1. Volume 20, Issue 9, Pages 1750-1766 (September 2012)
Secondary Sphere Formation Enhances the Functionality of Cardiac Progenitor Cells 
Hyun-Jai Cho, Ho-Jae Lee, Seock-Won Youn, Seok-Jin Koh, Joo-Yun Won, Yeon-Ju Chung, Hyun-Ju Cho, Chang-Hwan Yoon, Sae- Won Lee, Eun Ju Lee, Yoo-Wook Kwon, Hae-Young Lee, Sang Hun Lee, Won-Kyung Ho, Young-Bae Park, Hyo-Soo Kim 
Molecular Therapy 
Volume 20, Issue 9, Pages (September 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Generation of primary and secondary cardiospheres and their characteristics. (a) Timeline of primary CS, SDC, and secondary CS generation. Within 48 hours, secondary CSs were generated from SDCs. (b) Phase-contrast bright field images and step-by-step average cell numbers (n = 5). Bar: 500 µm. (c) The gene expressions of Oct4 and c-kit were measured by real-time PCR. (d) The protein expressions of Oct4 and c-kit were measured by western blots and quantified by densitometry. (e) Primary and secondary CSs were positive for alkaline phosphatase (ALP) staining, but SDCs were not. The protein expressions of Oct4 and c-kit were assessed by confocal imaging. Oct4 expression was verified using Oct4 promoter-driven GFP cells. Nuclear colocalization of Oct4 was also confirmed in the single, dissociated secondary CS cells (Supplementary Figure S1). Secondary CSs expressed Oct4 and c-kit more homogeneously and densely than primary CSs, whereas SDCs lacked expressions. TO-PRO-3 (nuclei). Bar: 50 µm. AU, arbitrary unit; CS, cardiosphere; FN, fibronectin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; PDL, poly-D-lysine; SDC, sphere-derived cell.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 1
Generation of primary and secondary cardiospheres and their characteristics. (a) Timeline of primary CS, SDC, and secondary CS generation. Within 48 hours, secondary CSs were generated from SDCs. (b) Phase-contrast bright field images and step-by-step average cell numbers (n = 5). Bar: 500 µm. (c) The gene expressions of Oct4 and c-kit were measured by real-time PCR. (d) The protein expressions of Oct4 and c-kit were measured by western blots and quantified by densitometry. (e) Primary and secondary CSs were positive for alkaline phosphatase (ALP) staining, but SDCs were not. The protein expressions of Oct4 and c-kit were assessed by confocal imaging. Oct4 expression was verified using Oct4 promoter-driven GFP cells. Nuclear colocalization of Oct4 was also confirmed in the single, dissociated secondary CS cells (Supplementary Figure S1). Secondary CSs expressed Oct4 and c-kit more homogeneously and densely than primary CSs, whereas SDCs lacked expressions. TO-PRO-3 (nuclei). Bar: 50 µm. AU, arbitrary unit; CS, cardiosphere; FN, fibronectin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; PDL, poly-D-lysine; SDC, sphere-derived cell.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 2
Differentiation potentials of secondary cardiospheres. (a) Cells were transduced with eNOS promoter-driven eGFP or VEGFR2 promoter-driven eGFP using lentivirus. Seven days after differentiation induction, secondary CSs expressed GFP but SDCs scarcely expressed. GFP-expressing cells were confirmed to have differentiated into the endothelial lineage by isolectin B4 staining. (b) Cardiomyogenic differentiation was induced by coculture with rat cardiomyocytes. Cells were from the heart of a β-actin promoter-driven eGFP-expressing mouse to distinguish them from rat cardiomyocytes or transduced with α-MHC promoter-driven eGFP reporter to validate cardiac differentiation. Cx43 was densely expressed between secondary CSs and rat cardiomyocytes. Bar: 50 µm. (c) Calcium transients showed the synchronous contractions between secondary CSs from the eGFP-expressing mouse and wild-type rat cardiomyocytes. The ratio of fluorescence intensity, F340/F380, was indicated as calcium transient of each cell. α-MHC, α-myosin heavy chain; CS, cardiosphere; eGFP, enhanced green fluorescent protein; eNOS, endothelial nitric oxide synthase; SDC, sphere-derived cell; VEGFR2, vascular endothelial growth factor receptor 2.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 3
Transplantation of secondary cardiospheres into ischemic myocardium. After MI, we transplanted three types of cells, namely, SDCs, singly dissociated secondary CS cells (2nd-S), secondary CS-hanging drop cells as spherical form (2nd-H). PBS was injected as a control. All types of cells were generated from heart explants of eGFP-expressing mice. (a) Echocardiographic parameters at 14 days after cell transplantation. Left ventricular end-systolic dimension (LVESD), LV end-diastolic dimension (LVEDD), LV fractional shortening (LVFS), and LV ejection fraction (LVEF) (n = 6 per group, *P < 0.01, PBS versus the two transplanted groups; **P < 0.05, 2nd-H group versus SDC and 2nd-S groups). (b) Masson's trichrome staining; 2nd-H transplantation markedly reduced infarct-related fibrosis length (*P < 0.01, 2nd-H group versus the other three groups); 2nd-H transplantation increased infarct thickness more than transplantation with SDCs or 2nd-S (*P < 0.05, PBS versus the SDC and 2nd-S groups; **P < 0.01, the 2nd-H group versus the SDC and 2nd-S groups). Bar: 500 µm. (c) Amplification plots of various sets of known number of female and male cells using Y chromosome-specific primers and probe (right panel). Standard curve to calculate unknown male cell numbers (left panel). (d) Transplantation of male cells to female infarcted heart. Real-time PCR for Y chromosome showed that cell transplantation as spherical form was superior cellular engraftment to single-dissociated cells from secondary CSs and SDCs (n = 4 per group, *P < 0.01, 2nd-H group versus the SDC or 2nd-S groups). (e) Transplantation of GFP-expressing cells to wild-type infarcted heart. Secondary CSs as spherical form (2nd-H group) enhanced engraftment at 7 days after transplantation (n = 6 per group, *P < 0.01, 2nd-H group versus the SDC or 2nd-S groups). No significanct difference was observed between the SDC and 2nd-S groups. Bar: 50 µm. (f) In the peri-infarct area at day 7 post-transplantation, engrafted SDCs and 2nd-S cells did not express cardiac transcription factor Nkx2.5. However, 2nd-H cells expressed Nkx2.5, and some Nkx2.5 (+) cells did not express GFP, suggesting that 2nd-H cells activated endogenous cardiac progenitor cells. Bar: 20 µm. (g) At day 14, transplanted GFP (+) secondary CSs differentiated into the cardiomyocyte (yellow arrow) and the endothelial cell (white arrow). Bar: 20 µm. α-SA, α-sarcomeric actinin; CS, cardiosphere; eGFP, enhanced green fluorescent protein; ILB4, isolectin B4; MI, myocardial infarction; PBS, phosphate-buffered saline; SDC, sphere-derived cell.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 3
Transplantation of secondary cardiospheres into ischemic myocardium. After MI, we transplanted three types of cells, namely, SDCs, singly dissociated secondary CS cells (2nd-S), secondary CS-hanging drop cells as spherical form (2nd-H). PBS was injected as a control. All types of cells were generated from heart explants of eGFP-expressing mice. (a) Echocardiographic parameters at 14 days after cell transplantation. Left ventricular end-systolic dimension (LVESD), LV end-diastolic dimension (LVEDD), LV fractional shortening (LVFS), and LV ejection fraction (LVEF) (n = 6 per group, *P < 0.01, PBS versus the two transplanted groups; **P < 0.05, 2nd-H group versus SDC and 2nd-S groups). (b) Masson's trichrome staining; 2nd-H transplantation markedly reduced infarct-related fibrosis length (*P < 0.01, 2nd-H group versus the other three groups); 2nd-H transplantation increased infarct thickness more than transplantation with SDCs or 2nd-S (*P < 0.05, PBS versus the SDC and 2nd-S groups; **P < 0.01, the 2nd-H group versus the SDC and 2nd-S groups). Bar: 500 µm. (c) Amplification plots of various sets of known number of female and male cells using Y chromosome-specific primers and probe (right panel). Standard curve to calculate unknown male cell numbers (left panel). (d) Transplantation of male cells to female infarcted heart. Real-time PCR for Y chromosome showed that cell transplantation as spherical form was superior cellular engraftment to single-dissociated cells from secondary CSs and SDCs (n = 4 per group, *P < 0.01, 2nd-H group versus the SDC or 2nd-S groups). (e) Transplantation of GFP-expressing cells to wild-type infarcted heart. Secondary CSs as spherical form (2nd-H group) enhanced engraftment at 7 days after transplantation (n = 6 per group, *P < 0.01, 2nd-H group versus the SDC or 2nd-S groups). No significanct difference was observed between the SDC and 2nd-S groups. Bar: 50 µm. (f) In the peri-infarct area at day 7 post-transplantation, engrafted SDCs and 2nd-S cells did not express cardiac transcription factor Nkx2.5. However, 2nd-H cells expressed Nkx2.5, and some Nkx2.5 (+) cells did not express GFP, suggesting that 2nd-H cells activated endogenous cardiac progenitor cells. Bar: 20 µm. (g) At day 14, transplanted GFP (+) secondary CSs differentiated into the cardiomyocyte (yellow arrow) and the endothelial cell (white arrow). Bar: 20 µm. α-SA, α-sarcomeric actinin; CS, cardiosphere; eGFP, enhanced green fluorescent protein; ILB4, isolectin B4; MI, myocardial infarction; PBS, phosphate-buffered saline; SDC, sphere-derived cell.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 4
Long-term benefits of secondary cardiosphere transplantation. Secondary CS transplantation in spherical form (2nd-H group) prevents detrimental cardiac remodeling and sustains beneficial effects in the long-term. (a) Echocardiographic parameters at 10 weeks (n = 5 per group, *P < 0.05, PBS versus all cell-transplanted groups; **P < 0.05, 2nd-H group versus SDC and 2nd-S groups). (b) Masson's trichrome staining (*P < 0.05, 2nd-H group versus the other three groups). Bar: 1,000 µm. 2nd-H, secondary CS-hanging drop cells as spherical form; 2nd-S, singly dissociated secondary CS cells; CS, cardiosphere; LVEDD, left ventricular end-diastolic dimension; LVEF, LV ejection fraction; LVESD, LV end-systolic dimension; LVFS, LV fractional shortening; PBS, phosphate-buffered saline; SDC, sphere-derived cell.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 5
The paracrine effects of secondary cardiospheres. (a) Microarray profiles of angiovasculogenic cytokines; 0 hour indicates SDCs before being introduced to secondary CS by a hanging-drop. (b) Real-time PCR of a representative cytokine, VEGF, of neovascularization. (c) The secretion of VEGF protein into culture supernatant after sphere formation as determined by ELISA (n = 4; *P < 0.01, 0 hour versus 24 or 48 hours after sphere formation). (d) Three days after MI and cell transplantation, tissue VEGF mRNA and protein expression was measured by real-time PCR and western blot (n = 3; *P < 0.01). (e) Colocalization with injected GFP (+) cells and VEGF expression at day 3. The GFP (+) transplanted cells were colocalized with VEGF (left panel) and also observed around native GFP(−) capillaries (right panel). (f) Capillary density at day 14. Secondary CS transplantation further enhanced neoangiogenesis (n = 4; *P < 0.05, PBS versus SDC; **P < 0.05, SDC versus 2nd CS). Isolectin B4 (ILB4) for capillary. Bar: 50 µm. α-SA, α-sarcomeric actinin; AU, arbitrary unit; bFGF, basic fibroblast growth factor; CS, cardiosphere; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IL, interleukin; MI, myocardial infarction; PBS, phosphate-buffered saline; SDC, sphere-derived cell; VEGF, vascular endothelial growth factor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 5
The paracrine effects of secondary cardiospheres. (a) Microarray profiles of angiovasculogenic cytokines; 0 hour indicates SDCs before being introduced to secondary CS by a hanging-drop. (b) Real-time PCR of a representative cytokine, VEGF, of neovascularization. (c) The secretion of VEGF protein into culture supernatant after sphere formation as determined by ELISA (n = 4; *P < 0.01, 0 hour versus 24 or 48 hours after sphere formation). (d) Three days after MI and cell transplantation, tissue VEGF mRNA and protein expression was measured by real-time PCR and western blot (n = 3; *P < 0.01). (e) Colocalization with injected GFP (+) cells and VEGF expression at day 3. The GFP (+) transplanted cells were colocalized with VEGF (left panel) and also observed around native GFP(−) capillaries (right panel). (f) Capillary density at day 14. Secondary CS transplantation further enhanced neoangiogenesis (n = 4; *P < 0.05, PBS versus SDC; **P < 0.05, SDC versus 2nd CS). Isolectin B4 (ILB4) for capillary. Bar: 50 µm. α-SA, α-sarcomeric actinin; AU, arbitrary unit; bFGF, basic fibroblast growth factor; CS, cardiosphere; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IL, interleukin; MI, myocardial infarction; PBS, phosphate-buffered saline; SDC, sphere-derived cell; VEGF, vascular endothelial growth factor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

10. Figure 6
Molecular mechanisms of sphere formation. (a) Primary CS and secondary CS highly expressed E-selectin, but SDC did not. Gene expressions were measured by RT-PCR and protein expressions were confirmed by immunofluorescent staining. (b) The cells from E-selectin KO cardiac explants were not able to generate CSs. CD44 (a ligand of E-selectin) is expressed in both KO and WT mice. Bar: 200 µm. (c) Western blots at 12 hours after sphere formation demonstrated that E-selectin expression was induced following sphere formation. Blocking ERK pathway (E) suppressed E-selectin expression, but blocking AKT (A) pathway did not. When ERK was blocked, Sp1 was downregulated. (d) After 24 hours, ERK inhibition significantly inhibited VEGF expression, indicating VEGF participates in the later stages of sphere formation (*P < 0.01, ERK block versus the WT and versus an AKT block). (e) Signal pathway blocking experiments demonstrated that the ERK pathway is responsible for sphere initiation and maturation. Average sphere areas were measured using pixel values (*P < 0.01, ERK block versus the WT; **P > 0.05, AKT block versus the WT). Bar: 200 µm. (f) Sphere maturation at 48 hours was markedly decreased by siRNA, targeting VEGF (*P < 0.01, control siRNA versus VEGF siRNA). Bar: 200 µm. AU, arbitrary unit; CS, cardiosphere; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KO, knock-out; RT-PCR, reverse transcription-PCR; SDC, sphere-derived cell; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor; WT, wild-type.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

11. Figure 7
The concept of secondary cardiospheres for repairing damaged heart. ERK/E-selectin and ERK/Sp1/VEGF signaling cascades underlie the molecular mechanisms of sphere initiation and maturation. The transplantation of secondary CSs enhanced cellular engraftment, cardiovascular differentiation, and humoral factor secretion. CS, cardiosphere; SDC, sphere-derived cell; VEGF, vascular endothelial growth factor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions