Fusion-independent expression of functional ACh receptors in mouse mesoangioblast stem cells contacting muscle cells

Abstract

Mesoangioblasts are vessel-associated fetal stem cells that can be induced to differentiate into skeletal muscle, both in vitro and in vivo. Whether this is due to fusion or to transdifferentiation into bona fide satellite cells is still an open question, for mesoangioblasts as well as for other types of stem cells. The early steps of satellite cell myogenic differentiation involve MyoD activation, membrane hyperpolarization and the appearance of ACh sensitivity and gap junctional communication. If mesoangioblasts differentiate into satellite cells, these characteristics should be observed in stem cells prior to fusion into multinucleated myotubes. We have investigated the functional properties acquired by mononucleated green fluorescent protein (GFP)-positive mesoangioblasts co-cultured with differentiating C2C12 myogenic cells, using the patch-clamp technique. Mesoangioblasts whose membrane contacted myogenic cells developed a hyperpolarized membrane resting potential and ACh-evoked current responses. Dye and electrical coupling was observed among mesoangioblasts but not between mesoangioblasts and myotubes. Mouse MyoD was detected by RT-PCR both in single, mononucleated mesoangioblasts co-cultured with C2C12 myotubes and in the total mRNA from mouse mesoangioblasts co-cultured with human myotubes, but not in human myotubes or stem cells cultured in isolation. In conclusion, when co-cultured with muscle cells, mesoangioblasts acquire many of the functional characteristics of differentiating satellite cells in the absence of cell fusion, strongly indicating that these stem cells undergo transdifferentiation into satellite cells, when exposed to a myogenic environment.

Figure 1. Mesoangioblasts contacting C2C12 myotubes respond to ACh

A, representative field of a co-culture of mesoangioblasts (GFP-positive, white cells) and C2C12 myotubes, as viewed under transmitted light and epifluorescence simultaneously. Arrowhead points to a binucleated mesoangioblast, arrows to mononucleated cells. The patch pipette is on the right. B, the mesoangioblast marked a is isolated, that marked b contacts a myotube through the process indicated by the arrow. Right panel, cell a does not respond to ACh (10 μm, black line), cell b shows an inward current (downward deflection). Holding potential, −70 mV. C, histogram of membrane resting potential of mesoangioblasts cultured alone or with C2C12 myotubes. Values were obtained with a CsCl-based intracellular solution, immediately after membrane rupture.

Figure 2. Mesoangioblasts are connected through gap junctions among them but not to C2C12 myotubes

A, mesoangioblast a (patched cell, patch pipette on the right, out of focus) is dye-coupled to mesoangioblast b but not to C2C12 myoblast c. The GFP-positive cells are green when simultaneously viewed under phase contrast and epifluorescence (top). At 7 min after membrane patch rupture, cell b but not cell c shows a strong Alexa Fluor 594 epifluorescence signal (in red, bottom panel). B, time course of dye loading in four mesoangioblast pairs, including that shown in A (•) at pH 7.3 or 5.3. C none of the GFP-positive mesoangioblasts (green) juxtaposed to a myotube became loaded by Alexa Fluor 594 within 12 min of myotube dialysis through the patch pipette (red halo on the right, bottom). D, the mesoangioblast indicated by the arrowhead in C is not coupled to the other mesoangioblasts. Picture taken 38 min after membrane patch rupture (patch pipette on the right). The myotube was still fluorescent. E, capacitative transient (grey trace), superimposed with the best fitting exponential curve (black line, τ = 0.22 ms) and ACh-response (30 μm) in the same cell as in D. Holding potential, −70 mV. All pseudocolor images were obtained digitally.

Figure 3. Nicotinic responses of mesoangioblasts co-cultured with C2C12 myotubes

A, responses to ACh (10 μm) and nicotine (100 μm) in the same mesoangioblast. B, currents evoked by ACh (10 μm) in a mesoangioblast, before (left) and after (right) lifting the cell. C, ACh dose–response relationships in mesoangioblasts (□, n = 3–7 cells) and C2C12 myotubes (▪, n = 4 cells), best fitted to Hill equations (lines). Inset: ACh-currents at the indicated concentrations in a single mesoangioblast. Note the acceleration in current decay increasing ACh concentrations. D, ACh-current response versus test potential (TP) in mesoangioblasts (□, n = 2–7 cells) and C2C12 myotubes (▪, n = 3 cells), with linear best fits. Inset: ACh-currents at the indicated test potentials in a mesoangioblast. Note the clear outward current evoked at +30 mV. ACh, 30 μm. E, responses to ACh (10 μm) in a mesoangioblast under whole-cell recording condition (left) and after excising an outside-out patch (right). In all panels, holding potential, −70 mV; current digital filtering, 200 Hz. Data in C and D: mean ±s.e.m., with error bars omitted if n = 2 or if smaller than symbol.

Figure 4. Unitary ACh-evoked events in mesoangioblasts

A, channel openings recorded in an outside-out patch from a mesoangioblast, superfused with ACh (10 μm). Holding potential, −70 mV. B amplitude and open time distributions of the events from the same patch. Superimposed lines: best fitting Gaussian, with mean = 2.7 pA and exponential curve, with time constant, τ = 2.1 ms. Single channel conductance, estimated considering a reversal potential of +4 mV for ACh-current, was 36.5 pS.

Figure 5. Expression of mRNAs for MyoD and nAChR α1 subunit in mesoangioblasts cultured with myogenic cells

A, RT-PCR with mouse specific oligonucleotides for the α1 subunit of the muscle nAChR and MyoD, as indicated, amplified from the RNA extracted from co-cultures of mouse mesoangioblasts and human myogenic cells (a and e), from cultures of mouse mesoangioblasts alone (b and f) or human myogenic cells alone (c and g) and from mouse C2C12 myotubes (d and h), as positive control. Molecular markers are shown in the middle. B, typical mononucleated mesoangioblast selected for single-cell RT-PCR analysis. C, results of nested RT-PCR amplifications with oligonucleotides for GAPDH (G) and MyoD (M) on a single C2C12 myotube, a non-responsive and an ACh-responsive mesoangioblast (top) and summary of all the tested cells (bottom).