Non-canonical Wnt signaling enhances differentiation of Sca1+/c-kit+ adipose-derived murine stromal vascular cells into spontaneously beating cardiac myocytes

Abstract

Recent reports have described a stem cell population termed stromal vascular cells (SVCs) derived from the stromal vascular fraction of adipose tissue, which are capable of intrinsic differentiation into spontaneously beating cardiomyocytes in vitro. The objective of this study was to further define the cardiac lineage differentiation potential of SVCs in vitro and to establish methods for enriching SVC-derived beating cardiac myocytes. SVCs were isolated from the stromal vascular fraction of murine adipose tissue. Cells were cultured in methylcellulose-based murine stem cell media. Analysis of SVC-derived beating myocytes included Western blot and calcium imaging. Enrichment of acutely isolated SVCs was carried out using antibody-tagged magnetic nanoparticles, and pharmacologic manipulation of Wnt and cytokine signaling. Under initial media conditions, spontaneously beating SVCs expressed both cardiac developmental and adult protein isoforms. Functionally, this specialized population can spontaneously contract and pace under field stimulation and shows the presence of coordinated calcium transients. Importantly, this study provides evidence for two independent mechanisms of enriching the cardiac differentiation of SVCs. First, this study shows that differentiation of SVCs into cardiac myocytes is augmented by non-canonical Wnt agonists, canonical Wnt antagonists, and cytokines. Second, SVCs capable of cardiac lineage differentiation can be enriched by selection for stem cell-specific membrane markers Sca1 and c-kit. Adipose-derived SVCs are a unique population of stem cells that show evidence of cardiac lineage development making them a potential source for stem cell-based cardiac regeneration studies.

Figure 1. Microscopic analysis of SVC-derived cardiac myocytes during in vitro differentiation

Adipose tissue derived SVCs were plated in methylcellulose media (GF M3534) and observed during the course of in vitro differentiation. Images were acquired at 10x magnification under a phase contrast microscope showing colonies which have differentiated to day 5 (A), day 10 (B), and day 15 (C). Spontaneous beating was observed as early as day 7 when colonies grew into an intercalated syncitium. Bar = 75 μm.

Figure 2. Western blot of sarcomeric proteins in SVCs isolated after 14 days of in vitro differentiation

Acutely isolated adipose derived SVCs were cultured in methylcellulose media (GF M3534) and allowed to differentiate for two weeks. Western blot was performed after microdissection of spontaneously beating colonies. Western blot controls include adult mouse heart, soleus (slow skeletal muscle), tibialis anterior (TA; fast skeletal muscle), adult rabbit heart, and embryonic mouse heart. By western blot, SVC-derived cardiac myocytes express slow skeletal troponin I, cardiac troponin I, α-sarcomeric actin, tropomyosin, and cardiac troponin T.

Figure 3. Raw data showing calcium transients and contractility in SVC-derived cardiac myocytes

After two weeks of in vitro differentiation, beating colonies of differentiated SVCs were loaded with the calcium indicator FURA 2AM in M199+. Using the Ionoptix imaging system colonies were analyzed for calcium transients during field stimulation at 80V and a rate of 0.5 Hz (A) or derived during spontaneous beating (B). Simultaneously, contractility was measured by edge detection during field stimulation (C). During analysis by edge detection, spontaneously beating SVC-derived cardiac myocytes were pharmacologically manipulated by acute addition of 2 mM muscarine chloride (arrow) (D).

Figure 4. Kinetics of calcium regulation in SVC-derived cardiac myocytes paced and spontaneously beating compared to murine cardiac myocytes

These data show that paced SVC-derived myocytes, spontaneously beating SVC-derived myocytes, and adult mouse myocytes have significantly different calcium transient kinetics including rate of calcium sequestration at time points of 50%, 75%, and 90% return to baseline normalized to time of calcium transient peak (A), baseline calcium (B), and calcium transient amplitude (C). Spontaneously beating SVC-derived myocytes (black bars), n=44; paced SVC-derived myocytes (white bars), n= 150; Mouse cardiac myocytes (gray bars), n=39. * = P<0.001 paced SVCs vs. mouse myocytes; # = P<0.001 spontaneously beating SVCs vs. mouse myocytes; + = P<0.001 spontaneously beating SVCs vs. paced SVCs. All values are mean ± s.e.m. Data were analyzed by one way ANOVA with Newman Keul’s post-hoc test.

Figure 5. Treatment of cultured SVCs with recombinant Wnt proteins and cytokines

(A) Acutely isolated adipose derived SVCs were cultured in methylcellulose media (GF M3534) with the addition of recombinant proteins including the non-canonical Wnt agonist Wnt5a (concentrations: 10 ng/mL, 20 ng/mL, 40 ng/mL, and 80 ng/mL), the canonical Wnt antagonist Dickkopf 1 (Dkk1) (concentrations: 40 ng/mL and 80 ng/mL), Wnt5a/Dkk1 together (50ng/mL of each protein), or the canonical Wnt agonist Wnt3a (80 ng/mL). * = P < 0.05 and ** = P < 0.01 compared to control. (B) Acutely isolated SVCs were cultured in cytokine replete minimum media (GF M3234) or complete media (GF M3534) enriched with cytokines Il-3 (10 ng/mL), Il-6 (10 ng/mL), and stem cell factor (SCF) (50 ng/mL). n = 5 cultures from 2 or more preparations. * = P < 0.05. All values are mean ± s.e.m. Data were analyzed by student’s t-test.

Figure 6. Analysis of Sca1 and c-kit selected SVCs

Flow cytometry analysis of purified SVCs prior to Sca1 selection (A), after three rounds of Sca1 selection (B), prior to c-kit selection (C), and after three rounds of c-kit selection (D). Comparison between Sca1⁺, Sca1⁻, and non-selected SVCs (E) as well as c-kit⁺, c-kit⁻, and non-selected SVCs (F) to form SVC-derived spontaneously beating cardiac myocytes after two weeks of in vitro differentiation. n = 5 cultures from 3 or more preparations. * = P < 0.01 for positive selected cells compared to negative selected cells and unselected cells. All values are mean ± s.e.m. Data were analyzed by student’s t-test.