Abstract
Stem/progenitor cells from multiple tissues have been isolated based on enhanced activity of cytosolic aldehyde dehydrogenase (ALDH) enzyme. ALDH activity has emerged as a reliable marker for stem/progenitor cells, such that cells from multiple tissues have been shown to possess enhanced stemness properties (self-renewal and multipotency). So far though, not much is known about ALDH activity in specific fetal organs. In this study, we sought to analyze the presence and activity of the ALDH enzyme in the stem cell antigen-1-positive (Sca-1+) cells of fetal human heart. Biochemical assays showed that a subpopulation of Sca-1+ cells (15%) possess significantly high ALDH1 activity. This subpopulation showed increased expression of self-renewal markers compared to the fraction. The fraction also exhibited significant increase in proliferation and pro-survival gene expression. In addition, only the and not the fraction could give rise to all the cell types of the original population, demonstrating multipotency. cells showed increased resistance against aldehyde challenge compared to cells. These results indicate that subpopulation of the cultured human fetal cells has enhanced self-renewal, multipotency, high proliferation, and survival, indicating that this might represent a primitive stem cell population within the fetal human heart.
1. Introduction
Stem cell antigen-1-positive (Sca-1+) cells from adult mouse hearts were shown to demonstrate increased proliferation and stemness along with potential to differentiate into multiple cardiac cell lineages [1–3]. Smits et al. have successfully isolated Sca-1+ cells from adult human heart and further demonstrated their ability to differentiate into cardiomyocytes [4]. These studies unequivocally suggest that Sca-1+ cells isolated from cardiac tissue are a subset of cardiac progenitor cells. Over the years, several approaches and strategies have been developed to enhance regeneration capacity of stem/progenitor cells by improving means of identification, expansion, pluripotency, self-renewal, and survival of these cells [5]. For instance, circulating progenitor cells, umbilical cord blood cells (UCBCs), hematopoietic stem cells (HSCs), and tissue-specific stem/progenitor cells are being identified based on aldehyde dehydrogenase (ALDH) activity [6–12]. Instead of solely relying on presence of cell surface markers, which may sometimes vary upon experimental processing during cell isolation, the functional cytosolic ALDH (ALDH1) activity assay is becoming more reliable and widely used [7, 13]. cells from multiple tissues have been shown to possess enhanced stemness properties, specifically self-renewal and differentiation [7, 11, 13]. stem cells are a small population of cells (0.5–5%) highly enriched for pluripotency [14–16]. In fact stem cells isolated from the blood are in clinical trials for ischemic heart failure [17]. Therefore in this study, we hypothesized that among the Sca-1+ cells from the human fetal heart, cells exhibit high self-renewal capacity, stemness, survival, and proliferation capacity compared to cells.
2. Materials and Methods
2.1. Isolation and Expansion of Fetal Sca-1+ Cells
To isolate fetal human Sca-1+ cells, anti-mouse Sca-1 antibody based magnetic separation was used, as described in a previous protocol [4]. The study protocol used here was approved by the Stanford Institutional Review Board. In brief, human fetal hearts (StemExpress, Diamond Springs, CA) were perfused using a Lagendorff apparatus, using Tyrode solution containing collagenase. Following this, fetal Sca-1+ cells were isolated by magnetic cell sorting (MACS, Miltenyi Biotec, Sunnyvale, CA), using Sca-1-coupled magnetic beads, according to the manufacturer’s protocol. Sca-1+ cells were eluted from the column by washing with PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA. The eluted Sca-1+ cells were cultured on 0.1% gelatin-coated dishes in M199 (Gibco)/EGM-2 (3 : 1) media, supplemented with 10% FBS (Gibco), 10 ng/mL basic fibroblast growth factor (bFGF), 5 ng/mL epithelial growth factor (EGF), 5 ng/mL insulin-like growth factor (IGF-1), 5 ng/mL vascular endothelial growth factor (VEGF), 5 ng/mL heparin, 5 ng/mL ascorbic acid, nonessential amino acids, β-mercaptoethanol, 1x Penicillin, and 1x streptomycin. All Sca-1+ cells used for this study were between passages 2 and 5.
2.2. Western Blotting
Protein samples (30 μg) were separated on SDS-polyacrylamide gels by electrophoresis and the proteins were transferred to immobilon-P membranes (GE Healthcare). Level of the ALDH1 protein was determined using anti-ALDH1A1 antibodies (Abcam) at a concentration of 1 : 1000. Anti-enolase antibody (Santa Cruz Biotechnology, Santa Cruz) was used as housekeeping marker at a concentration of 1 : 1000. The bound antibodies were visualized with horseradish peroxidase- (HRP-) coupled secondary antibody and chemiluminiscence detection system.
2.3. ALDH Activity Assay by Spectrophotometer
ALDH1 activity was measured in unsorted control and ALDH specific inhibitor, diethylaminobenzaldehyde- (DEAB-) treated cells by following the procedure as described elsewhere [6]. In brief, enzymatic activity of ALDH1 in cell lysate was determined spectrophotometrically by reductive reaction of NAD+ to NADH at 340 nm (Beckman).
10 mM phenyl acetaldehyde was used as the substrate for ALDH1. All the assays were carried out at 25°C in 0.1 M sodium pyrophosphate buffer, pH = 9.5. 2.4 mM NAD+.
2.4. Flow Cytometry Based Sorting of ALDHhigh and ALDHlow Sca-1+ Cells
Sca-1+ cells were sorted by flow cytometry into subpopulations having high or low ALDH enzyme activity using the Aldefluor kit (Stem Cell Technologies, Vancouver, Canada), as per manufacturer’s instructions. Briefly, cultured human fetal Sca-1+ cells were resuspended in Aldefluor assay buffer, and 1 × 106 cells/mL cell suspension was added to a tube containing 5 µL of the ALDH substrate, Aldefluor. 0.5 mL of this suspension was transferred to a second tube, containing the DEAB, which serves as a negative control. Following 30-minute incubation at 37°C and centrifugation, the cells were washed with the assay buffer, followed by resuspension in 0.3 mL of ice cold Aldefluor assay buffer for flow sorting (carried out on BD FACS Aria). Viability of the cells was determined by 7-amino actinomycin D (7-AAD). The DEAB negative control was used to set the gate. Cells having the highest fluorescence at 515 nm were collected as the population. The nonfluorescent population at 515 nm was collected as the cells. After sorting, the and Sca-1+ cells were cultured under identical culture conditions. After one week in culture, Aldefluor assay was carried out on the two populations as described above, followed by flow cytometric analysis of ALDH activity.
2.5. Quantitative (Real-Time) Reverse Transcriptase (RT) PCR (qRT-PCR)
Equal number of and cells at passage 2 was seeded in a 6-well plate and was harvested at 70–80% confluency. The cell pellet obtained after centrifugation was used for RNA isolation. Total RNA was purified from the and Sca-1+ cells using RNeasy Mini kit (Qiagen). 1 μg of total RNA was reverse transcribed using high capacity cDNA transcription kit (Applied Biosystems), followed by high throughput quantitative gene expression analysis using Taqman probes for all the genes analyzed. To assess the status of stemness markers, the following Taqman probes were used: Oct4 (Hs00982625_m1) for organic cation transporter-4 gene, Nanog (Hs02387400_g1) for the gene of nanog homeobox, GATA4 (Hs00171403_m1) for GATA binding protein 4 gene, Isl1 (Hs00158126_m1) for ISL1 transcription factor gene, and MEF2C (Hs00231149_m1) for myocyte enhancer factor 2C gene. Expression of two genes, the nuclear antigen Ki67 (Hs01032443_m1) and the antiapoptotic factor, B-cell CLL/lymphoma (Bcl) 2 (Hs00608023_m1) were assessed for comparative analysis of proliferation and cell death, respectively. The assay was carried out on the StepOne Plus Real-Time PCR platform (Applied Biosystems). 18S was used as the internal control gene for all samples and the ΔΔCt method was used to calculate fold change of gene expression.
2.6. Cell Death Assay after Aldehydic Challenge
First and Sca-1+ cells were treated with 50 μM acetaldehyde for 4 hr. Acetaldehyde-induced apoptotic cell death was determined using annexin V staining via flow cytometry following manufacturer’s instructions (Roche).
2.7. Statistical Analysis
All experiments were done in triplicate. FACS and real-time PCR data were analyzed using Graphpad Prism (GraphPad Software, Inc., CA, USA). All data have been presented as mean ± standard error over mean (SEM). For analysis of the FACS data, 1-way ANOVA, followed by Kruskal-Wallis test was used to calculate significance of the differences. For analysis of the RT-PCR data, unpaired -test was used to compare significance of differences between and cells for each individual gene. In all cases, differences were considered significant at values <0.05.
3. Results
3.1. ALDH1 Level and Activity in Cultured Sca-1+ Human Fetal Cells
Prior to isolating cells using Aldefluor kit, which is based on ALDH1 activity, we first determined ALDH1 presence/level in cultured human fetal cells by Western blot analysis. The results showed that these cells do express ALDH1A1 (Figure 1(a)). We also found significant ALDH1 activity in human fetal cell lysates by spectrophotometric assay using phenyl acetaldehyde as substrate (Figure 1(b)). In this activity assay, conversion of phenyl acetaldehyde into phenyl acetic acid by ALDH, generating NADH was measured spectrophotometrically at 340 nm. We found that DEAB (1.5 μM for 1 hour at 37°C) significantly decreased ALDH1 activity.
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3.2. Identification and Isolation of ALDHhigh Cells
We went ahead to identify and isolate the Sca-1+ human fetal cells subpopulation with the highest level of ALDH1 activity using the fluorescence based Aldefluor assay, which has been routinely utilized to identify and isolate primitive hematopoietic stem cells [7]. Using this assay, coupled with flow cytometry, we identified a subpopulation of ~15% cells within the Sca-1+ cells which demonstrated phenotype (Figure 2). Close to 98% of cells were viable with this procedure as determined by 7-AAD staining (data not shown). Both the and cells were sorted and expanded for downstream characterization.
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3.3. ALDHhigh Sca-1+ Cells Are Enriched in Stemness and Cardiac Development-Specific Genes
Equal number of first passage and Sca-1+ cells were seeded under identical culture conditions and harvested for real-time PCR after 48–72 hrs. cells demonstrated relatively high levels of stem cell-specific markers such as Oct4 and Nanog as compared to Sca-1+ cells () (Figure 3). In addition, multiple early mesoderm-specific transcripts, such as GATA4, Isl1 and MEF2C, were also enriched in Sca-1+ cells compared to Sca-1+ cells ( value < 0.05) (Figure 3).
3.4. Increase in Proliferation and Survival Potency in ALDHhigh Sca-1+ Cells
We also observed a difference in survival and proliferation status between the two populations, immediately after sorting and seeding them. The Sca-1+ cells had a large proportion of floating, dead cells when compared to the Sca-1+ cells. In order to confirm this, equal number of and Sca-1+ cells were seeded and harvested after 48 hours, followed by assessment for expression of a proliferation-specific gene, Ki67 and an antiapoptotic marker, Bcl2. The Sca-1+ cells demonstrated significant upregulation of both these markers when compared to the Sca-1+ cells ( value < 0.05) (Figure 4), indicating enhanced proliferation as well as survival advantage of Sca-1+ cells.
The apoptotic cell death was lower in Sca-1+ cells compared to Sca-1+ cells in both basal condition and acetaldehyde treatment as shown in Table 1.
3.5. ALDHhigh Sca-1+ Cells Produce Increased Number of ALDHhigh Sca-1+ Cells upon Culture
In order to test the potential of and Sca-1+ cells to give rise to all the cell types existing in the original population from which they were derived, the two populations were cultured. After two weeks in culture, both populations were subject to the Aldefluor assay to determine ALDH activity. While the Sca-1+ cells could only give rise to subpopulation (>97%; value < 0.05) (Figure 5), the Sca-1+ cells recapitulated the original population by giving rise to both the and populations, in addition to maintaining a high percentage of the cells with phenotype. This was consistently observed for multiple samples, and this observation held true even after 2-3 weeks of culture.
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4. Discussion
This is the first study which identifies a distinct subpopulation of cells in the fetal human heart having high ALDH activity and possessing enhanced potency for self-renewal, proliferation, survival and multipotency.
ALDH has been shown to be a functional marker of stem cells in multiple tissues and tumors of multiple organs [8–11]. The earliest studies of identification and isolation of live cells using a fluorescence based assay were carried out in cord blood cells [7] and gradually expanded to other types of stem cells [12–14]. In this in vivo cellular assay, BODIPY-aminoacetaldehyde (BAAA) is converted by intracellular isoform of ALDH, ALDH1 into a negatively charged reaction product, BODIPY-aminoacetate (BAA) which is retained inside cells, causing the cells expressing high levels of ALDH to become brightly fluorescent. Now this method, known as the “Aldefluor assay” is used routinely to enrich stem/progenitor cells from multiple tissues and tumor samples [9].
We confirmed biochemically that ALDH1 was expressed in human fetal Sca-1+ cells using immunoblotting technique and significant ALDH1 activity was found by enzymatic activity assay when phenyl acetaldehyde was used as a substrate (Figure 1). After confirming that human fetal Sca-1+ cells contain ALDH1, we used flow cytometry based Aldefluor assay to identify and sort the subpopulation. This fraction comprised ~15% of the human fetal heart cells (Figure 2). This is higher than the percentage reported from other tissues [15–17], that is, 0.5–5%. This increase in population may be because of fetal origin. Increase in numbers of cardiac progenitors in neonatal heart compared to adult heart has been reported in earlier studies [18, 19]. Further studies could demonstrate whether this is true for Sca-1+ cells too.
In addition to upregulation of typical stem cell markers (Oct4 and Nanog) in the Sca-1+ cells with respect to Sca-1+ cells, the Isl1 transcript, which is a well-established marker during cardiogenesis in the fetus, was seen to be enriched in the Sca-1+ cells. Another interesting observation in our study was the significant enrichment of MEF2C transcription factor exclusively in the Sca-1+ cells. MEF2C is known to be one of the earliest markers of cardiac lineage [20], validating the hypothesis that Sca-1+ cells might represent a primitive cardiac population in the fetal heart.
We observed higher expression of the proliferative marker Ki67 and antiapoptotic transcript Bcl2 in Sca-1+ cells. Considering that primitive progenitor cells typically possess high proliferative potential, this indicates yet again that Sca-1+ cells might represent the transit amplifying cells in the fetal heart, which proliferate extensively, and have robust prosurvival mechanisms, as indicated by the Bcl2 levels. The role of Bcl2 in stem cell survival and function has been extensively documented in hematopoietic stem cells (HSCs) [21, 22], intestinal stem cells [23], and melanocyte stem cells [24], to name a few. It has been shown that Bcl2 increases both number and self-renewal potential of HSCs by inhibiting their apoptosis [21]. In a study of embryonic stem cell (ESC) transplantation after cerebral ischemia in rats, overexpression of Bcl2 in the ESCs increased survival of the transplanted cells and brought about significant functional recovery in the brain [25]. In fact, acetaldehyde-induced apoptosis was less in Sca-1+ cells with respect to Sca-1+ cells.
Upon subsequent expansion, Sca-1+ cells gave rise to all population of cells including 28 to 40% of Sca-1+ cells and remaining Sca-1+ cells, thereby recapitulating the original population; however Sca-1+ cells primarily gave rise to Sca-1+ cells in culture (<3.5% of Sca-1+ cells). It was shown in case of prostate stem cells that cells proliferate much more efficiently compared to cells [8].
In accordance with earlier studies, our observations demonstrate that Sca-1+ cells represent a primitive stem/progenitor cell population in the human fetal heart. Even though existence of Sca-1+ cells in adult human heart remains to be addressed, based on our findings, we hypothesize that the adult heart also harbors an subpopulation, though their number might be much lower than the fetal ones. It would therefore be interesting to follow up this study in adult human heart samples and also investigate if there is a change in the number or characteristics of Sca-1+ cells in pathological heart samples.
In recent years, bone marrow-derived cells were recruited to the area of ischemia within hours of transplantation and continued accumulating for up to 1 week, increasing capillary formation in a hind limb ischemia model [16]. cell treatment enhanced the perfusion ratio continuously even after 3 weeks compared to cells [16]. In another study, human umbilical cord blood (UCB) derived cells with nanoparticle label were injected into mice 24 hrs after induction of myocardial infarction. The cells, but not the cells, preferentially homed to and increased blood vessel formation in the infarcted host myocardium [26]. These studies have provided a strong basis for the clinical potential of cells in regenerative medicine. In fact, two clinical trials were launched based on these studies [27, 28]. Therefore, our current study of identifying and partially characterizing Sca-1+ progenitor cells in human fetal heart is an important first step in moving forward to address multiple questions, including differentiation studies of these cells, understanding the role of different ALDH isozymes in the function of Sca-1+ cells, and testing the therapeutic potential of progenitor cells in preclinical models of cardiovascular diseases. More importantly, extrapolating this approach to other types of cardiac progenitor cells such as c-kit+ cells would be very useful for the advancement of cardiac regenerative medicine. In conclusion, our study reaffirms that high ALDH activity can be used as a functional biochemical marker to isolate progenitor cells within the heart, which possess enhanced self-renewal, proliferation, and survival potential.
Conflict of Interests
The authors have no conflict of interests to declare.
Acknowledgments
The authors would like to acknowledge Dr. Patricia De Almeida for the flow cytometry based experiments, as well as the very useful discussions about the results of this study, and Dr. Antje Dagmar Ebert for detailed discussions about ALDH biology.