From Hair to Heart: Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Can Simultaneously Differentiate Into Mature Cardiomyocytes and Atrial Myocytes

Hair-follicle-associated pluripotent (HAP) stem cells express nestin, and are located in the bulge area of hair follicles and can differentiate to numerous types of cells. In the present study, we demonstrate that rat HAP stem cells simultaneously differentiated to mature cardiomyocytes and atrial myocytes. The addition of isoproterenol, activin A, bone morphogenetic protein 4 (BMP 4), basic broblast growth factor (bFGF), and cyclosporin A (CSA), induced simultaneously differentiation of HAP stem cells to c-kit-positive cardiomyocytes and MLC-2a-expressing atrial myocytes. The results of the present study suggest that HAP stem cells differentiating to cardiomyocytes and atrial myocytes have future clinical potential for heart regeneration.


Introduction
Cardiomyocytes are a major target in regenerative medicine. In recent years, there are intense efforts have been made to induce cardiomyocyte differentiation from embryonic stem (ES) cells and induced pluripotent stem cells (iPSC). However, there are major issues in terms of maturation and functionality of the differentiated cardiomyocytes derived from these two types of stem cells. [1,2,3]. Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells are found in the hair-follicle bulge area and can differentiate into neurons, glia, keratinocytes, smooth muscle cells, melanocytes, and beating cardiac muscle cells [4,5,6,7,8]. HAP stem cells from mice have been used to repair the severed sciatic nerve in mouse models [9,10,11,12,13,14]. The implanted HAP stem cells differentiated into Schwann cells in the re-joined nerve and restored nerve and leg function. HAP stem cells have been used to repair the severed spinal cord in mice leading to improved hind limb locomotion [10,15]. Isoproterenol stimulated mouse HAP stem cells to differentiate to cardiac-muscle cells in large numbers in culture. The addition of activin A, BMP4, and bFGF, along with isoproterenol, stimulated the cardiac-muscle cells to form tissue sheets of beating heart muscle cells [5]. In the present study, we demonstrate that rat HAP stem cells can simultaneously differentiate into mature cardiomyocytes and atrial myocytes with regenerative potential.

F344 Rat
Four weeks old F344/jcl female rat (50 g or more) (CLEA Japan, Tokyo, Japan) were used to isolate vibrissa hair follicles. The experimental animals were kept in an animal housing system maintained at 24 ± 1℃, relative humidity of 50-60%, and 14 hours of light 10 hours of dark intervals. The experimental protocol was approved by the Kitasato University School of Medicine Animal Care Committee (No. 2020035). The study was conducted according to the ARRIVE guidelines for the reporting of animal experiments. All methods complied with the guidelines for the proper conduct of animal experiments of the Science Council of Japan.
Isolation and division of rat vibrissa hair follicles.
Isolation and division of rat vibrissa hair follicles were performed as previously reported [22]: For isolation of vibrissa hair follicles from the F344/jcl rats, the animals were anesthetized with a combination of 0.375 mg/kg medetomidine, 2.0 mg/kg midazolam and 2.5 mg/kg butorphanol. The upper lip containing the vibrissa pad was cut, and inner surface was exposed. All vibrissa hair follicles were gently pulled out from the pad, one by one, with ne forceps, under a binocular microscope. The tissues around the isolated hair follicles were removed, and the hair follicles were divided into thirds. All surgical procedures were performed in a sterile environment.

Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was extracted from 21day cardiomyocytes using the RNeasy Plus Mini Kit (#74134, Qiagen, Hilden, Germany) and then cDNA were synthesized with QuantiTect® Reverse Transcription (#205311, Qiagen) according to the manufacturer's instructions. GAPDH was used to normalize gene expressions. Quantitative PCR was performed using the Power SYBR® Green PCR Master mix (#4367659, Applied Biosystems, Waltham, MA, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and analyzed by the Delta Delta Ct method. Forward and reverse primer sequences are shown in Table. 1.

Ca 2+ Imaging
Intracellular calcium imaging was performed as previously reported [5]:

Statistical Analysis
The experimental data are expressed as the mean SD. Statistical analyses were performed with the unpaired Student's t-test. A probability (P) value of p ≤ 0.05 was considered signi cant.
Beating cardiomyocytes differentiated from HAP stem cells showed regular amplitude of intracellular calcium levels Intracellular Ca 2+ concentration changes were associated with beating of the cardiomyocytes differentiated from rat HAP stem cells (Fig. 3). Phase-contrast microscopy of the cardiomyocytes loaded with the calcium-sensitive dye Fluo 4-AM showed time-dependent intensity changes (S2 Movie).

Ultrastructural analysis of HAP stem-cell-derived mature cardiomyocytes
We con rmed features of cardiomyocytes differentiated from HAP stem cells at the ultrastructural level using transmission electron microscopy. Rat HAP stem cells cultured in supplemented medium, derived cardiomyocytes resembled native cardiomyocytes, showing myo brillar bundles with regularly aligned Zbands, A-and I-bands with a H-zone, enriched mitochondria, and T-tubule-like structures (Fig. 4).

Discussion
In the present report, we demonstrate that rat HAP stem cells cultured with isoproterenol, activin A, BMP 4, bFGF, and CSA can simultaneously differentiate into cardiomyocytes and atrial myocytes. Yan et al. previously demonstrated that addition of an immunosuppressant, cyclosporin-A (CSA), to mesoderm cells expanded cardiac progenitor cells and cardiomyocytes [16]. In the present report, cardiomyocytes differentiated from HAP stem cells were observed to contain T-tubules, sarcomere structures, and mitochondria, which are essential for mature cardiomyocytes [17,18]. Furthermore, cardiomyocytes differentiated from rat HAP stem cells showed intracellular Ca 2+ increasing with autonomous beating with a regular amplitude. Borysova et al. previously reported that right-atrial-appendage biopsy specimens from rats showed regular Ca 2+ waves of approximately every 0.2 seconds [19], suggesting that cardiomyocytes differentiated from rat HAP stem cells were electrophysiological similar to rat adult atrial muscle. Cardiomyocytes differentiated from HAP stem cells were c-kit-positive. c-kit is a cardiac stem cell marker, and c-kit-positive cardiac stem cells are abundant in the atrial muscle in humans [20]. Based on this evidence, we consider that c-kit-positive atrial myocytes differentiated from HAP stem cells have characteristics of cardiac stem cells. HAP stem cells also differentiated to MLC-2a expressing atrial myocytes. In the present study, we succeeded in simultaneously inducing c-kit-positive cardiomyocytes and MLC-2a-expressing atrial myocytes from HAP stem cells. Atrial myocytes differentiated from HAP stem cells have characteristics of both cardiac stem cells and adult cardiomyocytes, and may be useful for regeneration of damaged myocardium. The possibility of clinical use of HAP stem cells for cardiac regeneration is feasible and practical, since HAP stem cells are readily available from everyone and can be cryopreserved and banked without loss of pluripotency [8,21]. Tables   Table. 1     Scheme for the simultaneous differentiation of cardiomyocytes and atrial myocytes from HAP stem cells.

Declarations
HAP stem cells were initially cultured from the upper part of the rat vibrissa hair follicle (box in cartoon) in 10% FBS-DMEM supplemented with iso-proterenol, activinA, bone morphogenetic protein 4 (BMP4) and basic broblast growth factor (bFGF) for 7 days, which was changed to 10% FBS-DMEM supple-mented with cyclosporin A (CSA) for 14 days. (Total 21 days)

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. S1.movie.mp4 S2movienonsupplemented.avi S2moviesupplemented.avi