PDZD7 variants based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines from Korean families manifesting putative moderate-to-severe prelingual ARNSHL; these were c.490C>T (p.Arg164Trp), c.1669delC (p.Arg557Glyfs*13), and c.1526G>A (p.Gly509Glu), with p.Arg164Trp being a predominantly recurring variant. Given the recurring missense variant (p.Arg164Trp) from our cohort, we compared the genotyping data using six short tandem-repeat (STR) markers within or flanking PDZD7 between four probands carrying p.Arg164Trp and 81 normal-hearing controls. We observed an identical haplotype across three out of six STR genotyping markers exclusively shared by two unrelated hearing impaired probands but not by any of the 81 normal-hearing controls, suggesting a potential founder effect. However, STR genotyping, based on six STR markers, revealed various p.Arg164Trp-linked haplotypes shared by all of the affected subjects. In conclusion, PDZD7 can be an important causative gene for moderate to severe ARNSHL in Koreans. Moreover, at least some, if not all, p.Arg164Trp alleles in Koreans could exert a potential founder effect and arise from diverse haplotypes as a mutational hot spot.PDZD7, a PDZ domain-containing scaffold protein, is critical for the organization of Usher syndrome type 2 (USH2) interactome. Recently, biallelic PDZD7 variants have been associated with autosomal-recessive, non-syndromic hearing loss (ARNSHL). Indeed, we identified novel, likely pathogenic  USH2A, ADGRV1, WHRN, and PDZD7 genes are associated with USH2 in humans : th: thUSH2 p.G228R) ,26, are p.G228R) . TherefoPDZD7 have been associated with ARNSHL in the literature was in agreement with a previous study employing PDZD7-knockout mice. PDZD7 deletion led to different spatial effect in the USH2-complex organization between hair cells and photoreceptors, indicating that PDZD7 was essential for the development of USH2 complex at ankle links, exclusively in cochlear hair cells but not photoreceptors [PDZD7 variants manifest only hearing loss. Ebermann et al. demonstrated that PDZD7 showed dynamic interactions and differential binding affinities with members of the USH2 interactome, indicating that PDZD7 preferentially interacted with ADGRV1 rather than with USH2A [PDZD7 variants. For example, CADM1 is a PDZD7-binding protein that is highly expressed in the inner ear in mice, but Cadm1 knockdown did not elicit an abnormal hearing threshold, suggesting that its homologs might compensate for CADM1 loss in the inner ear [The observation that pathogenic biallelic variations in human eceptors ,27, it rth USH2A . The absth USH2A . SimilarAll procedures in this study were approved by the Institutional Review Boards of Seoul National University Bundang Hospital (IRB-B-1007–105–402). Written informed consent was obtained from both the affected and unaffected individuals included in the study. For children, written informed consent was obtained from their parents or guardians on their behalf.Four families , showing moderate-to-severe SNHL in an autosomal-recessive fashion, were enrolled in this study. Subjects underwent comprehensive phenotypic evaluations, including medical history interviews, physical examinations, imaging, and audiological assessments. Through a meticulous review of their medical histories, we found that none of the affected subjects had developed developmental delays, brain abnormalities, or cytomegalovirus infections. For imaging in all affected subjects, TBCT or IAC MRI analyses were conducted to identify phenotypic markers related to hearing loss, such as EVAS and incomplete partitions. Also, ophthalmologic and vestibular evaluations were performed in the affected subjects, where possible.ABRT, ASSR, and DPOAE analyses were performed with all affected subjects. Furthermore, age-appropriate behavior tests, such as play audiometry and PTA (pure tone audiogram), were performed to evaluate the hearing level before and after hearing aid application.PDZD7 variants, we attempted to document the following four properties of hearing based on PTA: severity, residual hearing at different frequencies, configuration, and symmetry. The hearing threshold was calculated by averaging the thresholds at 0.5, 1, 2, and 4 kHz, and the severity of hearing loss was classified as being mild (20–40 dB), moderate (41–55 dB), moderately severe (56–70 dB), severe (71–90 dB), and profound (>90 dB). The range of hearing loss, depending on the PTA results in available subjects, was described in the following manner: low frequency, 250–500 Hz; mid-frequency, 1–2 kHz; high frequency, 4–8 kHz. The audiogram configurations were classified as down-sloping, rising, or flat across various frequencies. Specifically, the down-sloping auditory configuration was defined by increasing thresholds from 0.25 to 8 kHz, and the difference between the thresholds at these frequencies was >20 dB hearing loss [To review the audiological characteristics of previous cases carrying ing loss . Furtherhttps://genome.ucsc.edu/), which is an interactive website offering access to genome sequence data from various vertebrate and invertebrate species (including major model organisms) that is integrated with a large collection of aligned annotations, with narrowed-down variants. Bioinformatics analysis was then performed to select candidate variants in autosomal-recessive genes related to non-syndromic SNHL, as follows.A molecular-genetic diagnosis was made for four families , as described previously. Paternal DNA samples from both families were available to investigate the segregation. For molecular-genetic testing, Exome Sequencing was employed on four proband samples. The obtained reads were aligned with the University of California, Santa Cruz (UCSC) hg19 reference genome browser (http://152.99.75.168/KRGDB/menuPages/firstInfo.jsp); (v) compatibility with clinical features. SNVs with MAFs ≤0.005 were chosen. Global MAFs were verified using several databases, including ExAC, 1000 Genomes, and ESP.In brief, the raw data generated by Exome sequencing were mapped onto the UCSC hg19 reference genome, and rare single-nucleotide variations (SNVs), indels, or splice-site variants were selected via the following filtering process: (i) a basic filtering step, which entailed excluding synonymous SNVs and selecting SNVs with a quality score of greater than 30 and a read depth of more than 20; (ii) compatibility with the autosomal-recessive inheritance pattern; (iii) confirmation of the presence of variants by Sanger sequencing; (iv) a segregation study and/or control study against the KRGDB consisting of 1722 Korean individuals  (https://hereditaryhearingloss.org/), deafness variation database (http://deafnessvariationdatabase.org/), OMIM (https://omim.org/). We have added this in the methods. To predict the pathogenic potential of each detected variant, and compatibility with inheritance and audiogram patterns, an in silico study using SIFT (http://sift.jcvi.org/), and PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) were performed to predict the missense variants. Additionally, to estimate the evolutionary conservation of the amino acid sequences, we referred to the GERP + + score from the UCSC Genome Browser (http://genome.ucsc.edu/).We constructed approximately 200 genes list related to hearing loss or deafness phenotype through hereditary hearing loss , were calculated and compared with the allele frequencies among Korean patients with prelingual ARSNHL. A schematic representation of the variant-filtering strategy employed with the Exome sequencing data is illustrated in PDZD7 p.Arg164Trp alleles in Korean subjects were determined to identify whether the mutant allele is a common founder variant or a mutational hot spot. For this purpose, we selected six STR markers flanking the PDZD7 gene, which included three markers  previously reported to be hypervariable and informative. Primers for the six STR markers flanking PDZD7 were generated based on the UCSC Genome Browser, as previously described. The sequence of the microsatellite markers was as follows: D10S185, D10S520, D10S192, PDZD7, D10S1697, D10S1268, and D10S597.The haplotypes of the Loci were amplified using AmpliTaq DNA polymerase  in a Perkin Elmer 9700 thermal cycler . The genotypes of these loci on 10 chromosomes were defined using the AmpliTaq Gold  touchdown protocol under the following conditions: 10 min at 95 °C; 10 cycles of 30 s at 94 °C, 30 s at 65 °C, and 30 s at 72 °C; a further 20 cycles under the same conditions but with the annealing temperature lowered to 60 °C for 5 cycles and then 55 °C for 15 cycles, and a final extension for 10 min at 72 °C. STR marker genotyping was performed, as described previously . We compPDZD7 can act as a disease-causing gene in Korean families, as evidenced by the segregation of recessive inherited moderate-to-severe hearing loss. All affected subjects demonstrated that the presence of biallelic PDZD7 variants is responsible for ARNSHL. Notably, p.Arg164Trp was frequently identified, and this variant was characterized as both a mutational hot spot and potentially having a founder effect by STR marker genotyping. Our results suggest that screening for p.Arg164Trp can narrow down the candidate population for subjects with moderate-to-severe ARNSHL for more efficient genetic diagnosis. Also, PDZD7 might serve as an appropriate candidate gene for personalized and customized auditory rehabilitation, based on a possible genotype–phenotype correlation observed in this study.In conclusion, we report here for the first time that


Childhood cancer patients treated with platinums often develop hearing loss and the degree is classified according to different scales globally. Our objective was to compare concordance between five well-known ototoxicity scales used for childhood cancer patients. Audiometric test results (n = 654) were evaluated longitudinally and graded according Brock, Chang, International Society of Pediatric Oncology (SIOP) Boston, Muenster scales and the U.S. National Cancer Institute Common Technology Criteria for Adverse Events (CTCAE) version 4.03. Adverse effects of grade 2, 3 and 4 are considered to reflect a degree of hearing loss sufficient to interfere with day-to-day communication . We term this “deleterious hearing loss”. A total number of 3,799 audiograms were evaluated. The prevalence of deleterious hearing loss according to the last available audiogram of each patient was 59.3% (388/654) according to Muenster, 48.2% (315/653) according to SIOP, 40.5% (265/652) according to Brock, 40.3% (263/652) according to Chang, and 57.5% (300/522) according to CTCAEv4.03. Overall concordance between the scales ranged from ĸ = 0.636 (Muenster vs. Chang) to ĸ = 0.975 (Brock vs. Chang). Muenster detected hearing loss the earliest in time, followed by Chang, SIOP and Brock. Generally good concordance between the scales was observed but there is still diversity in definitions of functional outcomes, such as differences in distribution levels of severity of hearing loss, and additional intermediate scales taking into account losses <40 dB as well. Regardless of the scale used, hearing function decreases over time and therefore, close monitoring of hearing function at baseline and with each cycle of platinum therapy should be conducted. Cisplatin and carboplatin are platinum compounds commonly used in the treatment of pediatric malignancies. Ototoxicity, a common adverse effect of treatment with cisplatin, is initially manifested as high frequency hearing loss commonly accompanied by tinnitus–4. HeariHearing status is usually measured by audiometry up to 8 kHz. For surveillance of hearing loss, the 5-point U.S. National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) scale is the most commonly used classification to grade hearing loss 18]. Mos. Mos18].The Brock grading system is a 5-point scale and was specifically designed for pediatric patients treated with cisplatin. This system is used to grade hearing loss progression from high to lower frequencies, taking into account the typical cisplatin-induced configuration of high frequency loss 19]. Hea. Hea19].A modification of the Brock scale resulted in the more clinically-sensitive Chang criteria that correlated with the expected course of treatment-induced ototoxicity in clinical trials 20]. Thi. Thi20].The 8-point Muenster scale is based on the World Health Organization classification and includes several subgroups . This grMore recently, the International Society of Pediatric Oncology (SIOP) Boston classification was released through international consensus to report hearing outcomes in international clinical trials for pediatric patients treated with platinum therapy 16]. Thi. Thi16].Audiograms can be graded for degree of hearing loss according to the hearing status, based on the audiogram alone. Alternatively, audiological datasets including additional information such as immittance, otoacoustic emission, and speech audiometry findings as well as patient history can be examined to determine the likelihood and extent of ototoxicity. The latter is important when an adverse drug effect is suspected and contribution to debilitation of additional etiologies must be eliminated. The objectives of this study are to summarize the baseline characteristics of a large cohort of pediatric childhood cancer patients treated with platinum compounds, and to compare five grading systems within this cohort.The analysis of the anonymized Canadian cohort was approved by the ethics committees of the University of British Columbia and British Columbia’s Children’s Hospital. The analysis of the anonymized Dutch cohort was approved by the ethics committee of the Erasmus Medical Center (MEC-2015-169).Patients were recruited from the BC Children’s Hospital , Alberta Children’s Hospital , Stollery Children’s Hospital , Winnipeg Health Sciences Centre , London Health Sciences Center , Hospital for Sick Children , Kingston General Hospital , Children’s Hospital of Eastern Ontario , Hôpital Saint-Justine , IWK Health Center , McMaster Children’s Hospital , and the Erasmus Medical Center—Sophia Children’s Hospital . Ethical approval was obtained from local research ethics boards. Serial audiological data were collected from 729 patients who received platinum chemotherapy between November 1981 and November 2016. Patients with previous hearing problems (n = 1) or missing data were excluded (n = 74). A total number of 654 patients were included and the total number of audiological evaluations was 3,799. Characteristics of the 654 patients studied are listed in Serial audiogram data was available from pure tone audiometry at frequencies of 0.5, 1, 2, 3, 4, 6, and 8 kHz. Testing was performed by the pediatric audiologists. For the first method, we retrospectively graded 3,789 audiograms from Canadian and Rotterdam patients for overall degree of sensorineural hearing loss according to the following grading systems:, the Muenster, the SIOFor the CTCAEv4.03, Brock and SIOP Boston, children with deleterious hearing loss were defined as those with grade 2 or greater. For Muenster, patients were classified as having deleterious hearing loss if the grade was 2b or greater. For Chang, children with deleterious hearing loss were defined as those with grade 2a or greater. To compare the longitudinal data of audiometric testing, we displayed the results of different ototoxicity classification systems. Due to the absence of standardized measuring timepoints after the start of treatment, adjacent measurements were combined at predetermined points. The prevalence of deleterious hearing loss was determined at the date of treatment discontinuation, one year after treatment discontinuation and 10 years after treatment discontinuation. Descriptive statistics included median and range for continuous variables, and frequencies including percentages for categorical variables.Two approaches were used to compare the different ototoxicity classification systems as the CTCAEv4.03 criteria were applied only on the last available audiogram of each patient. For the first analysis all available audiograms were compared and for the second analysis the ototoxicity scales of the last available audiogram of each patient were compared. Κ statistics were estimated and we consider a clinically meaningful ĸ to be at least 0.70. All analyses were performed using SPSS 24.0 software .Evaluation of the first post treatment audiogram showed that 282/549 patients (51.4%) had deleterious hearing loss according to Muenster, 231/548 (42.2%) according to SIOP, 182/549 (33.2%) according to Brock and 188/548 (34.3%) according to Chang. Median time after the end of platinum treatment was 0.3 years (range: 1 day–26.2 years). The prevalence of ototoxicity one year after treatment discontinuation  was 43% according to Brock and Chang, 56% according to Muenster and 49% according to SIOP. Ten years after treatment discontinuation (range: 9.01–10.98), the prevalence of ototoxicity was 46% according to Brock and Chang, 61% according to Muenster and 54% according to SIOP .There is debate about how much of a child’s high frequency ability can be damaged before the hearing loss has serious complications. For our comparison purposes we consider deleterious hearing loss for children, to be at grade 2 and higher degree on CTCAE, Brock and SIOP, grade 2b and higher on Muenster scale, and grade 2a and higher on Chang scale. As shown in The concordance among Muenster and Chang criteria was lowest (ĸ: 0.665) and the concordance among Chang and Brock criteria was highest  and concordance among Chang and Brock was highest  were graded by SIOP grade 3, and of the 684 audiograms graded as SIOP grade 3, 211 (31%) were graded by Chang as grade 2b.For grade 4 hearing loss, Brock and Chang are similar in coding as well. Of the 183 audiograms assigned a Brock grade 4 hearing loss, 181  were graded by Chang grade 4 as well, and of the 199 audiograms graded as Chang grade 4, 181 (91%) were graded by Brock as grade 4 as well.Figs Several grading scales have been developed for the purpose of describing hearing loss in pediatric oncology patients. In this study, the application of five commonly used ototoxicity grading scales was compared in a large cohort of cisplatin treated childhood cancer patients, namely: Brock, Chang, Muenster, SIOP Boston and CTCAEv4.03. The scales were applied to 3,799 audiograms of 654 platinum-treated pediatric patients and a generally good concordance between the scales was observed.The Brock scale is the oldest ototoxicity scale that was developed taking into account the course of high to low frequency loss. PlatinuThe SIOP Boston scale, validated on international multicenter audiological data, combineDetection of early stage hearing loss and early prediction for the need of hearing support was the motivation for the Muenster scale. This isThe CTCAE requires baseline testing to assess changes of hearing function. Since actual baseline thresholds were often not available, we assumed baseline of 0 dB hearing loss at all frequencies, and used hearing threshold levels. Our study showed lower concordance between CTCAE version 4.03 and the Brock scale ĸ = 0.681) than the study by Knight et al. (ĸ = 0.88)[1 than thWithin one year after end of treatment, 38% (Brock ≥grade 2), 38% (Chang ≥grade 2a), 62% (CTCAEv4.03 ≥grade 2), 44% (SIOP ≥grade 2) and 46% (Muenster ≥grade 2b) of patients had deleterious hearing loss. It is notable that ototoxicity may be occurring before the adverse drug effect is captured on a conventional audiogram which only tests up to 8000 Hz. Auditory access to the richness of the sound environment is diminished as a child losses hearing, first in the extended high frequency range, progressing into the conventional frequency range critical for understanding speech and for developing speech production and full language concepts. AudioloIn order to detect high frequency hearing loss at an early stage, extended high frequencies above 8 kHz should be measured. With high frequency audiometry, treatment protocols could be modified, or otoprotective medication such as sodium thiosulfate or amifostine could be administered, 28. WitA large sample size and audiological grading according to Brock, Chang, SIOP Boston and Muenster scales all performed by one researcher are strengths of this study. Since the ototoxicity grading systems limit grade definition to audiograms, although they may have reasonable concordance, they may be too simplistic for accurate phenotyping of hearing function. It can make a significant difference whether grading is based on audiogram alone or on audiological datasets that allow more effective elimination of other contributing factors to the hearing loss. By assessing a child’s overall hearing status, additional etiologies should be eliminated by taking into account as many factors as possible, as was the case for the CTCAEv4.03 criteria in our study.The lack of standardized time points relative to platinum dosing for audiometric testing during and after treatment for pediatric cancer is not only a limitation that we have encountered in our study, but is a general limitation in pediatric oncology. Protocols are essential for prospective monitoring of hearing function in childhood cancer patients receiving treatment. Ideally, hearing status will be measured after each cycle of cisplatin, although the medical treatment plan may not be alterable, adjustment to the hearing loss as it unfolds can be helped with suitable audiological counselling.In summary, we evaluated the concordance among five ototoxicity grading scales using Kappa values in a large cohort of pediatric patients that were treated with platinum chemotherapy. The analysis was not used to determine the best grading scale, but to compare the differences between them. Differences in concordances appeared to be related largely to differing distribution levels of severity of hearing loss, with some scales taking into account losses below 40 dB. Therefore, the classification scales are not interchangeable. Nevertheless, it is important to take into consideration that platinum-induced hearing loss, regardless which grading scale used, can have significant complications for children, 10. In It is our obligation as pediatric oncology professionals to consider the functional implications of hearing loss on educational and psychosocial scale, 34 and S1 FigThe yellow part depicts the area where hearing loss is defined.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


This study was designed to characterize the location, morphology and ultrastructure of telocytes (TCs) in human scalp tissue. After obtaining approval for this study and informed consent from the patient, a scalp specimen was obtained. The distribution and morphology of TCs in human scalp tissue was assessed by immunohistochemical staining of CD34 and CD117/c-KIT, and the ultrastructure of TCs was investigated using transmission electron microscopy (TEM). Immunohistochemical staining of CD34 revealed that TCs were located in the connective tissue of human scalp, and were concentrated around hair follicles (HFs), blood vessels, sweat glands, sebaceous glands and adipose lobules. Immunohistochemical staining of CD117 revealed that TCs were mainly located in the dermis of human scalp, surrounding the HFs and sweat glands. Under TEM, TCs were seen and confirmed by their special morphological features. These cells were spindle-shaped, had small cell bodies and long thin processes, and surrounded stem cell clusters in the bulge region of HFs. These results demonstrate that TCs in human scalp were positive for CD34 and CD117, and their strategic positioning surrounding stem cells suggests their possible involvement in local regeneration, remodeling and homeostasis of the skin. Tps consist of alternations of podomers (80–100 nm) and podoms (~300 nm)2, the latter of which may contain mitochondria, caveolae (Ca2+ uptake/release units) and endoplasmic reticulum (ER)4. In addition to the unique features of Tps , more and more evidence has emerged indicating that TCs differ from intersititial cells of Cajal and other types of interstitial cells  based on their expression of cell surface antigens and microRNA profiles, and their gene expression and proteomic signatures6. The shape of TCs depends upon the number of the Tps arising directly from their cell body: pyriform for one Tp, spindle for two Tps, triangular for three Tps and stellate for four or more Tps9. In recent years, researchers have used focused ion beam scanning electron microscopy to investigate the 3D imaging of TCs in human dermis, and have revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon-like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) – the podoms10. So far, TCs have been detected in many tissues and organs, including the heart12, lung14, skeletal muscle15, skin17, eye19, liver20, uterus21 and urinary system23.Telocytes (TCs) are a distinct type of interstitial cell that was first identified by Popescu’s group in 2005 and was officially named in 20101. TCs exist in various organs and tissues and are characterized by small cell bodies and one to five extremely long (ten to one hundred μm) and thin (less than 0.2 μm) processes called telopodes (Tps)25. Furthermore, TCs develop close relationships with other types of cells, including mast cells, fat cells, plasma cells, basophils, lymphocytes and connective fiber bundles (collagen and elastic fibers)27. TCs can build a 3D network throughout the entire stromal space, particularly by the branching of their long and bead-like Tps29. TCs can communicate with each other and with other cell types by homocellular and heterocellular junctions, respectively29. In addition, increasing evidence has indicated that TCs play significant roles in regulating tissue morphogenesis and homeostasis, the guidance of tissue-resident stem/progenitor cell self-renewal and differentiation, as well as in various pathologies34.TCs occupy strategic positions in tissues as they are located in the close vicinity of stem cell niches, vascular structures and nervesThere have been many studies about the existence, distribution, morphology, ultrastructure and the potential roles of TCs in different organs of the human body and in some animals in healthy and in diseased states. However, studies focused on the characteristics of TCs in human scalp are relatively rare. Therefore, this study was designed to investigate the existence, distribution, morphological characteristics and ultrastructure of TCs in the human scalp, using immunohistochemistry and transmission electron microscopy (TEM).This study was approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University and conformed with the basic principles of the Declaration of Helsinki. Written informed consent was obtained from the patient.A small piece of scalp tissue (2 cm × 3 cm) was obtained from a young man (21–35 years) undergoing a routine plastic surgery in the Department of Dermatology. The sample was kept under sterile conditions and was divided into small pieces (1 cm × 1 cm), which were fixed in 4% buffered formalin, then were dehydrated in a graded ethanol series and xylene, and finally were embedded in paraffin. Serial sections of 5 μm thick were obtained using a rotary microtome . The sections were deparaffinized and hydrated, then used for immunohistochemical analysis.An indirect immunoperoxidase method was used in the study. After having been subjected to routine immunohistochemical preparation, the sections were incubated overnight at 4 °C with primary antibodies specific for CD34  and CD117  diluted in 1% bovine serum albumin in PBS . The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) . The sections were finally counterstained with haematoxylin, then washed and mounted in an aqueous mounting medium. Double stain of CD34 and CD117 was performed in the same way. Negative controls were processed in the same manner except for replacing the primary antibody with PBS. Sections were examined using an IX73 fluorescence microscope  and images were obtained and analyzed.3) and was fixed in 2.5% glutaraldehyde (buffered to pH 7.3 with sodium cacodylate) for 4 h at 4 °C, and then was washed with 0.1 mol/L cacodylate buffer. The specimen was post-fixed in 1% osmium tetroxide in 0.1 mol/L cacodylate buffer at a pH of 7.3 for 1 h. Sequentially, the specimen was dehydrated using ethanol at concentrations of 30%, 50%, 70%, 80%, 90% and 100%, each phase for 10 min. This was followed by overnight impregnation with propylene and epoxyresin (Epon 812). Ultrathin sections (70 nm thick) were obtained using a Leica Ultracut UCT ultramicrotome . These sections were placed onto a copper grid and stained with lead and uranyl solutions, after which they were observed and photographed using a TEM .The scalp specimen was trimmed into a small piece  cells were predominantly distributed in the reticular dermis and subcutaneous tissue of normal scalp, although a few were spotted in the papillary dermis Fig. . CD34+ cTCs in the dermis of normal scalp had two or three extremely long and thin processes, which ran parallel and close to the basal layer of the epidermis Fig. . These cTCs had small oval bodies, which were mostly occupied by an ovoidal euchromatic nucleus and encircled by a small quantity of cytoplasm. The cytoplasm contained mitochondria, rough and smooth ER Fig. . TCs pre35. While specific immunohistochemical markers of TCs have not yet been identified, TCs in human dermis can be divided into two groups: one that expresses CD117 and another that expresses CD344. In our study, most CD34+ cells were detected in the reticular layer of the dermis and around vascular structures, sweat glands, sebaceous glands, HFs and fat lobules. CD34+ cells formed a 3D stromal network through homocellular and heterocellular junctions that is critical to maintaining the normal structure of the dermis. Only a few CD34+ cells were found in the papillary layer, results that are consistent with a previous study36. One slight difference noted in our study was that CD117+ cells appeared to be more pronounced around sweat glands in the scalp tissue.TCs have been regarded as a new type of cell. The immunohistochemical staining profile of TCs may be distinct between organs and may even be distinct within the same location1. Studies have revealed that TCs are positive for CD117/c-KIT, CD34 , vimentin, caveolin-1, platelet-derived growth factor receptor α, and so on8. Our immunohistochemical results showed that CD34+ cells are mainly located at the excretory ducts of eccrine sweat glands, blood vessels and the infundibulum of HFs, which suggests that TCs may reside in stem cell niches, and might participate in “nursing” the stem cells. The bulge of the HF is the best characterized stem cell compartment in the skin, located between the opening duct of sebaceous glands and the attachment point of arrector pili muscles. Based on the ‘bulge activation hypothesis’, bulge stem cells will proliferate and differentiate when they receive the necessary signals37. It is known that stem cells are not connected to each other by classical junctions, so the question arises: what is the carrier of those signals?Scalp tissue was chosen for our research since our purpose was to observe the spatial relationships between the locations of TCs and HFs. As far as we know, multiple stem cell niches have been observed in the skin, including the basal layer of the interfollicular epidermis, the bulge of HFs, the base of sebaceous gland dermal papillae and perivascular spaces34.At the ultrastructural level, TCs present as small cell bodies with several long and thin Tps whose lengths vary from ten to one hundred μm. The Tps extend with alternations of podomeres and podoms, and they surround stem cells to form niches. According to those locations and morphological characteristics, increasing evidence has revealed that TCs may be key cellular elements involved in regulating tissue morphogenesis and homeostasis, the guidance of tissue-resident stem/progenitor cell self-renewal and diferentiation, as well as in various pathologiesTCs can establish two distinct kinds of connections: long distance connections via Tps and short distance connections via EVs. TCs can establish homocellular contacts with each other and heterocellular contacts with different cell types and non-cellular elements. The homocellular contacts might be significant in exchanging, relaying and passing information between TCs, while the heterocellular contacts might help with distant signaling, which is essential in tissue renewal. Point contacts and planar contacts were observed between TCs and putative stem cells.33. Those vesicles may act as intercellular shuttles to deliver biological signals. Usually, EVs are involved in intercellular signaling in normal and in diseased states by sending important macromolecules  to adjacent cells. By these means, TCs integrate various types of information, from nerve cells, vascular cells, stem cells or immune system cells. In the future, it will be very important to determine the possible functions of TCs in stimulating resident stem cells to proliferate and differentiate for use in skin engineering and regenerative medicine. Recently, in consideration of the strategic location of TCs, some studies have been performed to account for their functions in pathologies and in regenerative medicine, including skin pathologies such as systemic sclerosis38, psoriasis39, basal cell carcinoma and squamous cell carcinoma40. Given the spatial relationship between stem cells and TCs in HFs, it may be promising to investigate variations of biological properties of melanocyte stem cells after co-culture with TCs since that may modulate their expression of the c-KIT receptor (CD117), their proliferation and migration and/or their ability to produce melanin. Exploring the mechanism of the effects of TCs on melanocyte stem cells may provide new approaches for the therapy of human pigmentary diseases.In addition to these atypical heterocellular junctions, TCs can contact other cells (including stem cells) via their shed vesicles. Previous studies have reported that TCs can release three kind of EVs: exosomes, ectosomes and multivesicular cargosIn summary, TCs in human scalp express both CD117 and CD34, and they form 3D interstitial networks contacting the surrounding cells/structures by their Tps and/or by EVs. The spatial relationship between TCs and stem cells in the bulge of the outer root sheath in HFs may imply an important role for TCs in regulating hair and skin regeneration. The number, distribution and normal ultrastructure of TCs varied in pathological conditions of the skin.in vitro and in vivo methods. Uncovering the potential functions of TCs in the skin to regulate the growth cycle of HFs may shed new light on their possible applications in hair regeneration.Thus, our results reveal that TCs are a distinct cellular entity in human scalp. Our study lays a foundation for studies on the roles of TCs in modulating the biological properties of melanocyte stem cells and even in some skin diseases, such as alopecia. To fully understand the roles of TCs in the skin, further study is warranted using dynamic