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. 2012 Sep 14;111(7):882-93.
doi: 10.1161/CIRCRESAHA.112.269001. Epub 2012 Jul 19.

Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation

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Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation

Mingxia Gu et al. Circ Res. .
Free PMC article

Abstract

Rationale: Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models.

Objective: To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.

Methods and results: Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery.

Conclusions: In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

Figures

Figure 1
Figure 1. Generation and characterization of porcine induced pluripotent stem cells (piPSCs)
A) Representative timeline of piPSC generation. Compact clones were observed on day 15. B) Immunofluorescence staining of pluripotent markers. Unlike the porcine adipose stromal cells (pASCs) from which they were derived, piPSCs stained positive for the traditional markers of pluripotency (e.g., Nanog, Oct4, Klf4 and SSEA-1). pASCs have a weak expression of c-Myc, which is consistent with other reports. C) Reverse transcription PCR analysis of pluripotent markers. Expression of Oct4, Klf4, Sox2, and Nanog is present in derived piPSCs, but absent from pASC and negative control (blank). D) Pearson correlation analysis for gene expression in piPSCs versus pASCs and porcine fibroblasts (pfibroblasts). Hierarchical clustering of whole genome expression showed that our generated piPSC lines were similar to reference piPSC lines (piPSC-IC1, piPSC-ID4, and piPSC-ID6) generated from another laboratory, and were distinct from pfibroblasts and pASCs. The range of Pearson correlation coefficients are displayed in the color bar (top).
Figure 2
Figure 2. Derivation and characterization of piPSC-derived endothelial cells
A) Bright-field images of endothelial cell differentiation from piPSCs using embryoid bodies. Differentiated cells were dissociated and sorted by FACS on day 12. B) Morphological and functional similarities between piPSC-ECs and endothelial cells harvested from the autologous porcine aorta (pAorta-ECs). Both piPSC-ECs and pAorta-ECs show positive immunofluorescence staining for the endothelial marker CD31 (green) and DAPI nuclear stain (blue). Similar to pAorta-ECs, piPSC-ECs take up acetylated LDL (red) and form a capillary-like network on Matrigel 24 hours after seeding the cells (far right). C) Microarray analysis confirmed that, similar to pAorta-ECs, piPSC-ECs express genes related to endothelial function.
Figure 3
Figure 3. Greater functional improvement noted in mice treated with piPSCs-ECs
A) Representative M-mode echocardiographic views of infarcted hearts receiving PBS, pASCs, pAorta-ECs, and piPSC-ECs (n=20 per group). B) Quantification of fractional shortening (FS) reveals significant improvement in systolic function of animals receiving piPSC-ECs at week 2 and week 4 post-MI compared to animals receiving PBS (week 2, 23.7±0.9% vs. 19.3±0.5%, *P<0.01; week 4, 27.2±1.3% vs. 22.3±1.1%, **P<0.001). Greater improvement was also seen in animals receiving piPSCs compared to pASCs although this did not reach statistical significance (week 2, 23.7±0.9% vs. 21.5±1.5%, P=NS; week 4, 27.2±1.3% vs. 24.2±2.5%, P=NS). Comparable improvement is observed between animals receiving piPSC-ECs and their endogenous counterpart, pAorta-ECs (week 2, 23.7±0.9% vs. 23.4±1.3%; week 4, 27.2±1.3% vs. 27.1±0.6%; P=NS). C) Representative BLI of an animal receiving 1×106 piPSC-ECs demonstrated robust cell engraftment at day 2 following injection. Progressive decrease in signal was observed over the next several weeks, but persistent cell engraftment is still noted at week 4.
Figure 4
Figure 4. Histological evaluation of piPSC-EC therapy in infarcted hearts at week 4
A) Representative triphenyltetrazolium chloride (TTC) gross histochemical analysis of infarcted hearts injected with PBS (control group) and piPSC-ECs. B) Quantitative analysis of the infarct size showed that the percent infarct size is significantly smaller in mice treated with piPSC-EC compared to PBS (25.2±1.5% versus 32.7±0.8%; *P<0.05). C) Representative histology of infarcted hearts injected with PBS and piPSC-ECs (hematoxylin and eosin, magnification 1.25x and 5x for the whole heart and left ventricular wall, respectively). D) Quantitative analysis of the left ventricular wall thickness showed thicker ventricular walls were present in the piPSC-EC compared to PBS control group (281±12 μm versus 161±6 μm; *P <0.01). E) Representative immunofluorescence staining of the murine endothelial marker CD31 in the peri-infarct area of mice treated with PBS, pASCs, pAorta-ECs, and piPSC-ECs. F) Quantitative analysis of capillary density (vessel/mm2) showed a significant increase in vessel density (# vessels per high power field) in animals treated with piPSC-ECs compared to PBS (395±28 vessels/mm2 versus 107±9 vessels/mm2,*P<0.001).
Figure 4
Figure 4. Histological evaluation of piPSC-EC therapy in infarcted hearts at week 4
A) Representative triphenyltetrazolium chloride (TTC) gross histochemical analysis of infarcted hearts injected with PBS (control group) and piPSC-ECs. B) Quantitative analysis of the infarct size showed that the percent infarct size is significantly smaller in mice treated with piPSC-EC compared to PBS (25.2±1.5% versus 32.7±0.8%; *P<0.05). C) Representative histology of infarcted hearts injected with PBS and piPSC-ECs (hematoxylin and eosin, magnification 1.25x and 5x for the whole heart and left ventricular wall, respectively). D) Quantitative analysis of the left ventricular wall thickness showed thicker ventricular walls were present in the piPSC-EC compared to PBS control group (281±12 μm versus 161±6 μm; *P <0.01). E) Representative immunofluorescence staining of the murine endothelial marker CD31 in the peri-infarct area of mice treated with PBS, pASCs, pAorta-ECs, and piPSC-ECs. F) Quantitative analysis of capillary density (vessel/mm2) showed a significant increase in vessel density (# vessels per high power field) in animals treated with piPSC-ECs compared to PBS (395±28 vessels/mm2 versus 107±9 vessels/mm2,*P<0.001).
Figure 5
Figure 5. Cytokine expression array demonstrate piPSC-ECs release paracrine factors with in vitro hypoxia stress
A) Angiogenesis and anti-apoptosis protein array data after hypoxia exposure for 24 hours. B) Quantitative analysis of the cytokine array confirmed significant up-regulation of several pro-angiogenic and anti-apoptotic related proteins in pASCs, pAorta-ECs, and piPSC-ECs compared to control (culture medium without cells under hypoxic conditions). Interestingly, piPSC-ECs released significantly more paracrine factors than pASCs. A similar pattern of paracrine secretion was noted between piPSC-ECs and their endogenous counterparts, pAorta-ECs. Two biological replicates per group (*P<0.05, **P<0.001 vs. pASCs).
Figure 6
Figure 6. Microfluidic single cell gene expression profiling demonstrates piPSC-ECs can release paracrine factors in vivo
A) Schematic outline of single cell expression profiling experiment demonstrating that paracine factors are released in response to the ischemic microenvironment. One-week post injection into normal vs. infarcted hearts, piPSC-ECs were harvested, sorted by FACS, and analyzed by single cell qRT-PCR. B) Comparison of fold-change obtained by single cell qRT-PCR showed a significant increase in the expression of 10 genes, which have pro-angiogenic and/ or anti-apoptotic effects, in the MI group compared to the sham group. Fold-change is defined as the Ct value of the MI group divided by the Ct value of the sham group. Results confirm that piPSC-ECs are able to respond to the local ischemic milieu by secreting pro-angiogenic and anti-apoptotic factors. C) Schematic outline of single cell expression experiment showing that the release of paracrine factors varied among different subpopulations of pASC and piPSC-ECs. One week post injection into infarcted hearts, injected cells were harvested, subpopulations of pASCs and piPSC-ECs were isolated using FACS and single cell qRT-PCR was performed. D) Comparison of fold-change obtained by single cell qRT-PCR showed patterns of paracrine release varied among three populations of piPSC-ECs and ASCs and cells: 1) CD31+/CD34+/CD144-, 2) CD31+/CD34-/CD144+, and 3) CD31+/CD34-/CD144-
Figure 6
Figure 6. Microfluidic single cell gene expression profiling demonstrates piPSC-ECs can release paracrine factors in vivo
A) Schematic outline of single cell expression profiling experiment demonstrating that paracine factors are released in response to the ischemic microenvironment. One-week post injection into normal vs. infarcted hearts, piPSC-ECs were harvested, sorted by FACS, and analyzed by single cell qRT-PCR. B) Comparison of fold-change obtained by single cell qRT-PCR showed a significant increase in the expression of 10 genes, which have pro-angiogenic and/ or anti-apoptotic effects, in the MI group compared to the sham group. Fold-change is defined as the Ct value of the MI group divided by the Ct value of the sham group. Results confirm that piPSC-ECs are able to respond to the local ischemic milieu by secreting pro-angiogenic and anti-apoptotic factors. C) Schematic outline of single cell expression experiment showing that the release of paracrine factors varied among different subpopulations of pASC and piPSC-ECs. One week post injection into infarcted hearts, injected cells were harvested, subpopulations of pASCs and piPSC-ECs were isolated using FACS and single cell qRT-PCR was performed. D) Comparison of fold-change obtained by single cell qRT-PCR showed patterns of paracrine release varied among three populations of piPSC-ECs and ASCs and cells: 1) CD31+/CD34+/CD144-, 2) CD31+/CD34-/CD144+, and 3) CD31+/CD34-/CD144-

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