Human hair-follicle associated pluripotent (hHAP) stem cells differentiate to cardiac-muscle cells

Abstract

We have previously demonstrated that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells are located in the bulge area. HAP stem cells have been previously shown to differentiate to neurons, glial cells, keratinocytes, smooth-muscle cells, melanocytes and 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 previous studies, we established an efficient protocol for the differentiation of cardiac-muscle cells from mouse HAP stem cells. In the present study, we isolated the upper part of human hair follicles containing human HAP (hHAP) stem cells. The upper parts of human hair follicles were suspended in DMEM containing 10% FBS where they differentiated to cardiac-muscle cells as well as neurons, glial cells, keratinocytes and smooth-muscle cells. 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.

Keywords: Hair follicle; cardiac-muscle cell; differentiation; stem cell.

Figure 1.

Isolated human hair follicle and culture of the upper follicle. a. Schema of a human scalp hair follicle shows the location of nestin-positive hHAP stem cells. b. The upper parts of human scalp hair follicles were isolated and cultured in DMEM containing 10% FBS.

Figure 2.

Differentiation of hHAP stem cells. Four weeks after culture in DMEM containing 10% FBS, the upper part of hair follicles differentiated to troponin (cTnT)-positive cardiac-muscle cells, nestin- and βIII-tubulin-positive neurons, GFAP-positive glial cells, K15-positive keratinocytes and smooth-muscle actin (SMA)-positive smooth-muscle cells. Scale bar = 100 µm.

Figure 3.

Production of hHAP stem-cell colonies. Human hair follicle culture protocol: isolated upper parts of human hair follicles were cultured in DMEM containing 10% FBS. Four weeks after culture growing cells from the upper parts of human hair follicle were transferred to DMEM/F12 without FBS. One week after culture, the growing cells formed many hHAP stem-cell colonies. Two days after transfer to DMEM containing 10% FBS, hHAP stem-cell colonies started to differentiate.

Figure 4.

Stem-cell marker expression in hHAP stem-cell colonies and cells differentiated from them. (A) Stem-cell marker expression in hHAP stem-cells colonies. B) Two weeks after transfer to DMEM containing 10% FBS, the nestin-expressing hHAP stem-cell colonies differentiated to troponin (cTnT)-positive cardiac-muscle cells, nestin and βIII-tubulin-positive neurons, GFAP-positive glial cells, K15-positive keratinocytes and smooth-muscle actin (SMA)-positive smooth-muscle cells. Scale bar = 100 μm.