iPSC-based approach for human hair follicle regeneration.

Hair follicles (HFs) are a multifunctional structure involved in physical protection, thermoregulation, sensational detection, and wound healing. Formation and cycling of HFs require dynamic interaction between different cell types of the follicles. Although the processes have been well studied, the generation of human functional HFs with a normal cycling pattern for clinical utilization has yet to be achieved. Recently, human pluripotent stem cells (hPSCs) serve as an unlimited cell source for generating various types of cells including cells of the HFs. In this review, HF morphogenesis and cycling, different cell sources used for HF regeneration, and potential strategies for HF bioengineering using induced pluripotent stem cells (iPSCs) are depicted. Challenges and perspectives toward the therapeutic use of bioengineered HFs for hair loss disorder are also discussed.

Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. METHODS: Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-ß signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. RESULTS: Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-ß signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. CONCLUSION: This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

Generation of an integration-free human induced pluripotent stem cell line MUSIi010-A from occipital scalp fibroblasts of a male patient with androgenetic alopecia.

An induced pluripotent stem cell (iPSC) line, MUSIi010-A, was established by Sendai virus (SeV) transduction of scalp fibroblasts from a 59-year-old male with androgenetic alopecia (AGA). Pluripotency of the iPSC line was verified by immunofluorescence staining of pluripotent markers and by in vitro trilineage differentiation. The MUSIi010-A line was shown to retain normal karyotype and free of SeV vectors at passage 17. This iPSC line can be used for studying pathological mechanisms of AGA.

Generation of two induced pluripotent stem cell lines (MUSIi011-A and MUSIi011-B) from peripheral blood T lymphocytes of a healthy individual.

Activated T lymphocytes of a healthy individual were reprogrammed to induced pluripotent stem cells (iPSCs) using Sendai viral vectors. Two iPSC lines, MUSIi011-A and MUSIi011-B, were established and characterized for the expression of pluripotent markers. Both iPSC lines were able to differentiate into cells of three embryonic germ layers via embryoid body formation, exhibited normal karyotypes and were free of viral genome and transgenes at passage 15. These T lymphocyte-derived iPSCs (T-iPSCs) represent a useful starting cell source for developing next-generation immune cells such as chimeric antigen receptor (CAR)-engineered iPSC-derived T lymphocytes for the application in adoptive immunotherapy.

Induced pluripotent stem cell line MUSIi006-A derived from hair follicle keratinocytes as a non-invasive somatic cell source.

In this study, we used hair follicle keratinocytes for reprogramming. Collection of plucked hairs offers advantages over other somatic cells because no medical professional or operation room is required. Keratinocytes were isolated from plucked hairs of a 21-year-old healthy woman and characterized for the expression of cytokeratin 14 (CK14). Reprogramming of keratinocytes was performed using Sendai virus. Further characterization of the keratinocyte-derived iPSC line (designated as MUSIi006-A) confirmed that the cell line was pluripotent, free from Sendai viral genome and transgenes, and retained normal karyotype. Our method represents an easy, non-invasive and efficient approach for iPSC generation from hair samples.

Derivation of an induced pluripotent stem cell line (MUSIi004-A) from dermal fibroblasts of a 48-year-old spinocerebellar ataxia type 3 patient.

Dermal fibroblasts were obtained from a 48-year-old female patient with spinocerebellar ataxia type 3 (SCA3). Fibroblasts were reprogrammed by nucleofection with episomal plasmids, carrying L-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1 and shRNA against p53. The SCA3 patient-specific iPSC line, MUSIi004-A, was characterized by immunofluorescence staining to verify the expression of pluripotent markers. The iPSC line exhibited an ability to differentiate into three germ layers by embryoid body (EB) formation. Karyotypic analysis of the MUSIi004-A line was normal. The mutant allele was still present in the iPSC line. This iPSC line represents a useful tool for studying neurodegeneration in SCA3.

Establishment of an integration-free induced pluripotent stem cell line (MUSIi005-A) from exfoliated renal epithelial cells.

Human induced pluripotent stem cells (iPSCs) were generated from exfoliated renal epithelial cells isolated from a urine sample of a 31-year-old healthy woman. Epithelial cells were characterized for the expression of E-cadherin and reprogrammed using non-integrating Sendai viral vectors. The urine-derived iPSC line (designated as MUSIi005-A) was karyotypically normal, expressed pluripotent markers, differentiated into cells of three embryonic germ layers, and showed no viral and transgene expressions at passage 29. Our protocol offers a non-invasive and efficient approach for iPSC generation from patients with genetic or acquired disorders.

Generation of a human induced pluripotent stem cell line (MUSIi001-A) from caesarean section scar fibroblasts using Sendai viral vectors.

We generated a human induced pluripotent stem cell (iPSC) line from caesarean section scar fibroblasts of a 33-year-old healthy woman using transgene-free Sendai viral vectors under feeder-free condition. The established iPSC line, designated as MUSIi001-A, exhibited a normal karyotype, expressed pluripotent markers, differentiated into cells of three embryonic germ layers. Further analyses showed that the Sendai viral genome was absent at passage 25. The MUSIi001-A line can serve as a control for studying developmental biology and phenotypic comparison with disease-specific iPSCs.

Genetic dissection of TrkB activated signalling pathways required for specific aspects of the taste system.

BACKGROUND: Neurotrophin-4 (NT-4) and brain derived neurotrophic factor (BDNF) bind to the same receptor, Ntrk2/TrkB, but play distinct roles in the development of the rodent gustatory system. However, the mechanisms underlying these processes are lacking. RESULTS: Here, we demonstrate, in vivo, that single or combined point mutations in major adaptor protein docking sites on TrkB receptor affect specific aspects of the mouse gustatory development, known to be dependent on BDNF or NT-4. In particular, mice with a mutation in the TrkB-SHC docking site had reduced gustatory neuron survival at both early and later stages of development, when survival is dependent on NT-4 and BDNF, respectively. In addition, lingual innervation and taste bud morphology, both BDNF-dependent functions, were altered in these mutants. In contrast, mutation of the TrkB-PLCγ docking site alone did not affect gustatory neuron survival. Moreover, innervation to the tongue was delayed in these mutants and taste receptor expression was altered. CONCLUSIONS: We have genetically dissected pathways activated downstream of the TrkB receptor that are required for specific aspects of the taste system controlled by the two neurotrophins NT-4 and BDNF. In addition, our results indicate that TrkB also regulate the expression of specific taste receptors by distinct signalling pathways. These results advance our knowledge of the biology of the taste system, one of the fundamental sensory systems crucial for an organism to relate to the environment.

Ablation of TrkB signalling in CCK neurons results in hypercortisolism and obesity.

Dysregulation of hypothalamic-pituitary-adrenal (HPA) axis activity leads to debilitating neuroendocrine or metabolic disorders such as Cushing's syndrome (CS). Glucocorticoids control HPA axis activity through negative feedback to the pituitary gland and the central nervous system (CNS). However, the cellular mechanisms involved are poorly understood, particularly in the CNS. Here we show that, in mice, selective loss of TrkB signalling in cholecystokinin (CCK)-GABAergic neurons induces glucocorticoid resistance, resulting in increased corticotrophin-releasing hormone expression, chronic hypercortisolism, adrenocortical hyperplasia, glucose intolerance and mature-onset obesity, reminiscent of the human CS phenotype. Interestingly, obesity is not due to hyperphagia or decreased energy expenditure, but is associated with increased de novo lipogenesis in the liver. Our study therefore identifies CCK neurons as a novel and critical cellular component of the HPA axis, and demonstrates the requirement of TrkB for the transmission of glucocorticoid signalling.