic differentiation medium (Millipore) or in the presence of TGF-β. In this condition, iCPC^Sca-1 were also able to up-regulate α-actinin expression when compared with cells subjected to basal, that is, cell-maintenance, conditions for the same time period (Fig. 2B). Furthermore, when iCPC^Sca-1 were transduced with a green fluorescent protein (GFP) reporter under the cardiac troponin T (cTnT) promoter and subjected to TGF-β [32] for 45 days, approximately 97% of cells displayed GFP expression (Supplementary Fig. S3). Since cTnT and α-actinin are also expressed in the developing skeletal muscle, we evaluated the possibility of iCPC^Sca-1 having a skeletal origin. The latter was clearly excluded by demonstrating the up-regulation of cardiac myosin heavy chain beta expression after differentiation, while no detectable expression of myogenic differentiation 1(MyoD), a marker of skeletal commitment, was found (Fig. 2B). Endothelial differentiation potential was assessed by priming iCPC^Sca-1 with EGM medium for 10 days followed by a classical Matrigel assay to evaluate the capacity of the cells to form tubular-like structures. Primed iCPC^Sca-1 seeded onto Matrigel exhibited tube-like interconnected structures, similar to the capillary assembly of human umbilical vein endothelial cells (HUVECs) (Fig. 2C, n=6), whereas unprimed iCPC^Sca-1 (iCPC^Sca-1 pre-cultured in α-MEM) were unable to assemble a fully organized capillary network (n=6). In fact, the tubular complex formed by unprimed cells presented a significantly smaller number of branch points (P<0.001) and tube length (P<0.05 and P<0.01) as compared with that assembled by EGM-primed iCPC^Sca-1 and HUVECs (Fig. 2C). An endothelial phenotype was further addressed by analyzing vWF and CD31 protein expression. vWF was only detected in iCPC^Sca-1 cells after EGM priming (Fig. 2C). Accordingly, CD31 was increased by 1.75% on primed cells when compared with iCPC^Sca-1 maintained in α-MEM for the same period (Fig. 2C).

FIG. 2.

iCPC^Sca-1 in vitro differentiation potential. (A) Immunostaining demonstrating an increase in GATA-4 expression and presence of Cx43 and α-actinin in Vybrant^™-stained iCPC^Sca-1 after 1 week of co-culture with neonatal cardiomyocytes. Scale bar: 20 μm. (B) iCPC^Sca-1 α-actinin expression is increased after 21 days of culture in cardiomyocytic differentiation conditions: transforming growth factor β or commercial medium (Millipore). Scale bar: 20 μm. RT-PCR representing the up-regulation of β-MHC in iCPC^Sca-1 cultured in Millipore medium, while no MyoD transcript was observed in basal conditions or after differentiation. (C) Calcein staining evidencing the tubular structures formed by human umbilical vein endothelial cells (HUVECs) and iCPC^Sca-1 [pre-conditioned in either α-minimum essential medium (α-MEM) or endothelial growth medium (EGM)] on Matrigel. Scale bar: 200 μm. The tube length and the number of branch points formed in each culture condition were assessed. Data are represented as mean±SEM [n=6, ***P<0.001; **P<0.01; *P<0.05 and not significant (ns) by one-way analysis of variance (ANOVA) with post hoc Tukey's test]. EGM-preconditioned iCPC^Sca-1 grown on Matrigel exhibit increased von Willebrand Factor (vWF) and CD31 expression as assessed by ICC and flow cytometry, respectively. Scale bars: 20 μm and 5 μm. Data are represented as mean±standard deviation (SD). (D) α-Smooth muscle actin (α-SMA) expression in control iCPC^Sca-1 and after 10 days of culture in differentiation medium containing PDGF-BB. Scale bar: 20 μm. Histogram plots show control in white and specific staining in black. Data are represented as mean±SD. qRT-PCR revealed an up-regulation in α-SMA, SM22, and Cnn1 mRNA levels; while no dramatic alteration in Desmin expression levels was found. No detectable levels of expression were observed for Myocd and Tcf21 (n.d.). Data are represented as a fold increase relative to basal condition (n=3). Color images available online at www.liebertpub.com/scd

The capacity to differentiate into smooth muscle cells was evaluated by subjecting iCPC^Sca-1 to PDGF-BB for 10 days. After this period, iCPC^Sca-1 became elongated and displayed α-SMA protein expression, as demonstrated by ICC and flow cytometry (27%) (Fig. 2D). In addition, the expression levels of the smooth muscle-affiliated genes, α-SMA, Myocardin (Myocd), Transgelin (SM22), Calponin 1 (Cnn1), and Desmin were also assessed by qRT-PCR (Fig. 2D). After 10 days of PDGF-BB treatment, iCPC^Sca-1 up-regulated α-SMA, SM22, and Cnn1; while no clear alteration was observed for Desmin mRNA levels. In addition, no detectable expression of Myocd was found, even on PDGF-BB stimulation. Since some of the proteins mentioned earlier are also displayed by fibroblasts/myofibroblasts, expression of the fibroblast marker Tcf21 was evaluated in order to discard a fibroblastic origin of the iCPC^Sca-1. Detectable expression of Tcf21 was not found, neither in basal conditions nor after PDGF-BB treatment (Figs. 1E and 2D). Overall, these results demonstrate that iCPC^Sca-1 were not derived from cardiac fibroblasts and that, after PDGF-BB stimulation, cells undergo an incomplete smooth-muscle differentiation program as the levels of Myocd and Desmin have not changed after PDGF-BB treatment.

Taken together, the data demonstrate that iCPC^Sca-1 preserved the in vitro phenotype and functional ability to moderately differentiate as described by our team [29,33] and others [5,12,13,16] for the native Lin⁻Sca-1⁺ counterparts.

iCPC^Sca-1-transplanted hearts show improved systolic function and lessened LV remodeling after MI

The beneficial effect of Lin⁻Sca-1⁺ CPCs administration into the MI heart has been clearly established. Notably, CPCs are able to improve cardiac function and attenuate LV remodeling after MI. However, the in vivo differentiation of the CPCs in CM and endothelial cells is minimal, and the amelioration observed on MI is primarily attributed to paracrine effects [5]. Aiming at demonstrating that iCPC^Sca-1 display the hallmarks of CPCs in the myocardium in the onset of injury, a proof-of-principle experiment was performed using a commonly reported experimental injury setting [16,40]. Female C57BL/6 mice subjected to MI via LAD coronary artery ligation were immediately injected on the peri-infarct region with either vehicle medium (n=9) or male-derived iCPC^Sca-1 (n=8). MI size measurement (based on collagen deposition) at 14 days post-infarction showed that MI extent was comparable between both experimental groups with 52% of the LV wall being affected (Fig. 3A). Notwithstanding, echocardiography at 14 days, post-surgery in animals injected with iCPC^Sca-1 showed improved LV function when compared with vehicle controls, as demonstrated by significantly increased (P<0.05) EF and FS in iCPC^Sca-1-transplanted hearts (Fig. 3B).

FIG. 3.

Functional and histological assessment of ischemic hearts transplanted with either iCPC^Sca-1 or vehicle. (A) Both iCPC^Sca-1 and vehicle-injected groups display similar myocardial infarction (MI) size. (B) Ejection fraction and fractional shortening measured in non-manipulated animals, vehicle-control and iCPC^Sca-1-injected hearts at 2 weeks post-intervention show a significant improvement in left ventricle (LV) contractile capacity in iCPC^Sca-1-transplanted hearts. (C) Increased LV wall thickness and decreased LV dilation in iCPC^Sca-1-injected animals, demonstrated by Masson's Trichrome staining of LV sections. Broken and continuous lines highlight the thickness of the infarcted LV wall in vehicle and iCPC^Sca-1 transplanted animals, respectively. (D) Measurements of LV wall thickness in non-manipulated hearts and in the ischemic region corroborate a significant difference between both experimental groups. Scale bar: 100 μm. Data are represented as mean±SEM (***P<0.001, **P<0.01, and *P<0.05 by Mann–Whitney test). Color images available online at www.liebertpub.com/scd

Altered cardiac loading after MI due to extensive loss of myocardial cells induces changes in the heart shape, size, and function that are commonly designated as cardiac remodeling. A stereoscopic view of representative cross-sections of infarcted hearts showed that iCPC^Sca-1 transplantation prevented the major LV wall thinning and LV chamber expansion observed in the vehicle control group (Fig. 3C). This was corroborated by a morphometric analysis of MT stains, evidencing an increase (P<0.01) in LV wall thickness of iCPC^Sca-1-transplanted hearts (Fig. 3D) to a value not statistically different from the non-manipulated animals (Fig. 3D). However and despite this increase, the LV contractile function of iCPC^Sca-1-injected animals was not comparable with that of non-manipulated animals (P<0.01, Fig. 3B).

Overall, the data suggest that iCPC^Sca-1 exerted a cardio-protective effect, translated on a reduction of the deleterious consequences of ischemia and a partial recovery of cardiac functional parameters at 2 weeks post-MI.

Transplanted iCPC^Sca-1 improve neovascularization of the infarcted myocardium, engraft, and differentiate into CM-, endothelial-, and smooth muscle-like cells

To address whether cardiac performance amelioration after iCPC^Sca-1 administration occurred at least partly via stimulation of the de novo vessel formation by paracrine mechanisms, the density of small blood vessels in the infarcted myocardium and in the border zone was quantified at 2 weeks post-surgery (Fig. 4A). Overall, iCPC^Sca-1 transplantation resulted in a significant increase in CD31⁺ cells per mm² when compared with the vehicle control (709±19 vs. 555±24, respectively, P<0.001, Fig. 4B). iCPC^Sca-1, identified by Y-Chr staining were consistently observed in close proximity to vessels, suggesting contribution to either endothelial precursors mobilization/proliferation and/or differentiation into endothelial cells. The latter possibility was evaluated by FISH combined with immunostaining for CD31, and, indeed, double positive events were observed in all analyzed animals although at very low frequencies (4.1±0.7 cells per transverse heart section) (Figs. 4C and 5I). In fact, the majority of small vessels were not formed by donor cells, hinting that iCPC^Sca-1 contribution to neovascularization is more likely to occur by stimulating the mobilization and/or proliferation of host endothelial precursors/cells (paracrine action).

FIG. 4.

Contribution of iCPC^Sca-1 to neovascularization post-MI. (A) Immunolocalization of CD31 in the ischemic region of control and iCPC^Sca-1-transplanted animals. Scale bar: 100 μm. (B) Quantification of CD31⁺ cells demonstrates a significant increase in small vessels number in iCPC^Sca-1-injected animals. (C) Immunostaining combined with fluorescence in situ hybridization (immuno-FISH) demonstrating iCPC^Sca-1 differentiation into CD31⁺ cells (arrowheads). Scale bar: 10 μm. Data are represented as mean±SEM (***P<0.001 by Mann–Whitney test). Color images available online at www.liebertpub.com/scd

FIG. 5.

In vivo characterization of iCPC^Sca-1 on transplantation into MI hearts. (A, B) Y-Chromosome detection (FISH) in vehicle (A) and male-derived iCPC^Sca-1-injected hearts (B) demonstrates the immortalized cell-line successful engraftment. (C–H) Functional communication between donor–donor [*, (C)] and donor–host [#, (D)] was revealed by the assembly of Cx43 at the cellular membrane. iCPC^Sca-1 expressing α-SMA (arrowheads) were found integrating small vessels [*, (E)] and scattered throughout the myocardium displaying a fibroblast-like morphology (F). α-Actinin⁺ iCPC^Sca-1 (arrowheads) in the infarcted tissue displayed an immature phenotype (G) and, less-frequently, well-defined cross-striations (H). Scale bar: 10 μm. (I) Graph displaying the number of cells, per transverse histological section, bearing the Y-chr and expressing α-SMA, α-actinin, or CD31. Data are represented as mean±SEM (***P<0.001 by one-way ANOVA with post hoc Tukey's test). Color images available online at www.liebertpub.com/scd

We next investigated iCPC^Sca-1 functional engraftment and contribution to the de novo cardiac cells formation. In vehicle-injected female mice, no Y-Chr⁺ cells were detected in the heart (Fig. 5A); whereas in transplanted animals, numerous iCPC^Sca-1 were observed throughout ischemic myocardium and MI border zone at 2 weeks post-infarction. This demonstrates that cells were able to survive and engraft the LV ischemic region (Fig. 5B). Moreover, iCPC^Sca-1 or their progeny were not found in healthy remote areas of the injury site. Ki67 staining was frequently associated with Y-Chr evidencing iCPC^Sca-1 proliferation within the host myocardium (data not shown). Assembly of the gap junction protein Cx43 at the cellular membrane confirmed that donor cells established functional communication to both donor and host cells (Fig. 5C, D), substantiating successful iCPC^Sca-1 engraftment within the host tissue.

Although the majority of grafted cells retained an undifferentiated morphology and Sca-1 expression (Supplementary Fig. S4), discrete differentiation events were observed. Co-localization of Y-Chr with α-SMA was observed in all analyzed animals (41.2±5.6 cells per transverse heart section) (Fig. 5I), either integrating small vessels (Fig. 5E) or displaying a fibroblast-like morphology (Fig. 5F). In fact, in addition to smooth muscle cells, α-SMA is also expressed by myofibroblasts, cells capable of establishing cell connections through gap junctions, and relevant players in formation of the contractile granulation tissue [41]. However, considering that Tcf21 is a marker of fibroblasts [42] and is also involved in the activation of myofibroblasts [43], and that iCPC^Sca-1 failed to express this marker even when specifically stimulated (Figs. 1E and 2D), there is little support for considering a myofibroblast fate. Furthermore, iCPC^Sca-1 contributed to the cardiomyocytic compartment as evidenced by α-actinin expression. The majority of α-actinin⁺ iCPC^Sca-1 presented an immature phenotype in three out of six analyzed animals (9.0±0.6 cells per transverse heart section) (Fig. 5I), suggesting an incomplete differentiation. Moreover, α-actinin⁺ iCPC^Sca-1 were found in close proximity with α-actinin⁺ host cells displaying a similar immature phenotype, suggesting an eventual iCPC^Sca-1—mediated recruitment/activation of endogenous precursors (Fig. 5G). Donor cells presenting well-defined cross-striations, characteristic of a more mature phenotype, were found in one animal (Fig. 5H; Supplementary Fig. S5). While we do not rule out the possibility that these scarce events resulted from cell fusion, differentiation into more mature CM seems a more consistent scenario (Supplementary Fig. S5).

Overall, these results are in agreement with the previous observations, that Lin⁻Sca-1⁺-mediated beneficial effects after MI are partially mediated by paracrine mechanisms [5,44] in detriment to a major contribution to cardiac cell types after transplantation.

Discussion

Different progenitor cell subsets have been so far suggested as candidates for the optimal cell population to be implanted in infarcted and/or failing hearts (Fig. 6), but a relationship among these populations remains undetermined. The absence of a specific molecular signature as well as the multiplicity of the isolation and characterization protocols do not enable an understanding of whether the described CPC populations constitute different subsets of a common progenitor or independent cell lineages of distinct ontogeny. Indeed, it has been suggested that the various identified CPCs partake in the same cell lineage, and the different phenotypes are a consequence of the particular culturing protocols used in different laboratories [21]. Importantly, other aspects account for an increased difficulty in the identification of CPCs and to understand their real efficiency. Namely, primary cells can easily change along time in culture as a consequence of genotypic and phenotypic drift and/or senescence [45 –47] and, thus, lead to a lack of reproducibility across studies.

FIG. 6.

Illustrative scheme of described multipotent progenitor cell populations isolated from the adult heart. Several isolation methods have been applied to separate CPCs based on the expression of specific cell-surface markers (eg, c-Kit⁺ and Sca-1⁺), in vitro functional assays (eg, cardiospheres, side population), or the expression of a specific genetic marker (Wt-1 epicardial progenitors). Regardless of the increasing number of putative CPCs reported, the functional, hierarchical, and anatomical relationship of the illustrated cell progenitor subsets is still elusive. Color images available online at www.liebertpub.com/scd

Taken together, the experience accumulated in the last decade indicates that only two strategies are used to implement a reliable cell therapy: to try and fail, that is, to implant whatever progenitor cell phenotype in the patient trusting to identify the perfect treatment, or to systematically dig the complexity of the stem/progenitor cell behavior and, based on novel data and concepts, to formulate a logically stringent procedure to transfer experimental observations to the clinical setting.

Novel tools should be developed to help understanding and, thus, governing the CPC complexity and phenotype variability and to enable the standardization of the experimental sets and the inter-laboratory comparison. Consistently, the present study has been carried out to create an immortalized CPC line retaining a constant phenotype corresponding to native features. CPC immortalization has been achieved by inducing TERT overexpression, in order to circumvent the limitations determined by the diminished mitotic capacity of somatic cells [48 –52]. The use of immortalized lines that retain the in vivo features of primary cells, by combining the advantages of a high proliferative cell source with a stable phenotype in long-term culture, is a major breakthrough in cell biology. Indeed, we have previously shown that bone marrow-derived mesenchymal stromal/stem cell (MSC) immortalized lines preserve multipotency in standard and three-dimensional conditions in vitro [49,53]. Although telomerase overexpression does not induce oncogenesis, it may provide cells with a greater opportunity to accumulate mutations, predisposing to later malignant transformation [54,55]. The iCPC^Sca-1 karyotype analysis showed aneuploidy, characteristic of malignant cells. However, an in vivo tumorigenicity assay [30,31], monitored for 5 months, did not reveal tumor formation either locally (subcutaneously) or in any of the analyzed organs (heart, spleen, lungs, and liver).

Consistently with adult Lin⁻Sca-1⁺ CPC, the iCPC^Sca-1 phenotype was characterized by the expression of high levels of Sca-1, stemness-related genes (Bcrp1, Bmi-1 and Nestin), mesenchymal-associated proteins (eg, CD29, CD44, CD105, CD106, and PDGFRα), and early cardiac transcription factors (Gata-4, Isl-1, Mef2c, and Tef-1). Conversely, immortalized cells did not express c-Kit while also lacking hematopoietic, endothelial, fibroblasts, mature contractile myofilaments (α-and β-Mhc), and pluripotency (Sox2, Nanog, and Oct4) markers.

iCPC^Sca-1 displayed the capability to generate in vitro cardiomyocyte-, smooth muscle-, and endothelial-like cells, with the differentiation toward smooth muscle-like cells being the most efficient. This differential in vitro potential of Sca-1⁺ cells has been previously demonstrated by others [16]. Whether this indicates a higher commitment of CPC^Sca-1 to smooth muscle-like cells or to the fact that the in vitro differentiation conditions are less favorable to activate endothelial and cardiomyocytic pathways, and, thus, not suitable to demonstrate the full potential of these cells, still needs to be determined.

iCPC^Sca-1 is a phenotypically stable cell line that provides investigators with the same unique cell model to dissect CPC behavior and differentiation mechanisms. Furthermore, iCPC^Sca-1 could represent a benchmark to compare with other cells posited to bear stemness characteristics. In fact, the immortalized CPCs retain the features of their ex vivo counterparts, that is, moderate differentiation into cardiomyocyte-, endothelial-, and smooth muscle-like cells, when subjected to appropriate stimuli in vitro as well as in vivo, in a myocardial injury setting. Furthermore, the cell line combines the advantages of a high proliferative cell source with a stable phenotype in long-term culture. Indeed, murine cells are prone to undergo spontaneous immortalization after prolonged in vitro culture [46,56,57], which may result in the accumulation of unpredictable cytogenetic modifications and malignant transformations [47]. The use of uncharacterized spontaneously immortalized cell clones as in vitro models for their native cell counterparts should be circumvented due to the dissimilar phenotypes generated by unpredictable cytogenetic alterations in overextended sub-culture in vitro.

The iCPC^Sca-1 capability to mimic their native non-immortalized corresponding cells has also been apparent in in vivo experiments. In fact, iCPC^Sca-1 were able to contribute toward repairing the myocardial injuries experimentally provoked by coronary ligation in vivo, as shown by minor LV chamber expansion and increased thickness of the LV-free wall. Importantly, echocardiography follow-up revealed improved systolic function after iCPC^Sca-1 transplantation into MI animals. iCPC^Sca-1 abundance in the host infarcted and peri-infarcted myocardium was particularly surprising, and cells were able not only to survive but also to engraft. Moreover, engrafted cells established donor–donor and donor–host Cx43-mediated connections. Indeed, Cx43 promotes MSC survival in the ischemic heart [58], and it is critical for protection against ventricular tachycardia, which is a resultant from the transplantation of embryonic CM in MI [59]. Accordingly, it can be conjectured that Cx43 is involved in iCPC^Sca-1 resistance to the adverse scenario of myocardial ischemia.

The implantation of immortalized cells into infarcted mouse hearts also induced a significant improvement in the vascularization of the damaged region, very likely, through a paracrine action. Since an improved vascularization is associated with a “pro-regenerative” response, the formation of new vessels in the infarcted tissue was carefully assessed. iCPC^Sca-1 transplanted hearts exhibited a denser capillary network, when compared with the vehicle control group. In addition, iCPC^Sca-1 contributed directly to de novo vessel formation, as demonstrated by CD31 and α-SMA expression on Y-Chr bearing cells. However, endothelial differentiation was modest; hence, direct contribution of engrafted cells to new vessel formation is minimal, suggesting that paracrine mechanisms are involved in the recruitment and/or proliferation of endothelial cells and/or precursors.

CPCs differentiation into CM has been frequently demonstrated by the expression of proteins of the contractile machinery [5,6,12,15]. Indeed, although CPCs appear to grasp a lot of expectations with regard to cardiac cell therapies, most studies still report an immature phenotype for the differentiated cells, to a great extent resembling fetal-neonatal CM [5,6,12,15]. In our in vivo setting, iCPC^Sca-1 differentiated into cardiomyocyte-like cells, though the majority displayed a disorganized structure with no detectable sarcomeres, characteristic of an immature phenotype. It should be noted that both primary CPC^Sca-1 and iCPC^Sca-1 express the transcription factor c-Myc, which has been previously shown to play a role in the blockage of cell differentiation [36] and in the survival and proliferation of CPCs [37]. Whether the levels of c-Myc are regulating CPCs cell-fate decision to differentiate at the expenses of cell proliferation remains to be determined and should be subject to further investigation. Since iCPC^Sca-1 showed an in vitro and in vivo differentiation potential similar to their native counterparts, it is our conviction that iCPC^Sca-1 constitutes a reliable easy-to-use model to dissect the key molecular mechanisms governing cardiomyocytic differentiation from adult CPCs.

In this work, an immortalized cell line for modeling mouse Lin⁻Sca-1⁺ CPCs was specifically generated by means of TERT overexpression. The immortalization did not appear to impair any important cellular property, as iCPC^Sca-1 were phenotypically and functionally similar to their primary cell counterparts. Overall, the results indicate that iCPC^Sca-1 are representative of the native Sca-1⁺ cardiac population, thus constituting a suitable tool for functional and mechanistic studies in need in the field. Hence, with regard to the heterogeneity and low frequency in the adult heart of described CPC populations (Fig. 6), iCPC^Sca-1 comes forth as a uniquely validated off-the-shelf line for in vitro high-throughput and bioengineering studies. The latter combined with the remarkable capacity of iCPC^Sca-1 to engraft and differentiate in loco makes this cell line a valuable model system to investigate in vivo the role of the Sca-1⁺ stem/progenitor cells resident in the adult heart.

Last but not the least, the widespread use of this and other similarly generated cardiac progenitor-subset cell lines will enable the reduction of animal usage and will contribute in a properly standardized manner to the definition of benchmark(s) for stem/progenitor cells from distinct organ systems and from different laboratories.

Footnotes

Acknowledgments

This work was supported by Fundação para a Ciência e a Tecnologia (SFRH/BD/64715/2009 to A.G.F., SFRH/BPD/42254/2007 to D.S.N., SFRH/BD/74218/2010 to M.V., SFRH/BPD/80588/2011 to T.P.R., and Ciência2007 to P.P.O.); Fundo Europeu de Desenvolvimento Regional, Programa Operacional Factores de Competitividade-COMPETE, Quadro de Referência Estratégico Nacional, and Fundo Social Europeu (PEst-C/SAU/LA0002/2011, PTDC/SAU-OSM-68473/2006, PTDC/SAU-ORG/118297/2010 and NORTE-07-0124-FEDER-000005 to D.S.N.); and European Regional Development Fund – Project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123). Travel funds were awarded by Boehringer Ingelheim Fonds to (T.P.R.); by American Portuguese Biomedical Research Fund to (P.P.O and D.S.N.).

The authors are indebted to Joana G. Guedes (CIBIO), Rui Fernandes (IBMC), Paula Sampaio (IBMC), Dina Leitão (IPATIMUP), and the members of MCA Lab (IBMC) for sharing scientific expertise and equipment.

Author Disclosure Statement

No competing financial interests exist.

References

Bui

, Gertz

and Wilensky

. (2010). Intracoronary delivery of bone-marrow-derived stem cells. Stem Cell Res Ther, 1:29

Muller-Ehmsen

, Krausgrill

, Burst

, Schenk

, Neisen

, Fries

, Fleischmann

, Hescheler

and Schwinger

. (2006). Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction. J Mol Cell Cardiol, 41:876–884.

Gnecchi

, He

, Liang

, Melo

, Morello

, Mu

, Noiseux

, Zhang

, Pratt

, Ingwall

and Dzau

. (2005). Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med, 11:367–368.

Kinnaird

, Stabile

, Burnett

, Shou

, Lee

, Barr

, Fuchs

and Epstein

. (2004). Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation, 109:1543–1549.

Wang

, Hu

, Nakamura

, Lee

, Zhang

, From

and Zhang

. (2006). The role of the sca-1+/CD31−cardiac progenitor cell population in postinfarction left ventricular remodeling. Stem Cells, 24:1779–1788.

Beltrami

, Barlucchi

, Torella

, Baker

, Limana

, Chimenti

, Kasahara

, Rota

, Musso

, et al. (2003). Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell, 114:763–776.

Chong

, Chandrakanthan

, Xaymardan

, Asli

, Li

, Ahmed

, Heffernan

, Menon

, Scarlett

, et al. (2011). Adult cardiac-resident msc-like stem cells with a proepicardial origin. Cell Stem Cell, 9:527–540.

Gambini

, Pompilio

, Biondi

, Alamanni

, Capogrossi

, Agrifoglio

and Pesce

. (2011). C-kit+ cardiac progenitors exhibit mesenchymal markers and preferential cardiovascular commitment. Cardiovasc Res, 89:362–373.

Hierlihy

, Seale

, Lobe

, Rudnicki

and Megeney

. (2002). The post-natal heart contains a myocardial stem cell population. FEBS Lett, 530:239–243.

10.

Martin

, Meeson

, Robertson

, Hawke

, Richardson

, Bates

, Goetsch

, Gallardo

and Garry

. (2004). Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart. Dev Biol, 265:262–275.

11.

Messina

, De Angelis

, Frati

, Morrone

, Chimenti

, Fiordaliso

, Salio

, Battaglia

, Latronico

, et al. (2004). Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res, 95:911–921.

12.

, Bradfute

, Gallardo

, Nakamura

, Gaussin

, Mishina

, Pocius

, Michael

, Behringer

, et al. (2003). Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A, 100:12313–12318.

13.

Pfister

, Mouquet

, Jain

, Summer

, Helmes

, Fine

, Colucci

and Liao

. (2005). CD31- but not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res, 97:52–61.

14.

Smart

, Bollini

, Dube

, Vieira

, Zhou

, Davidson

, Yellon

, Riegler

, Price

, et al. (2011). De novo cardiomyocytes from within the activated adult heart after injury. Nature, 474:640–644.

15.

Takamiya

, Haider

and Ashraf

. (2011). Identification and characterization of a novel multipotent sub-population of Sca-1⁺ cardiac progenitor cells for myocardial regeneration. PLoS One, 6:e25265

16.

Tateishi

, Ashihara

, Takehara

, Nomura

, Honsho

, Nakagami

, Morikawa

, Takahashi

, Ueyama

, Matsubara

and Oh

. (2007). Clonally amplified cardiac stem cells are regulated by Sca-1 signaling for efficient cardiovascular regeneration. J Cell Sci, 120:1791–1800.

17.

Yamahara

, Fukushima

, Coppen

, Felkin

, Varela-Carver

, Barton

PJR

, Yacoub

and Suzuki

. (2008). Heterogeneic nature of adult cardiac side population cells. Biochem Biophys Res Commun, 371:615–620.

18.

, Boyle

, Shih

, Sievers

, Zhang

, Prasad

, Su

, Zhou

, Grossman

, Bernstein

and Yeghiazarians

. (2012). Sca-1⁺ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury. PLoS One, 7:e30329

19.

Bolli

, Chugh

, D'Amario

, Loughran

, Stoddard

, Ikram

, Beache

, Wagner

, Leri

, et al. (2011). Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet, 378:1847–1857.

20.

Lim

. (2012). Stem cells: myocardial regeneration after infarction—promising phase I trial results. Nat Rev Cardiol, 9:187

21.

Di Nardo

, Forte

, Ahluwalia

and Minieri

. (2010). Cardiac progenitor cells: potency and control. J Cell Physiol, 224:590–600.

22.

Di Nardo

and Parker

. (2011). Stem cell standardization. Stem Cells Dev, 20:375–377.

23.

Dawn

and Bolli

. (2005). Cardiac progenitor cells. Circ Res, 97:1080–1082.

24.

Gonzalez

, Rota

, Nurzynska

, Misao

, Tillmanns

, Ojaimi

, Padin-Iruegas

, Müller

, Esposito

, et al. (2008). Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan. Circ Res, 102:597–606.

25.

Hooijberg

, Ruizendaal

, Snijders

PJF

, Kueter

EWM

, Walboomers

JMM

and Spits

. (2000). Immortalization of human CD8+ T cell clones by ectopic expression of telomerase reverse transcriptase. J Immunol, 165:4239–4245.

26.

Simonsen

, Rosada

, Serakinci

, Justesen

, Stenderup

, Rattan

SIS

, Jensen

and Kassem

. (2002). Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol, 20:592–596.

27.

Yang

, Chang

, Cherry

, Bangs

, Oei

, Bodnar

, Bronstein

, Chiu

C-P

and Herron

. (1999). Human endothelial cell life extension by telomerase expression. J Biol Chem, 274:26141–26148.

28.

Jiang

X-R

, Jimenez

, Chang

, Frolkis

, Kusler

, Sage

, Beeche

, Bodnar

, Wahl

, Tlsty

and Chiu

C-P

. (1999). Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet, 21:111–114.

29.

Forte

, Carotenuto

, Pagliari

, Cossa

, Fiaccavento

, Ahluwalia

, Vozzi

, Vinci

, et al. (2008). Criticality of the biological and physical stimuli array inducing resident cardiac stem cell determination. Stem Cells, 26:2093–2103.

30.

Schou

and Engel

A-M

. (2006). Assay of tumorigenicity in nude mice. In: Cell Biology: a laboratory handbook. Cells

, ed. Elsevier Science, pp 353–357.

31.

G-J

, Fu

, Wang

S-W

and Wu

M-WH

. (2008). Enforced expression of MCAM/MUC18 Increases in vitro motility and invasiveness and in vivo metastasis of two mouse melanoma K1735 sublines in a syngeneic mouse model. Mol Cancer Res, 6:1666–1677.

32.

Smits

, van Vliet

, Metz

, Korfage

, Sluijter

JPG

, Doevendans

and Goumans

M-J

. (2009). Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nat Protoc, 4:232–243.

33.

Pagliari

, Vilela-Silva

, Forte

, Pagliari

, Mandoli

, Vozzi

, Pietronave

, Prat

, Licoccia

, et al. (2011). Cooperation of biological and mechanical signals in cardiac progenitor cell differentiation. Adv Mater, 23:514–518.

34.

Michael

, Entman

, Hartley

, Youker

, Zhu

, Hall

, Hawkins

, Berens

and Ballantyne

. (1995). Myocardial ischemia and reperfusion: a murine model. Am J Physiol, 269:H2147–H2154.

35.

Nascimento

, Valente

, Esteves

, de Pina Mde

, Guedes

, Freire

, Quelhas

and Pinto-do-Ó

. (2011). MIQuant: semi-automation of infarct size assessment in models of cardiac ischemic injury. PLoS One, 6:e25045

36.

Bhattacharyya

, Kumar

and Lal Khanduja

. (2012). The voyage of stem cell toward terminal differentiation: a brief overview. Acta Biochim Biophys Sin (Shanghai), 44:463–475.

37.

Cottage

, Bailey

, Fischer

, Avitable

, Collins

, Tuck

, Quijada

, Gude

, Alvarez

, Muraski

and Sussman

. (2010). Cardiac progenitor cell cycling stimulated by Pim-1 kinase. Circ Res, 106:891–901.

38.

Garbade

, Schubert

, Rastan

, Lenz

, Walther

, Gummert

, Dhein

and Mohr

F-W

. (2005). Fusion of bone marrow-derived stem cells with cardiomyocytes in a heterologous in vitro model. Eur J Cardio-Thorac Surg, 28:685–691.

39.

Wurmser

and Gage

. (2002). Stem cells: cell fusion causes confusion. Nature, 416:485–487.

40.

Liang

, Tan

TYL

, Gaudry

and Chong

. (2010). Differentiation and migration of Sca1+/CD31− cardiac side population cells in a murine myocardial ischemic model. Int J Cardiol, 138:40–49.

41.

Baum

and Duffy

. (2011). Fibroblasts and myofibroblasts: what are we talking about?. J Cardiovasc Pharmacol, 57:376–379.

42.

Lajiness

and Conway

. (2013). Origin, development, and differentiation of cardiac fibroblasts. J Mol Cell Cardiol, pii:S0022–2828.

43.

Plotkin

and Mudunuri

. (2008). Pod1 induces myofibroblast differentiation in mesenchymal progenitor cells from mouse kidney. J Cell Biochem, 103:675–690.

44.

Matsuura

, Honda

, Nagai

, Fukushima

, Iwanaga

, Tokunaga

, Shimizu

, Okano

, Kasanuki

, Hagiwara

and Komuro

. (2009). Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice. J Clin Invest, 119:2204–2217.

45.

Macieira-Coelho

, Loria

and Berumen

. (1975). Relationship between cell kinetic changes and metabolic events during cell senescence in vitro . Adv Exp Med Biol, 53:51–65.

46.

Sherr

and DePinho

. (2000). Cellular senescence: minireview mitotic clock or culture shock?. Cell, 102:407–410.

47.

Tolar

, Nauta

, Osborn

, Panoskaltsis Mortari

, McElmurry

, Bell

, Xia

, Zhou

, Riddle

, et al. (2007). Sarcoma derived from cultured mesenchymal stem cells. Stem Cells, 25:371–379.

48.

Armstrong

, Saretzki

, Peters

, Wappler

, Evans

, Hole

, von Zglinicki

and Lako

. (2005). Overexpression of telomerase confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic lineage. Stem Cells, 23:516–529.

49.

Forte

, Franzese

, Pagliari

, Di Francesco

, Cossa

, Laudisi

, Fiaccavento

, Minieri

, Bonmassar

and Di Nardo

. (2009). Interfacing Sca-1(pos) mesenchymal stem cells with biocompatible scaffolds with different chemical composition and geometry. J Biomed Biotechnol, 2009:910610

50.

Geserick

, Tejera

, Gonzalez-Suarez

, Klatt

and Blasco

. (2006). Expression of mTert in primary murine cells links the growth-promoting effects of telomerase to transforming growth factor-beta signaling. Oncogene, 25:4310–4319.

51.

Lee

, Hande

and Sabapathy

. (2005). Ectopic mTERT expression in mouse embryonic stem cells does not affect differentiation but confers resistance to differentiation- and stress-induced p53-dependent apoptosis. J Cell Sci, 118:819–829.

52.

Steele

, Wu

, Kolb

, Gooz

, Haycraft

, Keyser

, Guay-Woodford

, Yao

and Bell

. (2010). Telomerase immortalization of principal cells from mouse collecting duct. Am J Physiol Renal Physiol, 299:F1507–F1514.

53.

Miranda

, Teixeira

, Liz

, Sousa

, Franquinho

, Forte

, Di Nardo

, Pinto-Do

and Sousa

. (2011). Systemic delivery of bone marrow-derived mesenchymal stromal cells diminishes neuropathology in a mouse model of Krabbe's disease. Stem Cells, 29:1738–1751.

54.

Harley

. (2002). Telomerase is not an oncogene. Oncogene, 21:494–502.

55.

Prescott

and Blackburn

. (1999). Telomerase: Dr. Jekyll or Mr. Hyde?. Curr Opin Genet Dev, 9:368–373.

56.

Denhardt

, Edwards

, McLeod

, Norton

, Parfett

CLJ

and Zimmer

. (1991). Spontaneous immortalization of mouse embryo cells: strain differences and changes in gene expression with particular reference to retroviral gag-pol genes. Exp Cell Res, 192:128–136.

57.

Fusenig

, Dzarlieva-Petrusevska

and Breitkreutz

. (1985). Phenotypic and cytogenetic characteristics of different stages during spontaneous transformation of mouse keratinocytes in vitro . Carcinog Compr Surv, 9:293–326.

58.

Wang

, Shen

, Zhang

, Chen

and Cao

. (2010). Connexin43 promotes survival of mesenchymal stem cells in ischaemic heart. Cell Biol Int, 34:415–423.

59.

Roell

, Lewalter

, Sasse

, Tallini

, Choi

B-R

, Breitbach

, Doran

, Becher

, Hwang

S-M

, et al. (2007). Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia. Nature, 450:819–824.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

4.18 MB

0.00 MB

0.14 MB

0.28 MB

0.24 MB

0.16 MB

0.11 MB

Stable Phenotype and Function of Immortalized Lin − Sca-1 + Cardiac Progenitor Cells in Long-Term Culture: A Step Closer to Standardization

Abstract

Introduction

Methods

Animals

Isolation and culture of Sca-1+ CPCs

Establishment of iCPCSca-1

In vivo tumorigenicity assay

Flow cytometry

Gene expression

Immunocytochemistry

iCPCSca-1 differentiation

MI, iCPCSca-1 delivery, and echocardiography

Echocardiographic assessment

Histological procedures

MI size calculation and morphometric analysis

Fluorescence in situ hybridization

Blood vessel density quantification

Data and statistical analysis

Results

Generation of an immortalized line representative of Lin−Sca-1+ adult CPCs

iCPCSca-1 display a typical tri-lineage potential and a stable phenotype after long-term in vitro culture

iCPCSca-1-transplanted hearts show improved systolic function and lessened LV remodeling after MI

Transplanted iCPCSca-1 improve neovascularization of the infarcted myocardium, engraft, and differentiate into CM-, endothelial-, and smooth muscle-like cells

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Supplementary Material

Isolation and culture of Sca-1⁺ CPCs

Establishment of iCPC^Sca-1

iCPC^Sca-1 differentiation

MI, iCPC^Sca-1 delivery, and echocardiography

Generation of an immortalized line representative of Lin⁻Sca-1⁺ adult CPCs

iCPC^Sca-1 display a typical tri-lineage potential and a stable phenotype after long-term in vitro culture

iCPC^Sca-1-transplanted hearts show improved systolic function and lessened LV remodeling after MI

Transplanted iCPC^Sca-1 improve neovascularization of the infarcted myocardium, engraft, and differentiate into CM-, endothelial-, and smooth muscle-like cells