A Hyper-Crosslinked Carbohydrate Polymer Scaffold Facilitates Lineage Commitment and Maintains a Reserve Pool of Proliferating Cardiovascular Progenitors

Abstract

Background: Cardiovascular progenitor cells (CPCs) have been cultured on various scaffolds to resolve the challenge of cell retention after transplantation and to improve functional outcome after cell-based cardiac therapy. Previous studies have reported successful culture of fully differentiated cardiomyocytes on scaffolds of various types, and ongoing efforts are focused on optimizing the mix of cardiomyocytes and endothelial cells as well as on the identification of a source of progenitors capable of reversing cardiovascular damage. A scaffold culture that fosters cell differentiation into cardiomyocytes and endothelial cells while maintaining a progenitor reserve would benefit allogeneic cell transplantation.

Methods: Isl-1 + c-Kit + CPCs were isolated as clonal populations from human and sheep heart tissue. After hyper-crosslinked carbohydrate polymer scaffold culture, cells were assessed for differentiation, intracellular signaling, cell cycling, and growth factor/chemokine expression using real time polymerase chain reaction, flow cytometry, immunohistochemistry, and calcium staining.

Results: Insulin-like growth factor 1, hepatocyte growth factor, and stromal cell derived factor 1α paracrine factors were induced, protein kinase B signaling was activated, extracellular signal-regulated kinase phosphorylation was reduced and differentiation into both cardiomyocytes and endothelial cells was induced by scaffold-based cell culture. Interestingly, movement of CPCs out of the G1 phase of the cell cycle and increased expression of pluripotency genes PLOU5F1 (Oct4) and T (Brachyury) within a portion of the cultured population occurred, which suggests the maintenance of a progenitor population. Two-color immunostaining and 3-color fluorescence-activated cell sorting analysis confirmed the presence of both Isl-1 expressing undifferentiated cells and differentiated cells identified by troponin T and von Willebrand factor expression. Ki-67 labeling verified the presence of proliferating cells that remained in situ alongside the differentiated functional derivatives.

Conclusions: Cloned Isl-1 + c-kit + CPCs maintained on a hyper-cross linked polymer scaffold retain dual potential for proliferation and differentiation, providing a scaffold-based stem cell source for transplantation of committed and proliferating cardiovascular progenitors for functional testing in preclinical models of cell-based repair.

FIGURE 1

CPCs express markers of early cardiogenic mesoderm.^- CPCs used for experiments uniquely express markers of early cardiogenic mesoderm. The expression of markers of early cardiogenic mesoderm was assessed using flow cytometry (A-B). Notably, 2-color FACS analysis indicates the co-expression of Isl-1 and c-kit (B; negative population inset). We visualized amplicons of genes of interest using gel electrophoresis to verify this expression (C). CPCs express Mesp-1, Bry, Isl-1, c-kit, and PDGFRα. To confirm the multipotency of the CPCs, 10 nM of dexamethasone was used to induce the development of cardiomyocytes (TropT and TropI), endothelium (vWF), and smooth muscle (SMA). Antibodies against these proteins were measured using flow cytometry and reported as the change in mean fluorescence intensity compared with undifferentiated CPCs (D). As shown in panel E, mesoderm differentiates into cardiogenic mesoderm, which has the capacity to become several types of cardiac cells, including cardiomyocytes, endothelium, and smooth muscle. These human cardiac progenitors may give rise to sinoatrial nodal cells; however, the timing and location of specification is currently undetermined. Values represent the mean ± S.E.M. n = 7.

FIGURE 2

Scaffold-cultured CPCs exhibit a reduced G1 phase and markers of differentiation. Human-derived CPCs were scaffold-cultured. Two- and 3-color FACS analysis (A-C) indicated the expression of only Isl1 under control culture conditions (red), but of Isl1 along with TropT and vWF under scaffold culture conditions (blue). Populations were defined using unstained cells (grey). Flow cytometry verified the expression of TropT and vWF (D, n = 5, P < 0.001). RT-PCR indicated a significant 52-fold and 27-fold increase in the expression of TNNT2 (TropT) and MLC2V, respectively (E, n = 3, P < 0.001). As indicated by FACS analysis, we also measured a 70-fold increase in POU5F1 (OCT4) expression (E, n = 3, P < 0.001) and a 69-fold increase in T (Bry) expression (E, n = 3, P < 0.05). To determine whether scaffold-cultured CPCs maintained their proliferative potential, we performed cell cycle analysis, which indicated a punctuated G1 phase (F, n = 3, P < 0.05). Mean values are reported, while error bars represent the S.E.M. *P < 0.05, n = 3; ***P < 0.001, n = 4.

FIGURE 3

After scaffold culturing, cells display rhythmic pattern of calcium flux. After incubation with Fluo4-AM, a calcium dye, scaffold-cultured CPCs were imaged. Regions of interest were drawn around cells using ImageJ and the intensity of fluorescence in each region of interest was quantified for each frame. The scaffold-cultured CPCs exhibit an auto-rhythmic trafficking of calcium, which is a feature of mature cardiomyocytes, whereas control CPCs do not (A; n = 5-7). Representative frames for sequential time points are shown for control (B) and scaffold-cultured (C) CPCs.

FIGURE 4

ERK phosphorylation decreases and AKT phosphorylation exhibits an increasing trend after scaffold culturing. Fluorophore-conjugated antibodies were used to detect the concentrations of (A) phosphorylated ERK1/2 (P-ERK+) and (B) phosphorylated AKT (P-AKT+) in cultured patient-derived CPCs. The phosphorylation of ERK1/2 was significantly reduced while the phosphorylation of AKT generally increased in the scaffold-cultured CPCs. Values represent the mean ± SEM. *P < 0.05, n = 4.

FIGURE 5

Scaffold-cultured progenitors show elevated expression of IGF-1, HGF, and SDF-1α. The expression of cytokines in human CPCs was assessed after culturing under scaffold or control conditions (A). We confirmed the size of the statistically significant products from (A) using a 1% agarose gel (B). Band sizes based upon primer sequences: Actin (130 bp), SDF-1α (226 bp), HGF (239 bp). *P < 0.05, **P< 0.01, n = 3.

FIGURE 6

Ovine-derived CPCs express markers of early cardiogenic mesoderm. The expression of markers of early cardiogenic mesoderm was assessed by visualizing amplicons of genes of interest using gel electrophoresis (A) and by flow cytometry (B). Similar to the profile of human-derived CPCs, gene and protein expression analysis indicates the presence of Bry, Isl-1, and c-kit in ovine CPCs.

FIGURE 7

Scaffold-cultured, ovine-derived CPCs maintain proliferative and stem-like potential while differentiating into cardiac derivatives. Using genetic and protein expression analysis, we measured the coexistence of differentiating and proliferating stem cells. FACS analysis and IHC were used to assess the expression of TropT and vWF. Scaffold-cultured, ovine-derived CPCs express increased levels of TropT (A-C) and significantly increased levels of vWF (D-F). RT-PCR and flow cytometry indicated the increased expression of Isl1 (G-I). Flow cytometry indicated a prostem cell shift in the cell cycle profile of scaffold-cultured ovine CPCs (J). Scaffold-cultured ovine CPCs stained positively for Ki-67, a marker of proliferation (K-L). Interestingly, the scaffold culture of CPCs induced differentiation yet a proportion of cells were maintained that express the stem cell marker Isl-1. *P < 0.05, n = 3; ***P < 0.001, n = 3.

FIGURE 8

Concomitant expression of Isl1, TropT, and vWF in ovine-derived, scaffold-cultured CPCs. In an effort to clarify the presence of both cardiac progenitor and derivative markers, we performed FACS analysis of CPCs cultured for 3 weeks under control (red) and scaffold (blue) conditions (A-C). Unlike control conditions, under which CPCs expressed only Isl1, scaffold conditions fostered both the maintenance of Isl1 as well as the expression of vWF (A) and TropT (B). Unstained cells are shown in grey. To confirm these observations, we performed IHC analysis of serial sections of paraffin-embedded scaffolds that were seeded with ovine-derived CPCs. Secondary only staining is shown in (D). Isl1 and TropT (E) as well as Isl1 and vWF (F) were all observed to be concomitantly expressed.