Tissue engineering the mechanosensory circuit of the stretch reflex arc with human stem cells: Sensory neuron innervation of intrafusal muscle fibers

Abstract

Muscle spindles are sensory organs embedded in the belly of skeletal muscles that serve as mechanoreceptors detecting static and dynamic information about muscle length and stretch. Through their connection with proprioceptive sensory neurons, sensation of axial body position and muscle movement are transmitted to the central nervous system. Impairment of this sensory circuit causes motor deficits and has been linked to a wide range of diseases. To date, no defined human-based in vitro model of the proprioceptive sensory circuit has been developed. The goal of this study was to develop a human-based in vitro muscle sensory circuit utilizing human stem cells. A serum-free medium was developed to drive the induction of intrafusal fibers from human satellite cells by actuation of a neuregulin signaling pathway. Both bag and chain intrafusal fibers were generated and subsequently validated by phase microscopy and immunocytochemistry. When co-cultured with proprioceptive sensory neurons derived from human neuroprogenitors, mechanosensory nerve terminal structural features with intrafusal fibers were demonstrated. Most importantly, patch-clamp electrophysiological analysis of the intrafusal fibers indicated repetitive firing of human intrafusal fibers, which has not been observed in human extrafusal fibers.

Keywords: Human; Intrafusal fiber; In vitro; Proprietary sensory circuit; Serum-free medium; Stem cells.

Fig. 1

Schematic diagram of the culture protocol and timeline.

Fig. 2

Phase contrast micrographs. A) A bag fiber in an induced culture after 16 days of differentiation. Note the equatorial distribution of multiple nuclei in the bag-shaped myofiber. Scale bar: 20 μm. B) A chain fiber in an induced culture after 16 days of differentiation. Note the linear assembly of the nuclei inside the myotube. Scale bar: 20 μm. C) A low magnification image of an induced culture after 17 days of differentiation. Scale bar: 80 μm. D) Graph comparison of the percentage of bag fibers when NRG/LMN/FN was added to the culture at different times during differentiation (* means P < 0.05).

Fig. 3

A&B) Immunocytochemistry of induced intrafusal muscle cultures stained with the bag fiber-specific antibody S46 (green), and co-stained with Phalloidin (red) which is a general marker for all myofibers. A) A representative bag fiber is highlighted with a yellow arrow and a blue arrow indicates a myofiber that was partially stained by S46 but did not present apparent bag morphology, suggests the possibility of a chain fiber or an emerging bag fiber formation. B) An image of a bag-fiber at higher magnification. C) A bag fiber immunostained with the BA-G5 antibody.

Fig. 4

Activation of Neuregulin signaling pathway demonstrated by immunocytochemistry. A) Co-immunostaining of Phalloidin and erbB2-Ⓟ. To visualize the erbB2-Ⓟ clusters on the cell membrane, two regions of the low magnification image were enlarged. Image a′ and b′ are the higher magnifications of regions a and b respectively. Abundant erbB2-Ⓟ signals (indicated by arrows) were observed only on multi-nuclei bag fibers (b and b′), and rarely observed on the others (a and a′). B) Immunostaining of Egr3 co-stained with S46. Egr3-positivity was only observed in S46-positive myofibers, confirming its specificity. C) A bag fiber under higher magnification.

Fig. 5

Immunocytochemical analysis of the co-culture of human sensory neurons and intrafusal fibers indicated connectivity. A) Phase image of a Day 10 co-culture. Note the pseudo-unipolar sensory neuron (orange arrow) and bipolar sensory neuron (red arrow) in the vicinity of a bag-shaped myofiber. B&C) Co-immunostaining of Peripherin and BA-G5 revealed two typical sensory terminal structures around intrafusal fibers: annulospiral wrappings (B) and flower spray endings (C), as indicated by the arrows in both images.

Fig. 6

Optical section of a sensory terminal structure indicating annulospiral wrappings on intrafusal fibers immunostained with Peripherin and BA-G5. A) Projected image of an intrafusal fiber with sensory axons. B) Series of optical sections of the intrafusal fiber demonstrated in A).

Fig. 7

Patch clamp recording from intrafusal fibers in the co-culture system. A) Phase micrograph of the recorded cell. B) Current clamp recording indicating repetitive firing of APs. C) An example trace of active Na⁺ and K⁺ currents from a voltage clamp recording. D) An example trace of an Action Potential (AP) elicited when the cell received a saturated stimulus (2 msec 200 pA inward current).

Fig. 8

Immunocytochemical analysis of the connection of human sensory neurons and intrafusal fibers from a day 5 co-culture by co-immunostaining with PICK1 and Neuro-filament antibodies. Co-localization of PICK1 expression on the myofiber at the neural terminal ending site is indicated by the arrow.