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)-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

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

Primers for qPCR.

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.</div

Beating cardiomyocytes differentiated from rat HAP stem cells.

Beating cardiomyocytes differentiated from rat HAP stem cells.</p

Rat HAP stem cells differentiated into atrial or ventricular cardiomyocytes.

(a-c) Immunofluorescence staining of the upper parts of rat vibrissa hair follicles, which were cultured for 21 days, differentiated into cTnI (a-upper; red) and cTnT (a-lower; red)-positive non-supplemented cardiomyocytes; cTnI (b-upper; red), cTnT (b-lower; red)- and MLC2a (b; green)-positive atrial cardiomyocytes; cTnI (c-upper; red), cTnT (c-lower; red)- and MLC2v (c; green)-positive ventricular cardiomyocytes. Nuclear staining with DAPI (blue). Scale bars; 100 μm. (d, e) qPCR analyses of cardiomyocytes differentiated from HAP stem cells. n = 4 per group for non-supplemented, atrial, and ventricular cardiomyocytes. Data are presented as mean ± SD. * P < 0.05, ** P < 0.005, two-sided Student’s t-test.</p

Shema for rat HAP stem cells differentiation into atrial or ventricular cardiomyocytes.

(a) The upper parts of rat vibrissa hair follicles were cultured and supplemented as described.</p

Regular contraction of atrial and ventricular cardiomyocytes differentiated from rat HAP stem cells.

(a-c) Intracellular Ca2+ (Fluo-4-AM) imaging of spontaneously beating cardiomyocytes differentiated from HAP stem cells. Lower, calcium transients shown at an expanded timescale taken from the region indicated (•) in the upper. (a) Irregular contractions of non-supplemented cardiomyocytes. (b, c) Representative waveforms of atrial and ventricular cardiomyocytes. (d, e) Dispersion in calcium transient duration of cardiomyocytes differentiated from HAP stem cells. n = 17 for number of standard deviation for non-supplemented cardiomyocytes beats (n = 51 for number of non-supplemented cardiomyocytes beats), n = 25 for number of standard deviation for atrial cardiomyocytes beats (n = 75 for number of atrial cardiomyocytes beats), n = 27 for number of standard deviation for ventricular cardiomyocytes beats (n = 81 for number of ventricular cardiomyocytes beats). Data are presented as mean ± SD. ** P < 0.001, two-sided Mann-Whitney U test. (f, g) Dispersion in calcium transient amplitude of cardiomyocytes differentiated from HAP stem cells. n = 17 for number of standard deviation for non-supplemented cardiomyocytes beats (n = 51 for number of non-supplemented cardiomyocytes beats), n = 25 for number of standard deviation for atrial cardiomyocytes beats (n = 75 for number of atrial cardiomyocytes beats), n = 27 for number of standard deviation for ventricular cardiomyocytes beats (n = 81 for number of ventricular cardiomyocytes beats). Data are presented as mean ± SD. ** P < 0.001, two-sided Welch’s t-test. (h, i) Dispersion in resting calcium concentration of cardiomyocytes differentiated from HAP stem cells. n = 17 for number of standard deviation for non-supplemented cardiomyocytes beats (n = 51 for number of non-supplemented cardiomyocytes beats), n = 27 for number of standard deviation for atrial cardiomyocytes beats (n = 81 for number of atrial cardiomyocytes beats), n = 27 for number of standard deviation for ventricular cardiomyocytes beats (n = 81 for number of ventricular cardiomyocytes beats). Data are presented as mean ± SD. * P < 0.005, two-sided Welch’s t-test.</p

SiR-actin live-cell imaging of beating cardiomyocytes differentiated from rat HAP stem cells.

SiR-actin live-cell imaging of beating cardiomyocytes differentiated from rat HAP stem cells.</p