Hair-follicle-associated Pluripotent Research Articles

Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db/db mice.

Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db/db) C57BL/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay in vitro showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate in vivo effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-β1 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-β1 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and α-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise.

LB1778 Wound closure in diabetic db/db mice is facilitated by autologous hair-follicle associated pluripotent (HAP)stem cells

LB1778 Wound closure in diabetic db/db mice is facilitated by autologous hair-follicle associated pluripotent (HAP)stem cells

1325 Extensive hair growth stimulation and follicle normalization with transplanted hair follicle associated pluripotent (HAP) stem cells in athymic nude mice

1325 Extensive hair growth stimulation and follicle normalization with transplanted hair follicle associated pluripotent (HAP) stem cells in athymic nude mice

Direct implantation of hair-follicle-associated pluripotent (HAP) stem cells repairs intracerebral hemorrhage and reduces neuroinflammation in mouse model.

Intracerebral hemorrhage (ICH) is a leading cause of mortality with ineffective treatment. Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into neurons, glial cells and many other types of cells. HAP stem cells have been shown to repair peripheral-nerve and spinal-cord injury in mouse models. In the present study, HAP stem cells from C57BL/6J mice were implanted into the injured brain of C57BL/6J or nude mice with induced ICH. After allo transplantation, HAP stem cells differentiated to neurons, astrocytes, oligodendrocytes, and microglia in the ICH site of nude mice. After autologous transplantation in C57BL/6J mice, HAP stem cells suppressed astrocyte and microglia infiltration in the injured brain. The mRNA expression levels of IL-10 and TGF-β1, measured by quantitative Real-Time RT-PCR, in the brain of C57BL/6J mice with ICH was increased by HAP-stem-cell implantation compared to the non-implanted mice. Quantitative sensorimotor function analysis, with modified limb-placing test and the cylinder test, demonstrated a significant functional improvement in the HAP-stem-cell-implanted C57BL/6J mice, compared to non-implanted mice. HAP stem cells have critical advantages over induced pluripotent stem cells, embryonic stem cells as they do not develop tumors, are autologous, and do not require genetic manipulation. The present study demonstrates future clinical potential of HAP-stem-cell repair of ICH, currently a recalcitrant disease.

Hair follicle associated pluripotent (HAP) stem cells jump from transplanted whiskers to pelage follicles and stimulate hair growth

Stimulation of hair growth in hair loss has been a difficult goal to achieve. Hair follicle-associated pluripotent (HAP) stem cells express nestin and have been shown to differentiate to multiple cell types including keratinocytes, neurons, beating cardiac muscles and numerous other cell types. HAP stem cells originate in the bulge area of the hair follicle and have been shown to migrate within and outside the hair follicle. In the present study, the upper part of vibrissa follicles from nestin-driven green-fluorescent protein (GFP) transgenic mice, containing GFP-expressing HAP stem cells, were transplanted in the dorsal area of athymic nude mice. Fluorescence microscopy and immunostaining showed the transplanted HAP stem cells jumped and targeted the bulge and hair bulb and other areas of the resident nude mouse pelage follicles where they differentiated to keratinocytes. These results indicate that transplanted nestin-GFP expressing HAP stem cells jumped from the upper part of the whisker follicles and targeted nude-mouse hair follicles, which are genetically deficient to grow normal hair shafts, and differentiated to keratinocytes to produce normal mature hair shafts. The resident nude-mouse pelage follicles targeted by jumping whisker HAP stem cells produced long hair shafts from numerous hair follicles for least 2 hair cycles during 36 days, demonstrations that HAP stem cells can stimulate hair growth. The present results for hair loss therapy are discussed.

Hair-follicle-associated pluripotent (HAP) stem cells differentiate into mature beating cardiomyocyte sheets on flexible substrates in vitro.

Cardiomyocytes have been differentiated from various stem cells such as human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), but it is difficult to produce mature cardiomyocytes. We showed rat hair-follicle-associated pluripotent (HAP) stem cells have pluripotency and produced mature beating cardiomyocyte sheets differentiated from rat HAP stem cells. The upper parts of rat vibrissa hair follicles were cultured in 10% FBS DMEM and stained with antibodies of the ectoderm, mesoderm, endoderm system to show the differentiation of multiple cell types. Moreover, HAP stem cells were cultured under three different conditions to decide the most suitable culture conditions for making beating cardiomyocyte sheets. The beating cardiomyocyte sheets were shown to be mature by staining sarcomere structures. Isoproterenol alone and the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) effectively induced beating long-fiber cardiomyocytes, which formed beating sheets, only in the presence of all four agents. Flexible substrates were essential for the differentiation of sheets of mature beating cardiomyocytes for HAP stem cells. The features of the cardiomyocytes differentiated from HAP stem cells demonstrate they have clinical potential for heart regeneration.

Chronic spinal cord injury functionally repaired by direct implantation of encapsulated hair-follicle-associated pluripotent (HAP) stem cells in a mouse model: Potential for clinical regenerative medicine.

Chronic spinal cord injury (SCI) is a highly debilitating and recalcitrant disease with limited treatment options. Although various stem cell types have shown some clinical efficacy for injury repair they have not for SCI. Hair-follicle-associated pluripotent (HAP) stem cells have been shown to differentiate into neurons, Schwan cells, beating cardiomyocytes and many other type of cells, and have effectively regenerated acute spinal cord injury in mouse models. In the present report, HAP stem cells from C57BL/6J mice, encapsulated in polyvinylidene fluoride membranes (PFM), were implanted into the severed thoracic spinal cord of C57BL/6J or athymic nude mice in the early chronic phase. After implantation, HAP stem cells differentiated to neurons, astrocytes and oligodendrocytes in the regenerated thoracic spinal cord of C57BL/6J and nude mice. Quantitative motor function analysis, with the Basso Mouse Scale for Locomotion (BMS) score, demonstrated a significant functional improvement in the HAP-stem-cell-implanted mice, compared to non-implanted mice. HAP stem cells have critical advantages over other stem cells: they do not develop teratomas; do not loose differentiation ability when cryopreserved and thus are bankable; are autologous, readily obtained from anyone; and do not require genetic manipulation. HAP stem cells therefore have greater clinical potential for SCI repair than induced pluripotent stem cells (iPSCs), neuronal stem cells (NSCs)/neural progenitor cells (NPCs) or embryonic stem cells (ESCs). The present report demonstrates future clinical potential of HAP-stem-cell repair of chronic spinal cord injury, currently a recalcitrant disease.

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.

Hair-follicle-associated pluripotent stem cells derived from cryopreserved intact human hair follicles sustain multilineage differentiation potential

The bulge area of the hair follicle contains hair-follicle-associated pluripotent (HAP) stem cells. Here, we present effective cryopreservation procedures of the human hair follicle that preserve the differentiation potential of HAP stem cells. Whole hair follicles isolated from human scalp were cryopreserved by a slow-rate cooling medium and stored in liquid nitrogen. A careful thawing method was used to collect the upper parts of the human hair follicles which were cultured for four weeks in a Dulbecco’s Modified Eagle’s Medium with fetal bovine serum (FBS). Proliferating hair follicle cells were then shifted to DMEM/Ham’s Nutrient Mixture F-12 medium without FBS and allowed to grow for one week. These proliferating cells were able to produce HAP stem cell colonies with multilineage differentiation capacity. They produced keratinocytes, smooth muscle cells, cardiac muscle cells, neurons and glial cells. Interestingly, these cryopreserved hair follicles produced pluripotent HAP stem cell colonies similar to fresh follicles. These findings suggest that the cryopreserved whole human hair follicle preserves the ability to produce HAP stem cells, which will enable any individual to preserve a bank of these stem cells for personalized regenerative medicine.

Expression of anti-aging type-XVII collagen (COL17A1/BP180) in hair follicle-associated pluripotent (HAP) stem cells during differentiation

Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells.

Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Encapsulated on Polyvinylidene Fluoride Membranes (PFM) Promote Functional Recovery from Spinal Cord Injury.

Our previous studies showed that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells, which reside in the bulge area of the hair follicle, could restore injured nerve and spinal cord and differentiate into cardiac muscle cells. Here we transplanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell colonies enclosed on polyvinylidene fluoride membranes (PFM) into the severed thoracic spinal cord of nude mice. After seven weeks of implantation, we found the differentiation of HAP stem cells into neurons and glial cells. Our results also showed that PFM-captured GFP-expressing HAP stem-cell colonies assisted complete reattachment of the thoracic spinal cord. Furthermore, our quantitative motor function analysis with the Basso Mouse Scale for Locomotion (BMS) score demonstrated a significant improvement in the implanted mice compared to non-implanted mice with a severed spinal cord. Our study also showed that it is easy to obtain HAP stem cells, they do not develop teratomas, and do not loose differentiation ability when cryopreserved. Collectively our results suggest that HAP stem cells could be abetter source compared to induced pluripotent stem cells (iPS) or embryonic stem (ES) cells for regenerative medicine, specifically for spinal cord repair.

1394 Hair-follicle-associated pluripotent (HAP) stem cells captured on polyvinylidene fluoride membranes promote functional recovery of spinal cord injury

1394 Hair-follicle-associated pluripotent (HAP) stem cells captured on polyvinylidene fluoride membranes promote functional recovery of spinal cord injury

Hair Follicle-Associated Pluripotent (HAP) Stem Cells in Gelfoam® Histoculture for Use in Spinal Cord Repair.

The stem cell marker, nestin, is expressed in the hair follicle, both in cells in the bulge area (BA) and the dermal papilla (DP). Nestin-expressinghair follicle-associated-pluripotent (HAP)stem cells of both the BA and DP have been previously shown to be able to form neurons, heart muscle cells, and other non-follicle cell types. The ability of the nestin-expressing HAP stem cells from the BA and DP to repair spinal cord injury was compared. Nestin-expressing HAP stem cells from both the BA and DP grew very well on Gelfoam®. The HAP stem cells attached to the Gelfoam® within 1h. They grew along the grids of the Gelfoam® during the first 2 or 3days. Later they spread into the Gelfoam®. After transplantation of Gelfoam® cultures of nestin-expressing BA or DP HAP stem cells into the injured spinal cord (including the Gelfoam®) nestin-expressing BA and DP cells were observed to be viable over 100days post-surgery. Hematoxylin and eosin (H&E) staining showed connections between the transplanted cells and the host spine tissue. Immunohistochemistry showed many Tuj1-, Isl 1/2, and EN1-positive cells and nerve fibers in the transplanted area of the spinal cord after BA Gelfoam® or DP Gelfoam® cultures were transplanted to the spine. The spinal cord of micewas injuredto effect hind-limb paralysis. Twenty-eight days after transplantation with BA or DPHAP stem cells on Gelfoam® to the injured area of the spine, the mice recoverednormal locomotion.

Nerve Growth and Interaction in Gelfoam® Histoculture: A Nervous System Organoid.

Nestin-expressing hair follicle-associated pluripotent (HAP) stem cells reside mainly in the bulge area (BA) of the hair follicle but also in the dermal papilla (DP). The BA appears to be origin of HAP stem cells. Long-term Gelfoam® histoculturewas established of whiskers isolated from transgenic mice, in which there is nestin-driven green fluorescent protein (ND-GFP). HAP stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam® forming nerve-like structures. These fibers express the neuron marker β-III tubulin-positive fibers and consisted of ND-GFP-expressing cells and extended up to 500 mm from the whisker nerve stump in Gelfoam® histoculture. The growing fibers had growth cones on their tips expressing F-actin indicating that the fibers were growing axons. HAP stem cell proliferation resulted in elongation of the follicle nerve and interaction with other nerves in 3D Gelfoam® histoculture, including the sciatic nerve, trigeminal nerve, and trigeminal nerve ganglion.

Hair-Shaft Growth in Gelfoam® Histoculture of Skin and Isolated Hair Follicles.

Human scalp skin with abundant hair follicles in various stages of the hair growth cycle was histocultured for up to 40days on Gelfoam® at the air/liquid interface. The anagen hair follicles within the histoculture scalp skin produced growing hair shafts. Hair follicles could continue their cycle in histoculture; for example, apparent spontaneous catagen induction was observed both histologically and by the actual regression of the hair follicle. In addition, vellus follicles were shown to be viable at day 40 after initiation of culture. Follicle keratinocytes continued to incorporate [3H]thymidine for up to several weeks after shaft elongation had ceased. Intensive hair growth was observed in the pieces of shaved mouse skin histocultured on Gelfoam®. Isolated human and mouse hair follicles also produced growing hair shafts. By day 63 in histoculture of mouse hair follicles, the number of hair follicle-associated pluripotent (HAP) stem cells increased significantly and the follicles were intact. Gelfoam® histoculture of skin demonstrated that the hair follicle cells are the most sensitive to doxorubicin which prevented hair growth, thereby mimicking chemotherapy-induced alopecia in Gelfoam® histoculture.

Hair Follicle-Associated Pluripotent(HAP) Stem Cells.

The hair follicle has been known, since 1990, to contain stem cells located in the bulge area. In 2003, we reported a new type of stem cell in the hair follicle that expresses the brain stem-cell marker nestin. We have termed these cells as hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells can differentiate into neuronal and glial cells, beating cardiac-muscle cells, and other cell types in culture. HAP stem cells can be used for nerve and spinal-cord repair such that locomotor activity is recovered. A major function in situ of the HAP stem cells is for growth of the hair follicle sensory nerve. HAP stem cells have critical advantages over embryonic stem cells and induced pluripotent stem (IPS) cells for regenerative medicine in that they are highly accessible, require no genetic manipulation, are nontumorigenic, and do not present ethical issues.

Beating Heart Cells from Hair-Follicle-Associated Pluripotent (HAP) Stem Cells.

The neural stem-cell marker nestin is expressed in hair follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that HAPstem cells could affect nerve and spinal cord regeneration in mouse models. We subsequently demonstrated that HAP stem cells differentiated into beating cardiac muscle cells. The differentiation potential to cardiac muscle is greatest in the upper part of the mouse whisker follicle. The beat rate of the cardiac muscle cells differentiated from HAP stem cells was stimulated by isoproterenol and inhibited by propanolol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. Under hypoxic conditions, HAP stem cells differentiated into troponin-positive cardiac-muscle cells at a higher rate that under normoxic conditions. Hypoxia did not influence the differentiation to other cell types. This method is appropriate for future use with human hair follicles to produce hHAP stem cells in sufficient quantities for future heart, nerve, and spinal cord regeneration in the clinic.

Hair follicle associated pluripotent (HAP) stem cell from young mice have the greatest potential to differentiate to cardiac muscle cells

Hair follicle associated pluripotent (HAP) stem cell from young mice have the greatest potential to differentiate to cardiac muscle cells

Hypoxia Enhances Differentiation of Hair Follicle-Associated-Pluripotent (HAP) Stem Cells to Cardiac-Muscle Cells.

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair-follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells from mouse and human could form spheres in culture, termed hair spheres, which are keratin 15-negative and nestin-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that nestin-expressing stem cells could effect nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We recently observed that isoproterenol directs HAP stem cells to differentiate to cardiac-muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. In the present study, we report that, under hypoxic conditions, HAP stem cells differentiated to troponin-positive cardiac-muscle cells at a higher rate that under normoxic conditions. Hypoxia did not influence the differentiation to other cell types. For future use of HAP stem cells for cardiac muscle regeneration, hypoxia should enhance the rate of differentiation thereby providing patients more opportunities to use their own HAP stem cells which are easily accessible, for this purpose. J. Cell. Biochem. 118: 554-558, 2017. © 2016 Wiley Periodicals, Inc.