Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Can Extensively Differentiate to Tyrosine-Hydroxylase-Expressing Dopamine-Secreting Neurons

Hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of hair follicles from mice and humans and have been shown to differentiate to neurons, glia, keratinocytes, smooth muscle cells, melanocytes and beating cardiac muscle cells in vitro. Subsequently, we demonstrated that HAP stem cells could effect nerve and spinal-cord regeneration in mouse models, differentiating to Schwann cells and neurons in this process. HAP stem cells can be banked by cryopreservation and preserve their ability to differentiate. In the present study, we demonstrated that mouse HAP stem cells cultured in neural-induction medium can extensively differentiate to dopaminergic neurons, which express tyrosine hydroxylase and secrete dopamine. These results indicate that the dopaminergic neurons differentiated from HAP stem cells may be useful in the future to improve the symptoms of Parkinson’s disease in the clinic.


Introduction
Parkinson's disease is progressive and decreases the quality of life of the patient over time, and is often causing death. Standard of care is a carbidopa and levodopa combination, which often poorly control the symptoms of Parkinson's disease. Cell transplantation into Parkinson's disease patients to replace the lost dopaminergic neurons of the substantia nigra pars compacta has been studied since the 1980s.
HAP stem cells from mice have been used to repair the severed sciatic nerve in mouse models [7][8][9][10][11][12]. The implanted HAP stem cells differentiated into Schwann cells in the re-joined nerve and restored nerve and leg function. Human HAP stem cells, which have a similar differentiation potential as mouse HAP stem cells, have also been used to restore the structure and function of the severed sciatic nerve in mice [4,5,13]. HAP stem cells have been used to repair the severed spinal cord in mice, leading to improved hindlimb locomotion [8,14]. The implanted HAP stem cells in the re-joined spinal cord differentiated to oligodendrocytes and βIII-tubulin-positive neuron-like cells [15].
In the present study, we demonstrate that mouse HAP stem cells can extensively differentiate to tyrosine-hydroxylase expressing, dopamine-secreting neurons. The dopaminergic neurons differentiated from HAP stem cells have future potential to improve the symptoms of Parkinson's disease, as they can be readily isolated and banked from everyone.

Isolation of Vibrissa Hair Follicles and Induction of Dopaminergic Neurons from HAP Stem Cells In Vitro
Vibrissa hair follicles from mice were isolated as previously reported [16]: To isolate the vibrissa hair follicles from C57BL/6 mice, the animals were anesthetized with a combination anesthetic of 0.75 mg/kg medetomidine, 4.0 mg/kg midazolam and 5.0 mg/kg butorphanol [17] and their upper lip containing the vibrissa pad was cut, and the inner surface was exposed. Intact vibrissa hair follicles were dissected under a binocular microscope and plucked from the pad by pulling them gently by the neck with fine forceps. All surgical procedures were performed in a sterile environment. The upper part of vibrissa hair follicles was separated under a binocular microscope. HAP stem cells from the isolated upper part of the vibrissa hair follicle were initially cultured in fresh DMEM (Sigma, MO, USA) containing 10% fetal bovine serum (FBS), 50 µg/mL gentamicin (GIBCO, NY, USA), 2 mM L-glutamine (GIBCO), 10 mM HEPES (MP Biomedicals, OH, USA) for 7 days [16]. For differentiation to dopaminergic neurons, HAP stem cells growing from the upper part of vibrissa hair follicle were switched to neural-induction medium (STEMdiff Dopaminergic Neuron Differentiation Kit, STEMCELL Technologies, British Columbia, Canada) containing neural-progenitor-cells-induction medium, dopaminergic-neuron-differentiation medium, dopaminergic-neuron-maturation medium-1, and dopaminergic-neuron-maturation medium-2 and cultured according to the instructions ( Figure 1). As a control, HAP stem cells were cultured in non-induction medium (10% FBS DMEM). The cells differentiated from HAP stem cells were prepared for immunofluorescence staining, FACS, and for measurement of dopamine secretion.

Ca 2+ Imaging
Ca 2+ imaging was performed on cells grown in 35 mm glass bottom microwell dishes (MatTek, Ashland, MA, USA) with the calcium-sensitive dye Fluo 4-AM (DOJINDO) 1 µM and AM ester-dissolving reagent Pluronic F-127 (0.04%) (FUJIFILM Wako, Osaka, Japan) in HEPES buffer (NaCl 145 mM; MgCl 2 1 mM; KCl 5 mM; glucose 5.5 mM; CaCl 2 1 mM; HEPES 10 mM; pH 7.4) [2]. Fluo-4 fluorescence images (488 nm excitation) were collected and recorded at 100 frames. After 10 s of image acquisition, ATP was added. We examined real-time movie files of continuously recorded data to assess changes in cell fluorescence that occur in response to ATP stimulation. Fluorescence images were collected and recorded using an LSM 710 microscope System with ZEN software (Carl Zeiss, Oberkochen, Germany).

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 significant.

HAP Stem Cells Differentiate Efficiently to Dopaminergic Neurons
HAP stem cells were cultured in neural-induction medium for 45 days. As a control, HAP stem cells were cultured in non-induction medium for 28 days. HAP stem cell differentiated to βIII tubulin-positive, tyrosine-hydroxylase-positive cells, which secreted dopamine by day 7 after switching to neural-induction medium, indicating they were dopaminergic neurons (Figure 1). Tyrosine-hydroxylase, DAT and Nurr1 in the differentiated dopaminergic neurons were observed by immunofluorescence staining (Figure 2). Fluorescence-activated cell sorting (FACS) analysis showed that the percentage of cells differentiating to dopaminergic neurons in neural-induction medium was 48.90 ± 4.64% compared to 15.53 ± 7.47% in non-induction medium (p = 0.0014) ( Figure 3A). The percentage of cells differentiating to βIII tubulin-positive neurons in neural-induction medium was 62.64% ( Figure 3A) and in non-induction medium it was 15.4% [1].

HAP Stem Cells Differentiated to Dopaminergic Neurons That Extensively Proliferate
HAP stem cells, differentiated to dopaminergic neurons, extensively proliferated in neural-induction medium to 6.67 ± 3.06 × 10 3 cells/hair follicle.

HAP Stem Cells Differentiate Efficiently to Dopaminergic Neurons
HAP stem cells were cultured in neural-induction medium for 45 days. As a control, HAP stem cells were cultured in non-induction medium for 28 days. HAP stem cell differentiated to βIII tubulin-positive, tyrosine-hydroxylase-positive cells, which secreted dopamine by day 7 after switching to neural-induction medium, indicating they were dopaminergic neurons (Figure 1). Tyrosine-hydroxylase, DAT and Nurr1 in the differentiated dopaminergic neurons was observed by immunofluorescence staining (Figure 2). Fluorescence-activated cell sorting (FACS) analysis showed that the percentage of cells differentiating to dopaminergic neurons in neural-induction medium was 48.90 ± 4.64% compared to 15.53 ± 7.47% in non-induction medium (p = 0.0014) ( Figure 3A). The percentage of cells differentiating to βIII tubulin-positive neurons in neural-induction medium was 62.64% ( Figure 3A) and in non-induction medium it was 15.4% [1].

HAP Stem Cells Differentiated to Dopaminergic Neurons Secreted Dopamine at High Levels
HPLC analysis showed that HAP stem cells differentiated to dopaminergic neurons that secreted dopamine at 3.73 ± 0.41 ng/hair follicle and 3,4-dihydroxyphenylacetic acid (DOPAC) at 1.96 ± 0.04 ng/hair follicle in neural-induction medium. Significantly more dopamine was secreted in neural-induction medium compared to non-induction medium (p = 0.0002) ( Figure 3B,C).

HAP Stem Cells Differentiated to Dopaminergic Neurons Have Increased Ca 2+ Levels When Treated with ATP
HAP stem cells differentiated to dopaminergic neurons increased their Ca 2+ levels when treated with 300 µM ATP (Figure 4, Supplementary Materials Video S1).

HAP Stem Cells Differentiated to Dopaminergic Neurons that Extensively Proliferate
HAP stem cells, differentiated to dopaminergic neurons, extensively proliferated in neural-induction medium to 6.67 ± 3.06 × 10 3 cells/hair follicle.

HAP Stem Cells Differentiated to Dopaminergic Neurons Secrete Dopamine at High Levels
HPLC analysis showed that HAP stem cells differentiated to dopaminergic neurons secreted dopamine at 3.73 ± 0.41 ng/hair follicle and 3,4-dihydroxyphenylacetic acid (DO-PAC) at 1.96 ± 0.04 ng/hair follicle in neural-induction medium. Significantly more dopamine was secreted in neural-induction medium compared to non-induction medium (p = 0.0002) ( Figure 3B,C).

HAP Stem Cells Differentiated to Dopaminergic Neurons Have Increased Ca 2+ Levels When Treated with ATP
HAP stem cells differentiated to dopaminergic neurons increased their Ca 2+ levels when treated with 300 µM ATP (Figure 4, Supplementary Materials Video S1).

Discussion
In 2020 a case report was published on a patient with dopamine-producing iPSC derived from the patient's stem cell. The dopamine-producing iPSC transplanted into the putamen of the patient showed stability and improvement [19]. However, it took the next steps to produce the dopamine-producing iPSC cells, inducing forced microRNAs expression, transfect with reprogramming factors, and eliminating progenic and tumorigenic

Discussion
In 2020 a case report was published on a Parkinson's patient transplanted with dopamine-producing iPSC derived from the patient's skin cells. The dopamine-producing iPSC were transplanted into the putamen of the patient who showed stability and improvement [19]. However, it took seven steps to produce the dopamine-producing iPSC, including forced miRNAs expression, transfect with reprogramming factors, and chemical elimination of potential tumorigenic un-differentiated cells [20]. None of these genetic steps to induce differentiation and chemical treatment to remove potential tumorigenic cells are necessary with dopamine-producing HAP stem cells.
The present report demonstrates that HAP stem cells can efficiently differentiate to dopaminergic neurons, which proliferate well and secrete high amounts of dopamine. Furthermore, HAP stem cells matured as neurons when cultured in neural induction medium demonstrated by increased levels of Ca 2+ upon treatment with ATP. Future studies need to shorten the culture period and increase the differentiation rate of dopamineproducing neurons.
Doi et al. [21] and Kikuchi et al. [22] demonstrated that implanted human induced pluripotent-stem-cell (hiPSC)-derived dopaminergic progenitor cells improved the motor behavior of 6-OHDA lesioned rats. Narytnyk et al. [23] demonstrated that human epidermal neural-crest stem cells could form dopaminergic neurons. Alizadeh et al. [24] demonstrated that when cultured olfactory bulb neural stem cells, from brain-dead donors, were supplemented with sonic hedgehog, fibroblast growth factor-8, and glial-derived neurotrophic factor, they differentiated to dopaminergic neurons. The differentiated dopaminergic neurons expressed dopaminergic markers tyrosine-hydroxylase and aromatic L-amino acid decarboxylase (AADC). Nakagawa et al. [25] demonstrated that human embryonic stem cells and hiPSC, cultured in xeno-free medium, could differentiate to dopaminergic neurons. Hartfield et al. [26] demonstrated that hiPSC differentiated to mature substantia nigra pars compacta dopaminergic neurons. Human iPSC-derived dopaminergic progenitor cells transplanted to the putamen of Macaca fascicularis monkeys that had Parkinson's symptoms alter neurotoxic treatment, improved their spontaneous movement. The transplantation required immunosuppression [22]. 4.0 − 8.0 × 10 4 TH positive neurons in the human brain may be required to achieve a meaningful therapeutic effect [27].
HAP stem cells, located in the hair follicle bulge area, used in the present study, are the most accessible stem cells compared to other stem cell types. As shown in the present report, HAP stem cells differentiated efficiently to dopaminergic neurons that extensively proliferated, expressed tyrosine-hydroxylase and secreted large amounts of dopamine without genetic manipulations or added growth factors, and do not form tumors [8]. Furthermore, human HAP stem cells differentiated into 5.0 ± 1.7 × 10 4 cells/hair follicle for 4 weeks [5]. HAP stem cells, which were discovered by Li et al [28], may be most useful in the future to improve the symptoms of Parkinson's disease. The possibility of clinical use of HAP stem cells for Parkinson's disease is feasible and practical, since HAP stem cells are readily available from everyone and can be cryopreserved and banked without loss of pluripotency [5,16].