Potency of Human Cardiosphere-Derived Cells from Patients with Ischemic Heart Disease Is Associated with Robust Vascular Supportive Ability

Abstract

Cardiosphere-derived cell (CDC) infusion into damaged myocardium has shown some reparative effect; this could be improved by better selection of patients and cell subtype. CDCs isolated from patients with ischemic heart disease are able to support vessel formation in vitro but this ability varies between patients. The primary aim of our study was to investigate whether the vascular supportive function of CDCs impacts on their therapeutic potential, with the goal of improving patient stratification. A subgroup of patients produced CDCs which did not efficiently support vessel formation (poor supporter CDCs), had reduced levels of proliferation and increased senescence, despite them being isolated in the same manner and having a similar immunophenotype to CDCs able to support vessel formation. In a rodent model of myocardial infarction, poor supporter CDCs had a limited reparative effect when compared to CDCs which had efficiently supported vessel formation in vitro. This work suggests that not all patients provide cells which are suitable for cell therapy. Assessing the vascular supportive function of cells could be used to stratify which patients will truly benefit from cell therapy and those who would be better suited to an allogeneic transplant or regenerative preconditioning of their cells in a precision medicine fashion. This could reduce costs, culture times and improve clinical outcomes and patient prognosis. Stem Cells Translational Medicine 2017;6:1399-1411.

Keywords: Cell-based and tissue-based therapy; Coronary artery disease; Humans; Myocardial ischemia; Tissue-specific progenitor cells.

Figure 1

Cardiosphere‐derived cells (CDCs) vary in their supportive ability. (A): The immunophenotype of human CDCs extracted from patients with ischemic heart disease (IHD) was analyzed by flow cytometry. The total tubule length (TTL) of GFP‐labeled human umbilical vein endothelial cells (HUVECs) was measured after 14 days of coculture with CDCs samples from a total of 43 IHD patients. TTL compared with control supportive cells BMSC was recorded as relative tubule length (RTL). CDC samples patients were subgrouped into three tertiles based on their TTL and RTL, the first tertile was defined as high tubule formation (n = 14), the second as moderate tubule formation (n = 14), and the third as low tubule formation (n = 15) (B–D). (B): Representative images of HUVEC tubule formation after 14 days of coculture with CDCs with high, medium and low tubule formation. Scale bars are equal to 500 µm. Quantification of TTL (C) and RTL (D) for the CDC tertiles with the ranges shown below each graph. Data is presented as mean and standard error of the mean. ***, p value ≤ .001.

Figure 2

Good and poor supporter CDCs may differ in their structural organization and cytokine release profile. Genes and transcripts highlighted by RNA‐sequencing (RNA‐seq) as having a differential expression with a posterior probability cut‐off of > .3 were analyzed by the online tool DAVID. The top 10 significantly upregulated (p value ≤ .05) biological processes (A, B) and molecular functions (C, D) categories are shown. Full RNA‐seq data can be accessed at gene expression omnibus; accession number GSE81827 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=mfupcoyovzyxdub&acc=GSE81827). Abbreviations: CDCs, cardiosphere‐derived cells; ECM, extracellular matrix; GO, gene ontology; PDGF, platelet derived growth factor.

Figure 3

Poor supporter cardiosphere‐derived cells (CDCs) have an enhanced inflammatory profile. The expression of 102 cytokines was determined by a human cytokine antibody array, 11 cytokines detected by the array had a differential expression between good and poor supporters in the RNA‐sequencing (RNA‐seq) array (posterior probability >.3) Gene / transcript levels (A) and protein levels (B) of the 11 overlapping cytokines are shown for good and poor supporter CDCs. (C): Six additional cytokines were significantly different between good and poor supporter CDCs as detected by the cytokine array. For figure A gene levels are shown for all cytokines except IL‐32 where transcript data is shown. Data is presented as mean and standard error of the mean. +,posterior probability ≥ .3; ++, posterior probability ≥ .5; *, p value ≤ .05; **, p value ≤ .01; ***, p value ≤ .001. Full RNA‐seq data for (A) can be accessed at gene expression omnibus; accession number GSE81827 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=mfupcoyovzyxdub&acc=GSE81827). Abbreviations: PDGF‐AB/BB, platelet derived growth factor AB/BB; uPAR, urokinase receptor.

Figure 4

Poor supporter cardiosphere‐derived cells (CDCs) have reduced proliferation and cell cycle progression but do not differ in migratory ability compared to good supporter CDCs. (A): Migratory ability of CDCs as shown by the mean number of CDCs which had migrated through a transwell with 8 µm pores toward a serum gradient after 24 hours of culture, with representative images (×100). (B): Quantification of the percentage of EdU positive CDCs with representative images (×200) as measured by a Click‐iT proliferation assay. (C): Quantification of Ki67 positive CDCs with representative images (×400). (D): Quantification of the phosphorylated and nonphosphorylated form of Rb in CDCs with good and poor supportive potential, with representative images. Scale bars are equal to 100 µm. Data is presented as mean and standard error of the mean. *, p value ≤ .05. Abbreviations: DAPI, 4′,6‐ diamino‐2‐phenylindole; NS, non‐significant; EdU, 5‐ethynyl‐2′‐deoxyuridine; Rb, retinoblastoma.

Figure 5

Poor supporters are resistant to apoptosis and have increased levels of senescence. Early and late apoptosis were measured in good and poor supporter cardiosphere‐derived cells (CDCs) by analysis of total and cleaved caspase 3 and PARP protein levels. Apoptosis was induced by hygromycin treatment and compared to control DMSO treated CDCs. (A): Representative images of caspase 3 and PARP Western blots. Quantification of full length [35 kDa, (B)] and cleaved [19 kDa (C), 17 kDa (D)] caspase 3. Quantification of full length [110 kDa, (E)] and cleaved [89 kDa, (F)] PARP. (G): Quantification of senescence associated β‐galactosidase (SABG) positive CDCs with representative images (×100). Scale bars are equal to 200 µm. Data is presented as mean and standard error of the mean. *, p value ≤ .05; **, p value ≤ .01. Abbreviations: DMSO, dimethyl sulfoxide; PARP, poly(ADP‐ribose) polymerase; SABG, senescence associated β‐galactosidase.

Figure 6

Effect of cardiosphere‐derived cell (CDC) transplantation on LV function, capillary density and infarct size. Adult athymic (HIH‐Foxn1rnu) rats had the left anterior descending LAD artery ligated. Animals were split into three groups which received either CDCs from good supporters, CDC from poor supporters or saline as a control. Echocardiography was used to measure heart function at baseline and 15 days. (A): Percentage of fractional area change (FAC %). (B): Percentage of fractional shortening (FS %) change. (C): Percentage of anterior wall thickening (AWT %). (D): Percentage change in left ventricular ejection fraction (LVEF %). 30 days following transplantation of CDCs or saline the hearts were excised, sectioned and stained with anti‐caveolin antibody or with Masson's trichrome stain. (E): Quantification of capillary density (vessels/mm²) in the hearts of animals infused with good supporters, poor supporters or saline control as measured by caveolin immunostaining, with representative images (×200, scale bars are equal to 50 µm). (F): Quantification of infarct size (% of LV wall) in the hearts of animals infused with good supporters, poor supporters or saline control as measured by Masson's trichrome stain, with representative images (scale bars are equal to 2 mm). In (F), the fibrotic tissue around the infarct stains blue against the healthy myocardium in red. Data is presented as mean and standard error of the mean. *, p value ≤ .05; **, p value ≤ .01. Abbreviations: AWT, aortic wall thickness; FAC %, percentage of fractional area change; FS %, percentage of fractional shortening; LVEF, left ventricular ejection fraction.