# Sca-1+ Cells from Fetal Heart with High Aldehyde Dehydrogenase Activity Exhibit Enhanced Gene Expression for Self-Renewal, Proliferation, and Survival

Authors: Devaveena Dey; Guodong Pan; Nadimpalli Ravi S. Varma; Suresh Selvaraj Palaniyandi
Journal: Oxidative Medicine and Cellular Longevity
 (2015)

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## 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 thatALDHbright/high 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 ALDHlow fraction. The ALDHhigh fraction also exhibited significant increase in proliferation and pro-survival gene expression. In addition, only the ALDHhigh and not the ALDHlow fraction could give rise to all the cell types of the original population, demonstrating multipotency. ALDHhigh cells showed increased resistance against aldehyde challenge compared to ALDHlow cells. These results indicate that ALDHhigh 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.


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## Body

## 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]. ALDHhigh cells from multiple tissues have been shown to possess enhanced stemness properties, specifically self-renewal and differentiation [7, 11, 13]. ALDHhigh stem cells are a small population of cells (0.5–5%) highly enriched for pluripotency [14–16]. In fact ALDHhigh 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, ALDHhigh cells exhibit high self-renewal capacity, stemness, survival, and proliferation capacity compared to ALDHlow 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 (30μg) 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 ALDHhigh population. The nonfluorescent population at 515 nm was collected as the ALDHlow cells. After sorting, the ALDHhigh and ALDHlow 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 ofALDHhigh and ALDHlow 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 ALDHhigh and ALDHlow 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) forISL1 transcription factor gene, andMEF2C (Hs00231149_m1) for myocyte enhancer factor 2C gene. Expression of two genes, the nuclear antigenKi67 (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

FirstALDHhigh and ALDHlow 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, unpairedt-test was used to compare significance of differences between ALDHhigh and ALDHlow cells for each individual gene. In all cases, differences were considered significant at P values <0.05.


## 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 (30μg) 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 ALDHhigh population. The nonfluorescent population at 515 nm was collected as the ALDHlow cells. After sorting, the ALDHhigh and ALDHlow 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 ofALDHhigh and ALDHlow 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 ALDHhigh and ALDHlow 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) forISL1 transcription factor gene, andMEF2C (Hs00231149_m1) for myocyte enhancer factor 2C gene. Expression of two genes, the nuclear antigenKi67 (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

FirstALDHhigh and ALDHlow 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, unpairedt-test was used to compare significance of differences between ALDHhigh and ALDHlow cells for each individual gene. In all cases, differences were considered significant at P values <0.05.


## 3. Results

### 3.1. ALDH1 Level and Activity in Cultured Sca-1+ Human Fetal Cells

Prior to isolatingALDHhigh 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.Figure 1
Level and activity of ALDH1. (a) Western immunoblot bands of ALDH1 and enolase, the loading control for cytosolic proteins, and (b) measurement of ALDH activity with phenyl acetaldehyde as substrate from total cell lysates (below) of control (open bar) and DEAB (1.5μM) treated (filled bar) cells. P** value < 0.006.
(a)
(b)


### 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 ALDHhigh phenotype (Figure 2). Close to 98% of cells were viable with this procedure as determined by 7-AAD staining (data not shown). Both the ALDHhigh and ALDHlow cells were sorted and expanded for downstream characterization.Figure 2
Identification ofALDHhigh subpopulation in cultured fetal Sca-1+ cells using activity based flow cytometric sorting. There is a distinct and consistent ALDHhigh Sca-1+ cell population (10–15%) in fetal heart. Dot plots have been shown for three human fetal Sca-1+ cell samples. Left panel represents negative control for each sample. Right panel is the test sample in the absence of the inhibitor, DEAB. ALDH activity is measured in the FITC channel, represented along x-axis; forward scatter (FSC) is on the y-axis.
(a)
(b)


### 3.3. ALDHhigh Sca-1+ Cells Are Enriched in Stemness and Cardiac Development-Specific Genes

Equal number of first passageALDHhigh and ALDHlow Sca-1+ cells were seeded under identical culture conditions and harvested for real-time PCR after 48–72 hrs. ALDHhigh cells demonstrated relatively high levels of stem cell-specific markers such asOct4 andNanog as compared to ALDHlow Sca-1+ cells (P<0.05) (Figure 3). In addition, multiple early mesoderm-specific transcripts, such asGATA4,Isl1 andMEF2C, were also enriched in ALDHhigh Sca-1+ cells compared to ALDHlow Sca-1+ cells (P value < 0.05) (Figure 3).Figure 3
Real-time PCR analysis ofALDHhigh and ALDHlow Sca-1+ cells. Increased expression of markers for stem cells, early cardiac, and self-renewal exclusively in ALDHhigh cells compared to ALDHlow Sca-1+ cells. (n=4; P* value < 0.05).


### 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. TheALDHlow Sca-1+ cells had a large proportion of floating, dead cells when compared to the ALDHhigh Sca-1+ cells. In order to confirm this, equal number of ALDHhigh and ALDHlow 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 ALDHhigh Sca-1+ cells demonstrated significant upregulation of both these markers when compared to the ALDHlow Sca-1+ cells (P value < 0.05) (Figure 4), indicating enhanced proliferation as well as survival advantage of ALDHhigh Sca-1+ cells.Figure 4
Gene expression analysis of proliferation and prosurvival genes inALDHhigh and ALDHlow Sca-1+ cells. Quantitative real-time PCR based gene expression analysis of a proliferation-specific gene (Ki67) and antiapoptotic gene,Bcl2 to compare ALDHhigh and ALDHlow Sca-1+ cells. Fold change in gene expression is with respect to ALDHlow Sca-1+ cells. (n=3; P* value < 0.05).The apoptotic cell death was lower inALDHhigh Sca-1+ cells compared to ALDHlow Sca-1+ cells in both basal condition and acetaldehyde treatment as shown in Table 1.Table 1
Reduced apoptotic death in ALDHhigh cells with acetaldehyde treatment.
Cell type
Basal
Acetaldehyde-treated
ALDHhigh cells
ALDHlow cells
ALDHhigh cells
ALDHlow cells
% apoptotic death
102 ± 12
197 ± 2*
163 ± 32
338 ± 5**##
*
P value < 0.001 versus ALDHhigh cells in basal conditions; **P value < 0.005 versus ALDHhigh cells with acetaldehyde treatment; ##P value < 0.0001 versus ALDHlow cells in basal conditions. N: 4 repeats.


### 3.5. ALDHhigh Sca-1+ Cells Produce Increased Number of ALDHhigh Sca-1+ Cells upon Culture

In order to test the potential ofALDHhigh and ALDHlow 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 ALDHlow Sca-1+ cells could only give rise to ALDHlow subpopulation (>97%; P value < 0.05) (Figure 5), the ALDHhigh Sca-1+ cells recapitulated the original population by giving rise to both the ALDHhigh and ALDHlow populations, in addition to maintaining a high percentage of the cells with ALDHhigh phenotype. This was consistently observed for multiple samples, and this observation held true even after 2-3 weeks of culture.Figure 5
Analysis of the potential ofALDHhigh and ALDHlow Sca-1+ cells to recapitulate the original population. (a) Representation of flow cytometric analysis of ALDHhigh Sca-1+ cells (left panel) and ALDHlow Sca-1+ cells (right panel) one week afterin vitro culture. (b) Graphical representation of the percentage of ALDHhigh Sca-1+ cells in the original population, arising from ALDHhigh Sca-1+ cells and ALDHlow Sca-1+ cells. (n=3; P* value < 0.001).
(a)
(b)


## 3.1. ALDH1 Level and Activity in Cultured Sca-1+ Human Fetal Cells

Prior to isolatingALDHhigh 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.Figure 1
Level and activity of ALDH1. (a) Western immunoblot bands of ALDH1 and enolase, the loading control for cytosolic proteins, and (b) measurement of ALDH activity with phenyl acetaldehyde as substrate from total cell lysates (below) of control (open bar) and DEAB (1.5μM) treated (filled bar) cells. P** value < 0.006.
(a)
(b)


## 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 ALDHhigh phenotype (Figure 2). Close to 98% of cells were viable with this procedure as determined by 7-AAD staining (data not shown). Both the ALDHhigh and ALDHlow cells were sorted and expanded for downstream characterization.Figure 2
Identification ofALDHhigh subpopulation in cultured fetal Sca-1+ cells using activity based flow cytometric sorting. There is a distinct and consistent ALDHhigh Sca-1+ cell population (10–15%) in fetal heart. Dot plots have been shown for three human fetal Sca-1+ cell samples. Left panel represents negative control for each sample. Right panel is the test sample in the absence of the inhibitor, DEAB. ALDH activity is measured in the FITC channel, represented along x-axis; forward scatter (FSC) is on the y-axis.
(a)
(b)


## 3.3. ALDHhigh Sca-1+ Cells Are Enriched in Stemness and Cardiac Development-Specific Genes

Equal number of first passageALDHhigh and ALDHlow Sca-1+ cells were seeded under identical culture conditions and harvested for real-time PCR after 48–72 hrs. ALDHhigh cells demonstrated relatively high levels of stem cell-specific markers such asOct4 andNanog as compared to ALDHlow Sca-1+ cells (P<0.05) (Figure 3). In addition, multiple early mesoderm-specific transcripts, such asGATA4,Isl1 andMEF2C, were also enriched in ALDHhigh Sca-1+ cells compared to ALDHlow Sca-1+ cells (P value < 0.05) (Figure 3).Figure 3
Real-time PCR analysis ofALDHhigh and ALDHlow Sca-1+ cells. Increased expression of markers for stem cells, early cardiac, and self-renewal exclusively in ALDHhigh cells compared to ALDHlow Sca-1+ cells. (n=4; P* value < 0.05).


## 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. TheALDHlow Sca-1+ cells had a large proportion of floating, dead cells when compared to the ALDHhigh Sca-1+ cells. In order to confirm this, equal number of ALDHhigh and ALDHlow 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 ALDHhigh Sca-1+ cells demonstrated significant upregulation of both these markers when compared to the ALDHlow Sca-1+ cells (P value < 0.05) (Figure 4), indicating enhanced proliferation as well as survival advantage of ALDHhigh Sca-1+ cells.Figure 4
Gene expression analysis of proliferation and prosurvival genes inALDHhigh and ALDHlow Sca-1+ cells. Quantitative real-time PCR based gene expression analysis of a proliferation-specific gene (Ki67) and antiapoptotic gene,Bcl2 to compare ALDHhigh and ALDHlow Sca-1+ cells. Fold change in gene expression is with respect to ALDHlow Sca-1+ cells. (n=3; P* value < 0.05).The apoptotic cell death was lower inALDHhigh Sca-1+ cells compared to ALDHlow Sca-1+ cells in both basal condition and acetaldehyde treatment as shown in Table 1.Table 1
Reduced apoptotic death in ALDHhigh cells with acetaldehyde treatment.
Cell type
Basal
Acetaldehyde-treated
ALDHhigh cells
ALDHlow cells
ALDHhigh cells
ALDHlow cells
% apoptotic death
102 ± 12
197 ± 2*
163 ± 32
338 ± 5**##
*
P value < 0.001 versus ALDHhigh cells in basal conditions; **P value < 0.005 versus ALDHhigh cells with acetaldehyde treatment; ##P value < 0.0001 versus ALDHlow cells in basal conditions. N: 4 repeats.


## 3.5. ALDHhigh Sca-1+ Cells Produce Increased Number of ALDHhigh Sca-1+ Cells upon Culture

In order to test the potential ofALDHhigh and ALDHlow 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 ALDHlow Sca-1+ cells could only give rise to ALDHlow subpopulation (>97%; P value < 0.05) (Figure 5), the ALDHhigh Sca-1+ cells recapitulated the original population by giving rise to both the ALDHhigh and ALDHlow populations, in addition to maintaining a high percentage of the cells with ALDHhigh phenotype. This was consistently observed for multiple samples, and this observation held true even after 2-3 weeks of culture.Figure 5
Analysis of the potential ofALDHhigh and ALDHlow Sca-1+ cells to recapitulate the original population. (a) Representation of flow cytometric analysis of ALDHhigh Sca-1+ cells (left panel) and ALDHlow Sca-1+ cells (right panel) one week afterin vitro culture. (b) Graphical representation of the percentage of ALDHhigh Sca-1+ cells in the original population, arising from ALDHhigh Sca-1+ cells and ALDHlow Sca-1+ cells. (n=3; P* value < 0.001).
(a)
(b)


## 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 ALDHhigh 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 thisin 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 ALDHhigh 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 (Figure1). After confirming that human fetal Sca-1+ cells contain ALDH1, we used flow cytometry based Aldefluor assay to identify and sort the ALDHhigh subpopulation. This fraction comprised ~15% of the human fetal heart cells (Figure 2). This is higher than the ALDHhigh percentage reported from other tissues [15–17], that is, 0.5–5%. This increase in ALDHhigh 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 ALDHhigh Sca-1+ cells too.In addition to upregulation of typical stem cell markers (Oct4 and Nanog) in theALDHhigh Sca-1+ cells with respect to ALDHlow Sca-1+ cells, the Isl1 transcript, which is a well-established marker during cardiogenesis in the fetus, was seen to be enriched in the ALDHhigh Sca-1+ cells. Another interesting observation in our study was the significant enrichment of MEF2C transcription factor exclusively in the ALDHhigh Sca-1+ cells. MEF2C is known to be one of the earliest markers of cardiac lineage [20], validating the hypothesis that ALDHhigh 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 inALDHhigh Sca-1+ cells. Considering that primitive progenitor cells typically possess high proliferative potential, this indicates yet again that ALDHhigh 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 ALDHhigh Sca-1+ cells with respect to ALDHlow Sca-1+ cells.Upon subsequent expansion,ALDHhigh Sca-1+ cells gave rise to all population of cells including 28 to 40% of ALDHhigh Sca-1+ cells and remaining ALDHlow Sca-1+ cells, thereby recapitulating the original population; however ALDHlow Sca-1+ cells primarily gave rise to ALDHlow Sca-1+ cells in culture (<3.5% of ALDHhigh Sca-1+ cells). It was shown in case of prostate stem cells that ALDHhigh cells proliferate much more efficiently compared to ALDHlow cells [8].In accordance with earlier studies, our observations demonstrate thatALDHhigh Sca-1+ cells represent a primitive stem/progenitor cell population in the human fetal heart. Even though existence of ALDHhigh Sca-1+ cells in adult human heart remains to be addressed, based on our findings, we hypothesize that the adult heart also harbors an ALDHhigh 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 ALDHhigh Sca-1+ cells in pathological heart samples.In recent years, bone marrow-derivedALDHhigh 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]. ALDHhigh cell treatment enhanced the perfusion ratio continuously even after 3 weeks compared to ALDHlow cells [16]. In another study, human umbilical cord blood (UCB) derived ALDHhigh cells with nanoparticle label were injected into mice 24 hrs after induction of myocardial infarction. The ALDHhigh cells, but not the ALDHlow 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 ALDHhigh 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 ALDHhigh 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 ALDHhigh 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.



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