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Force-triggered density gradient sedimentation and cocktail enzyme digestion treatment for isolation of single dermal papilla cells from follicular unit extraction harvesting human hair follicles

Stem Cell Research & Therapy volume 15, Article number: 416 (2024) Cite this article

Abstract

Background

Hair follicles (HFs) are dynamic structures which are readily accessible within the skin that contain various pools of stem cells with broad regenerative potential, such as dermal papilla cells (DPCs), dermal sheath cells, and epithelial HF stem cells. DPCs act as signalling centres for HF regeneration. The current method for isolating human DPCs are inefficient. These methods struggle to obtain freshly isolated original DPCs and do not maintain the characteristics of DPCs effectively.

Methods

In this study, two simple but more efficient methods were explored. Force-triggered density gradient sedimentation (FDGS) and cocktail enzyme digestion treatment (CEDT) were used to isolate purified DP spheres from human HFs, obtaining purified freshly isolated original DPCs from DP spheres. The expression profiles of isolated DPCs were tested, and gene expression of DPC-specific markers were analyzed using immunofluorescence staining, RT-qPCR and western blot.

Results

The 10% Ficoll PM400 was determined as the optimal concentration for FDGS method. Primary DPCs, DSCs and HFSCs were isolated simultaneously using the FDGS and CEDT method. The expression profiles of fresh DPCs isolated using the FDGS and CEDT methods were similar to those of traditionally isolated DPCs. DP-specific markers were expressed at significantly higher levels in freshly isolated DPCs than in traditionally isolated DPCs.

Conclusions

Compared to traditional methods, the presented laboratory protocols were able to isolate fresh DPCs with high efficiency, thereby improving their research potential.

Background

Hair follicles (HF) serve as versatile models for studying many fundamental biological questions because they contain several distinct stem cell populations, which support significant regenerative capacity. HF regeneration relies on the interaction between epithelial precursors that reside in the “bulge” [1, 2] and specialised mesenchymal cells located at the base of the follicle called the dermal papilla (DP). DP cells (DPCs) provide necessary signals to induce epithelial bulge cell proliferation, initiating anagen follicle growth [3, 4]. Freshly isolated DPCs or in vitro cultured low-passage DPCs can induce de novo HF formation when implanted elsewhere, with the hair type determined by the DP source [3, 5, 6]. Different molecular characteristics of DPCs, such as partial expression of specific markers (i.e. alkaline phosphatase [ALP], β-catenin, versican, and neural cell adhesion molecules), as well as pluripotent markers (i.e. Sox2), indicate that DPCs have a remarkable level of subpopulation heterogeneity [7, 8]. Thus, isolating single DPCs from tissues is crucial biological issue for both tissue engineering and cell subset research. The most used method for obtaining human DP spheres is microdissection of excised tissue. However, microdissection is a laborious and operator-dependent task. Drawbacks such as low yield, difficult adhesion, low survival rates, and poor growth of papillary plants hinder the isolation of large numbers of DP spheres [9]. Previous studies have reported that microdissection combined with enzyme digestion decreases the operating difficulty and improves the adhesion rate of DP spheres [9, 10]. However, a disadvantage of the common enzymatic digestion method is that isolated DP spheres are still contaminated with cells. Even when repeated low-speed centrifugation was used to primarily pellet the DP spheres, the sediment still contained unwanted non-DP cell impurities, such as dermal sheath cells (DSCs) and vascular endothelial cells. Furthermore, a previous study demonstrated that the unique extracellular matrix (ECM) composition of human DP renders them indigestible in a single-cell suspension using standard digestion method [11,12,13]. To obtain human DPCs in vitro, the DP must first be isolated via a microdissection approach from the HF, and it takes days for DPCs to migrate and expand from the isolated DP explants during in vitro culture. However, unlike rodent HF DPCs, cells cultured from human DP spheres are notably dissimilar to intact human DP [14], and their molecular characteristics change rapidly within 1 day of their immigration from DP spheres in a two dimensional (2D) culture microenvironment [15]. This limitation hampers their application in tissue engineering and further molecular studies of DP heterogeneity, such as single-cell sequencing. A method for the direct isolation of fresh DPCs from human DP spheres has not yet been reported, highlighting the need for improved isolation techniques.

Herein, we demonstrate two simple yet efficient methods used in our laboratory: force-triggered density gradient sedimentation (FDGS) to purify DP spheres, and a new two-step cocktail enzyme digestion treatment (CEDT) to obtain purified freshly isolated single DPCs from human DP spheres (Fig. 1). FDGS is based on the different biological characteristics (such as cellularity and density) of DP-sphere and other non-DP cells. After specific enzyme treatment, DP and non-DP cells were thoroughly separated using different density and centrifugal force gradients. Freshly isolated human DPCs were obtained from the resulting DP spheres using CEDT. Single DSCs and epithelial HF stem cells (HFSCs) can also be obtained during the isolation process, if required.

Fig. 1
figure 1

Schematic illustration of isolating original human dermal papilla cells using ficoll density gradient sedimentation combined with cocktail enzyme digestion treatment

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Methods

Ethics approval and consent to participate

The Medical Ethics Committee of Southern Medical University approved all the studies described here. The study was conducted in accordance with the principles of the Declaration of Helsinki, and written informed consent was obtained from all patients.

Extraction of human HFs

HFs from 12 different individuals (male, age range from 25 to 35 yrs) were used for all experiments. As the current follicular unit extraction (FUE) method is more widely used than follicular unit transplantation (FUT), and for better applicability and reproducibility of the experiments, intact HFs were originally obtained during microscopic hair transplantation for FUE.

Randomization

All of the HF samples were randomly grouped separately in either fDPC-P0, tDPC-P0 or tDPC-P3 group for further research. The samples were examined and microdissected under a Leica MZ8 dissecting microscope (Leica Microsystems, Wetzlar, Germany) with fiberoptic cool illumination using sterile equipment and plasticware. Only HFs in the anagen stage were selected and removed with fine forceps. HF samples.

Intervention methods

DPCs were isolated by FDGS and the FDG + CEDT method in the fDPC-P0 group, and by traditional DPCs in the tDPC-P0 or tDPC-P3 group. The fDPC-P0 group used freshly isolated primary cells for subsequent experiments, while tDPC-P0 performed subsequent experiments using primary DPCs cultured from DP spheres. The tDPC-P3 group isolated DPCs by traditional methods and passaged to three generations for subsequent experiments. Each experiment was repeated at least in triplicate. The work has been reported in line with the ARRIVE guidelines 2.0.

Experimental approaches for isolating purified DPCs from human HFs using FDGS and CEDT

An overview of the experimental approaches used is shown in Fig. 1, and the detailed protocol is as follows (no registered protocol was prepared before the study):

Separating the dermis and epidermis

  1. (1)

    HFs were cut at the level of the arrector pili muscle insertion point (1–2 mm below the sebaceous gland) using a scalpel blade to separate the upper (containing HFSCs) and lower parts (containing DPCs and DSCs). The lower part was transferred to a new plate containing a 0.25% dispase solution and incubated at 37 °C for 30 min to 1 h. If HFSCs are required, the upper part is treated according to the traditional method for obtaining HFSCs.

  2. (2)

    The dermis was gently separated using the pasteur pipette (Sigma-Aldrich, St. Louis, MO, USA) from the lower part to eliminate unwanted epidermis. The dermis was transferred to a new plate containing 0.1% dispase/0.2% collagenase I mixture and incubated at 37 °C for 60 min. Briefly, the dermis was pipetted every 10 min using a 1-mL pipettor in the enzyme solution during digestion to obtain a mixed suspension of DP spheres and DSCs. Tip: Care must be taken to ensure that the DP does not stick to the inside of the pipette, so the pipette tip is equilibrated in the dissection medium before the DP is taken up into the pipette.

Separating DP spheres and DSCs using FDGS

  1. (1)

    The suspension containing the DPCs and DSCs was transferred to another centrifuge tube. The dermis was resuspended in 10% foetal bovine serum (FBS)/Dulbecco’s Modified Eagle Medium (DMEM) and mixed with an equal volume of 10% Ficoll PM400 (Sigma-Aldrich, St. Louis, MO, USA). The mixed cell suspension was layered over an equal volume of 10% Ficoll and centrifuged at 80 × g for 5 min.

  2. (2)

    The upper layer of 10% Ficoll containing a single DSCs was absorbed. The DSCs were washed twice by resuspension in 10% FBS/DMEM and centrifuged at 1000 rpm for 3 min. Isolated DSCs were resuspended in 20% FBS/DMEM and plated into T25 flasks.

  3. (3)

    The lower layer was discarded, the precipitate containing the DP spheres was preserved, and the DP spheres were washed twice by resuspension in 10% FBS/DMEM and centrifuged at 200 rpm for 3 min.

Isolating DPCs from DP spheres using CEDT

  1. (1)

    To immediately obtain single fresh DPCs, the isolated DP spheres were incubated in 0.1% dispase/0.2% collagenase IV mixture at 37 °C for 30 min. The solution was pipetted every 10 min during digestion.

  2. (2)

    The suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the DP spheres were incubated with 0.25% trypsin for 5 min and gently pipetted into the enzyme solution during digestion to dissociate cell aggregates. Next, DPCs were washed twice by resuspension in 10% FBS/DMEM and centrifuged at 200 rpm for 3 min. Freshly isolated DPCs were resuspended in 20% FBS/DMEM and plated into a T25 flask.

Histological analysis

For histological analysis, the HFs were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Paraffin-embedded HFs were cut at 5-µm intervals. Sections were stained with haematoxylin and eosin (H&E) using a standard protocol.

Immunofluorescence staining

To observe the distribution of ECM molecules in human HFs, the sections were fixed for 30 min in 4% PFA at 25 °C, blocked in 10% bovine serum albumin (BSA) and 0.5% Triton in PBS for 1 h at RT, and incubated with collagen I antibody (1:100; Abcam, Cambridge, UK) and collagen IV antibody (1:100; Abcam) overnight at 4 °C. The samples were then incubated with the secondary antibodies, Alexa-Fluor-488-conjugated anti-rat and Alexa-Fluor-594-conjugated anti-rabbit, for 1 h at RT.

To further determine the accuracy of the resulting DP spheres and DSCs isolated via FDGS, the expression of the DP-specific genes ALP, β-catenin, versican, and NCAM1 in DP spheres were confirmed by immunofluorescence staining, and the expression of α-SMA in DSCs and K15 in HFSCs were also detected. The freshly isolated DP spheres, HFSCs, and DSCs were seeded into a Confocal Dish (Corning, Corning, NY, USA) and fixed for 30 min in 4% PFA at RT, and blocked in 5% BSA and 0.5% Triton in PBS for 1 h at RT. The sections were then incubated with the following primary antibodies overnight at 4 °C: anti-ALP (1:200; Abcam), anti-β-catenin (1:250; Abcam), anti-NCAM1 (1:100; Abcam), anti-versican (1:100; Abcam), anti-α-SMA (1:250; Abcam), and anti-K15 (1:100; Abcam). Subsequently, the sections were incubated with secondary antibodies, Alexa-Fluor-488-conjugated anti-rabbit and Alexa-Fluor-594-conjugated anti-rabbit antibodies (both 1:300; Abcam), for 2 h at RT. Images were obtained using an Olympus FLUOVIEW FV10i confocal laser-scanning microscope (Olympus, Tokyo, Japan). Z-stacks were acquired at 100 Hz with an optimal stack distance and a 1024 × 1024 dpi resolution. Z-stack projections were generated using the Olympus FV10-ASW software package. The author calculated the average fluorescence density in five fields per sample in a single blind way using ImageJ software (NIH, Bethesda, MD, USA).

Flow cytometry analysis

To characterise the isolated HFSCs, the expression of CD200 were examined using flow cytometry. Freshly isolated HFSCs were treated with 0.25% trypsin and strained through a 40-µm cell strainer, which were then incubated with biotin-conjugated CD200 antibody (1:100; eBioscience, San Diego, CA, USA) at 4 °C for 30 min, followed by treatment for 15 min at 4 °C with phycoerythrin-conjugated streptavidin (1:200; eBioscience). After staining, cells were sorted using a FACS Aria Flow cytometer (BD Biosciences, San Jose, CA, USA).

Preparation of DPCs and cell viability assay

To obtain DPCs cultured in a 2D environment, DP spheres isolated from randomly allocated HFs using FDGS were washed twice with PBS (centrifuged at 30 × g for 3 min) and cultured in DMEM supplemented with 10% FBS. All cells were incubated in 5% CO2 at 37 °C. The culture medium was changed every three days. The cells were passaged in 0.25% trypsin/0.01% EDTA (Gibco, Billings, MT, USA) when they began to merge. The viability of adhered DPCs isolated by the traditional method in a 2D environment (tDPC-P0) and fresh DPCs isolated using CEDT (fDPC-P0) was determined using a LIVE/DEAD cell staining kit (Invitrogen, Waltham, MA, USA).

Reverse transcription quantitative real-time PCR (RT-qPCR)

To compare the differences in the characteristics of DPCs between fDPC-P0 to tDPC-P0 and tDPCs on passage 3 (tDPC-P3), the expression levels of ALP, β-catenin, versican, NCAM1, and α-SMA were detected using RT-qPCR. Total RNA was isolated using TRIzol reagent (Life Technologies, Carlsbad, CA, USA), and cDNA was generated using the PrimeScript RT-PCR Kit (Takara, Dalian, China) according to the manufacturer’s instructions. RT-qPCR was performed using the SYBR PrimeScript RT-PCR Kit (Takara), Power SYBR Green PCR Master Mix (Life Technologies), and the ABI Prism 7900HT Sequence Detection System (Life Technologies). The PCR cycling conditions were as follows: denaturation for 10 min at 95 °C, 40 cycles of denaturation (95 °C for 15 s), annealing (60 °C for 20 s), and extension (72 °C for 10 s). GAPDH expression (4352932E) was used to normalise data using the ΔCt method. Fold changes in relative gene expression were identified using the 2-ΔΔCt method. The primer sequences used in this study are listed in Supplementary Table 1.

Western blot analysis

We then assayed the expression of ALP, β-catenin, versican, and NCAM1 by western blotting in freshly isolated DPCs, primary DPCs, and tDPC-P3. Protein extracts were isolated from DPCs using RIPA (Radio Immunoprecipitation Assay) protein lysis buffer containing 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein (10 μg/lane) were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis under non-reducing conditions, and the gels were transferred onto nitrocellulose membranes (Pierce, Waltham, MA, USA) in an electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). The membranes were blocked using 5% non-fat milk at 4 °C overnight and then incubated at RT for 1 h with the following primary antibodies: anti-ALP (1:100; Abcam), anti-β-catenin (1:100; Abcam), anti-versican (1:100; Abcam), and anti-NCAM1 (1:100; Abcam). After three washes with PBS-T (pH 7.4, 0.1% Triton), the membranes were incubated with the secondary antibody, goat anti-rabbit IgG conjugated to HRP (NeoBioscience, Shenzhen, China), for 1 h at RT, followed by three washes with PBS-T. The band density was determined using the Bio-Rad Quality One Software.

RNA extraction and detection

For sample size calculation, we calculated that the sample size of 3 achieves over 90% power in fDPC-P0, tDPC-P0 and tDPC-P3 with a significance level (α) of 0.05 using a two-sided paired t-test according to our pre-experimental results. Three samples per group were used. Total RNA from fDPC-P0, tDPC-P0, and tDPC-P3 was extracted and RNA quality was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries for mRNA sequencing were generated using the NEBNext Multiplex RNA Library Prep Kit for the Illumina platform (NEB, Ipswich, MA, USA), according to the manufacturer’s instructions. The final purified libraries were evaluated using a BioAnalyzer 2100 and quantified. The mRNA libraries were sequenced using the Illumina HiSeq platform (Illumina, San Diego, CA, USA). Three biological replicates were used for each group.

RNA sequencing data processing

Quality control was performed on the raw sequence data using the FastQC tool. To obtain high-quality clean data for analysis, adaptor sequence trimming and removal of low-quality reads were performed. Based on the ultra-high-throughput short-read aligner Bowtie, TopHat aligned RNA-seq reads to reference genomes and analysed the mapping reads to determine the possible splice junctions between exons. RNA expression levels were determined as reads per kilobase per million mapped reads (FPKM). Hierarchical clustering of representative mRNA expression was performed to reveal reproducibility in biological replicates, and the DESeq package was used to detect differentially expressed mRNAs (DEMs) between the fDPC-P0, tDPC-P0, and tDPC-P3 groups, with thresholds of a two-fold change (the ratio between three fDPC-P0, tDPC-P0, and tDPC-P3 samples averaged signal values) and a false discovery rate (FDR) of < 0.05.

The top 100 DEMs were visualised using a heatmap. The heatmap and unsupervised cluster analysis of the top 100 DEMs were plotted using OmicShare tools, which is an online platform for data analysis (www.omicshare.com/tools). To identify patterns of similarity across fDPC-P0, tDPC-P0, and tDPC-P3, the sample correlations – also known as Pearson (product-moment) correlation coefficients – were analysed and a correlation heatmap was drawn using OmicShare tools. Principal components analysis (PCA) was performed with the R package VEGAN ([http://cran.r-project.org/web/packages/vegan/index.html] version 2.5–6) and Pearson correlation coefficients between samples were performed using the OmicShare tools. For gene expression pattern analysis, a Short Time-series Expression Miner (STEM) was utilised [16]. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed using the OmicShare tools to identify the critical GO terms and signal pathways of the overlapping genes [17]. GO terms and KEGG pathways (p-values < 0.05) were considered significantly enriched. Overlapping genes were mapped to the STRING database (https://string-db.org/) to screen for protein–protein interactions (PPI) [18], and networks were visualised using Cytoscape [19]. The most significant modules were identified using the plug-in MCODE of Cytoscape with a cut-off MCODE score > 5 [20]. Hub genes were identified using the CytoHubba plug-in of Cytoscape, with the top 10 nodes ranked by Maximal Clique Centrality [21].

Statistical analysis

Statistical analyses were performed using SPSS software (version 19.0; SPSS, Inc., Chicago, IL, USA). Comparisons between groups were performed using the t-test, with a p-value of < 0.05 considered statistically significant. Experimental data were expressed as the means ± standard error of the mean. All experiments were repeated at least in triplicate.

Results

Type IV collagen expression in ECM of dermal papilla

H&E staining of human HFs revealed a dense group of dermal fibroblasts at the base of the follicle, the DP, and the dermal sheath which was wrapped around the outside of the HF (Fig. 2A). To observe the distribution of ECM molecules in human HFs, we stained human HFs with collagen I and IV (Fig. 2B). This staining demonstrated that DPCs synthesise type IV collagen, which resembles the basement membrane matrix. Consequently, collagenase I was used to digest type I collagen in the dermis, followed by collagenase IV to digest the resulting DP spheres.

Fig. 2
figure 2

Distribution of collagen in human hair follicles (HFs). A Haematoxylin and eosin staining of follicular unit extraction harvesting human HFs. Scale bar: 50 μm. B Extracellular matrix in anagen HFs contains an abundance of basement membrane components. Dermal papilla cells are embedded in abundant type IV collagen. Scale bar: 50 μm

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Isolation and purification of DP spheres and DSCs using FDGS method

A mixed cell suspension of follicle dermal components containing DP spheres and DSCs was successfully obtained after traditional microdissection and enzymatic digestion (Fig. 3A) (Fig. S1). To isolate and purify the DP spheres from the mixed cell suspension, we used the FDGS method (Fig. 3A-B), in which the core was subjected to density gradient centrifugation. Ficoll PM400 is the most used dense-gradient medium. Based on their different volumes and densities, DP spheres and DSCs were distributed in the upper and lower layers of the solution. To determine the optimal concentration of Ficoll PM400 for isolating DP spheres and DSCs using FDGS, 5%, 10%, and 15% Ficoll were tested (Fig. 3C). At a 5% concentration, some DSCs settled in the lower layer together with the DP spheres, while at a 15% concentration, both the DP spheres and DSCs were suspended in the upper layer. Finally, the DSCs and DP spheres were completely separated at a 10% concentration of Ficoll PM400.

Fig. 3
figure 3

Harvesting of single-cell components from human scalp hair follicles by ficoll density gradient sedimentation (FDGS). A Graphic of isolating original human dermal papilla cells (DPCs) using FDGS combined with cocktail enzyme digestion treatment. B Sketch of two major parts of FDGS for isolating DPCs. C Freshly isolated dermal papilla spheres and dermal sheath cells after treatment with different concentrations of Ficoll PM400 density separation. Scale bar: 200 µm

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Characteristics of isolated DP spheres and singe cells

Immunostaining and three-dimensional reconstruction imaging results confirmed that ALP, β-catenin, NCAM1, and versican expression were restricted to the DP spheres isolated using FDGS (Fig. 4A). DSCs showed high expression levels of α-SMA (Fig. 4B). Immunofluorescence staining of HFSCs showed that > 50% (53.34 ± 4.58%) of isolated HFSCs expressed the specific marker K15 (Fig. 4B) (n = 3; *p < 0.05). Flow cytometry analysis revealed positive reactions (37.53 ± 2.29%) to the upper-bulge specific marker CD200 of HFSCs from the upper part (Fig. 4C). Stem cells from the lower part were used as the control (2.83 ± 0.32%; *p < 0.05).

Fig. 4
figure 4

Characteristics of isolated dermal papilla (DP) spheres, dermal sheath cells (DSCs), and hair follicle stem cell (HFSCs). A Three dimensional reconstruction immunostaining imaging of DP spheres isolated using ficoll density gradient sedimentation with DP-specific markers alkaline phosphatase, β-catenin, versican, and NCAM1. Cells were counterstained with DAPI (4,6-Diamidino-2-phenylindole, blue). B Immunostaining of DSCs with specific marker α-SMA and HFSCs with specific marker K15. Cells were counterstained with DAPI (blue). C Flow cytometry analysis showed positive reactions to specific marker CD200 of HFSCs in upper bulge. Lower bulge cells were used as the control. Scale bars: 100 µm

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Characteristics of DPCs freshly isolated using CEDT

As shown in Fig. 5A, compared to tDPC-P0 (Fig. 5Ac, Ad), CEDT had a significantly higher efficiency in obtaining fDPC-P0 (Fig. 5Aa, Ab) from DP spheres isolated using FDGS. The results of LIVE/DEAD staining showed no significant difference in cell viability between adhered fDPC-P0 (Fig. 5B, D) and tDPC-P0 (p > 0.05). EDU (5-Ethynyl-2’-deoxyuridine) cell staining (Fig. 5C, E) also showed no significant differences in cell proliferation (p > 0.05). The cell migration assay (Fig. 5F, G) revealed no significant differences between the two groups. Immunofluorescence staining revealed that both tDPCs and fDPCs expressed ALP, a DP-specific marker (Fig. 5H). To compare the differences in the characteristics of DPCs between fDPC-P0, tDPC-P0, and tDPC-P3, the expression levels of ALP, β-catenin, versican, and NCAM1 of fDPC-P0, tDPC-P0, and tDPC-P3 were detected. As shown in Fig. 5I-K, RT-qPCR and western blot analysis showed that the DP-specific markers ALP, β-catenin, versican, and NCAM1 were expressed in fDPCs at significantly higher levels than in tDPC-P0 and tDPC-P3.

Fig. 5
figure 5

Characteristics of freshly isolated dermal papilla cells (DPCs) obtained using cocktail enzyme digestion treatment (CEDT). A DPCs were freshly isolated using CEDT (fDPC) a and adhered in 12 h b. Compared to primary DPCs traditionally migrated and expanded from dermal papilla spheres (tDPC) (c) after 4 days (d). B, C Live (green) / Dead (red) and EDU (5-Ethynyl-2’-deoxyuridine) cell staining were performed to examine viability and proliferation of adhered freshly isolated dermal papilla cell (fDPCs) and primary traditionally isolated dermal papilla cell (tDPCs). D, E Rate of dead cells and EDU+ cells in primary fDPCs and tDPCs showed no significant differences (p > 0.05). F, G Analysis of fDPCs and tDPCs in wounded area during migration assay using IBIDI chamber after incubation for 0 h, 12 h, and 24 h. H Immunofluorescence staining of alkaline phosphatase (ALP) in primary tDPCs and fDPCs. I, J, K Reverse transcription quantitative real-time PCR and western blot analysis of DPCs freshly isolated using CEDT (fDPC-P0), traditionally cultured primary DPCs (tDPC-P0), and tDPCs in passage 3 (tDPC-P3) with DP-specific markers ALP, β-catenin, versican, and NCAM1. Relative mRNA and protein expression levels were normalized to GAPDH. *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bars: 50 µm. All values are expressed as means ± SD (n = 3 individual experiments). Full-length blots/gels are presented in Fig. S4

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Identification of mRNA profiling data in fDPC-P0, tDPC-P0, and tDPC-P3

To further explore the influence of the CEDT method on DPC isolation, we investigated the gene expression profiles of fDPC-P0, tDPC-P0, and tDPC-P3 using high-throughput sequencing. Nine Samples (n = 3 per group) were collected from three individuals. PCA results showed that the three samples in each group shared common genetic expression characteristics and clustered closely together (Fig. 6B), indicating a small variance within each other in the same group on the first principal component (PC1) and PC2. The first three principal components cumulatively represented > 95% of the variation in the analysis (PC1: 81.4%; PC2: 10.7%; and PC3: 3.1%) and segregated the samples into three discrete groups, supporting the idea that the gene expression characteristics of DPCs varied and the resulting partitions fDPC-P0, tDPC-P0, and tDPC-P3 into three different groups. We also performed a Pearson’s correlation coefficient analysis between the samples (Fig. 6C). Based on the similarity matrix in Fig. 6C, the results showed that the parallel samples had good reproducibility, and the differences among the sample groups were significant. Expression pattern changed progressively from fDPC-P0 to tDPC-P3. For comparison, sample groups analysis demonstrated correlations ranging 0.73–0.80 between fDPC-P0 and tDPC-P0 samples, and 0.57–0.72 between fDPC-P0 and tDPC-P3 samples, suggesting that the gene expression of tDPCs are dissimilar to fDPCs.

Fig. 6
figure 6

mRNA profiling data demonstrates that freshly isolated dermal papilla cell (fDPCs) and traditionally isolated dermal papilla cell (tDPCs) are transcriptionally dissimilar. A Heatmap showing relative expression levels of 56 progressively downregulated differentially expressed genes (DEGs) and 30 progressively upregulated DEGs. Relative expression levels are colour-coded as colour key. B Principal component analysis of gene expression profiles of nine samples (three each of fDPC-P0, DPC-P0, and DPC-P3). C Pearson rank correlations were computed for every pair of transcriptome profiling samples. Resulting colour-coded correlation matrix revealing similarity of transcriptomes of fDPCs and tDPCs. D, E Expression patterns representing genes that were progressively up- or downregulated. F Venn diagram showing 148 overlapping DEGs by at least twofold in fDPC-P0, tDPC-P0, and tDPC-P3. G KEGG pathway enrichment for 148 DEGs. H RT-qPCR validation of mRNA expression of fDPCs and tDPCs. Abundances of mRNAs were normalized to that of GAPDH

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In our subsequent investigations, we focused on changes in cellular gene expression patterns using the CEDT method. Genes with the same expression patterns were identified using STEM (Fig. 6D). Using |log2 (fold change)|> 1 and FDR < 0.05 as the threshold cutoffs, four expression patterns, expression patterns 3, 0, 4, and 7 which consisted of 7,146 progressively upregulated (FPKM: fDPC-P0 > 2tDPC-P0 > 4tDPC-P3, FDR < 0.05) and 5,546 progressively downregulated (FPKM: fDPC-P0 < 2tDPC-P0 < 4tDPC-P3, FDR < 0.05) genes, exhibited significant enrichment (p ≤ 0.05) (Fig. 6D). Among these progressively regulated genes, 148 overlapping differentially expressed genes (DEGs) across fDPC-P0, tDPC-P0, and tDPC-P3 were identified (Fig. 6E) (Supplementary Table 2). Using KEGG pathway analysis to mine canonical signalling pathways, we found that overlapping mRNAs were strongly associated with cell adhesion molecules, ECM-receptor interactions, and focal adhesion (Fig. 6F) (Supplementary Table 3). GO analysis showed that changes in biological process of the overlapping mRNAs were significantly associated with GO terms such as “cellular process” and “biological regulation” (Fig. S2). To explore the interactions among the overlapping mRNAs, a PPI network of the 148 overlapping mRNAs was constructed, and the most important module with the highest score (9.0), including 10 nodes, was screened using MCODE (Fig. S3A). These genes may play critical roles in the loss of DP characteristics. Genes may interact with each other through specific genes or hub genes. COL1A1 was identified by CytoHubba as the most significant hub gene in this module (Fig. S3A). Among these overlapping genes, we found that the expression of DP-specific markers such ALP, β-catenin, versican, NCAM1, and NCAM2, was progressively downregulated and that the expression of the dermal sheath-specific marker α-SMA was progressively upregulated (Fig. 6A and S3B) (Supplementary Table 4), which indicates that the cell characteristics of DPCs were better maintained in fDPC group. To highlight the differences between the groups, unsupervised cluster analysis relative to the 56 progressively downregulated DEGs and 30 upregulated DEGs was performed (Fig. 6A), which was consistent with the trend of the Pearson correlation analysis, indicating that the characteristics of DPCs were better maintained in fDPC than in 2D-cultured tDPC. To validate the profiling data, we performed RT-qPCR on the same samples used for mRNA-seq to confirm the altered expression of 12 randomly selected overlapping DEGs. Consistent with the mRNA-Seq data, RT-qPCR results showed similar trends in DEGs expression (Fig. 6G). These data indicate that the screened overlapping mRNAs were reliable and feasible.

Thus, the results illustrate that fDPC-P0, tDPC-P0, and tDPC-P3 are highly dissimilar at the transcriptional level and show a progressive change from fDPC-P0 to tDPC-P3. This suggests that the characteristics of DPCs were better maintained in fDPC than in 2D-cultured tDPC-P0 and tDPC-P3.

Discussion

Recent discoveries demonstrating the multipotent capabilities of human HFSCs and their easy access to skin tissue make HFs an attractive source for isolating stem cells and their subsequent applications in tissue engineering and regenerative medicine [22, 23]. DPCs, one of the centres of HF research, display functional properties predicted by dermal stem cells and contribute to dermal maintenance, wound healing, and HF morphogenesis [24]. However, research on the physiology and bioinformatics of original human DPCs is lacking because of the difficulties in obtaining fresh DPCs. In this study, we report FDGS combined with CEDT as a unique method for isolating and purifying single DPCs from human HFs harvested using FUE. The hallmark of dermal fibroblasts is their ability to synthesise an extensive ECM, especially type I and III collagen, to fill the intercellular spaces in the dermis [25]. However, DPCs can synthesise a type IV collagen that resembles the basement membrane matrix [26, 27]. Collagenase I digests everything except the DP which remains intact with only a minor disruption to its ECM [28]. Thus, we used collagenase I to digest type I collagen in the dermis, from which DP spheres and single DSCs were obtained.

Furthermore, because of the differences between single non-DPCs and DP spheres (such as cellularity and density), non-DP cells and DP spheres can be separated and purified using centrifugal force and density gradient centrifugation. Repeated low-speed centrifugation primarily pellets non-DP cells and DP spheres. However, the sediment contained fibroblasts and vascular endothelial cells. Density gradient centrifugation is another technique that is used to separate cells based on their density. Ficoll PM400, a highly branched polymer formed by the copolymerisation of sucrose and epichlorohydrin, is the most used density gradient medium [29, 30]. In this study, DSCs and DP spheres were separated by 10% Ficoll gradient centrifugation. Additionally, after separation and purification, the resulting DP spheres and DSCs showed favourable adherent capacity, DP spheres isolated using FDGS expressed their specific markers ALP, β-catenin, versican, and NCAM1, and DSCs expressed their specific marker α-SMA. HFSCs can also be obtained during the isolation process by following the traditional method [31], and the resulting HFSCs show positive reactions to the specific markers, K15 and CD200. This method maximises the use of human HFs extracted using FUE and achieves efficient stem cell acquisition. FDGS has several significant advantages over the current methods. Primarily, FDGS is not limited by the volume of the object and can obtain many purified DP spheres that remove non-DP cell impurities. Moreover, FDGS can shorten the air exposure time during isolation which may reduce potential contamination, ensuring the greatest survival ratio and maintaining the correlated characteristics of isolated cells.

It takes nearly 1 week for DPCs to migrate and expand from DP spheres using traditional methods of culture in a 2D environment. With the increase in culturing time, the expression levels of DP-markers ALP, β-catenin, versican, and NCAM1 decreased, whereas the expression levels of α-SMA increased gradually, which indicates that DPCs rapidly lost their characteristics and began to differentiate after in vitro culture [32, 33]. In particular, the expression of NCAM1, which is involved in the aggregation of DPCs and formation of DP explants, was significantly downregulated after in vitro culture [34]. The unique matrix composition of DPCs makes it more difficult to digest them into single cells compared to other cells [27]. Obtaining original fresh DPCs has remained a challenge in the study of DPC subsets and HF single-cell sequencing. However, CEDT demonstrated high efficiency in obtaining original fresh DPCs, which showed good cell viability. The time for DPCs to migrate from DP spheres in a 2D environment was also reduced using CEDT, which helps to maintain the characteristics of DPCs. Transcriptome sequencing revealed that the gene expression profiles of tDPC-P0 and tDPC-P3 were significantly different from those of original fDPCs. To better understand the similarity across fDPC-P0, tDPC-P0, and tDPC-P3 samples, PCA and Pearson correlation coefficients were analysed [35, 36]. The results suggested significant partitioning of the overall fDPC-P0, tDPC-P0, and tDPC-P3 groups (Fig. 2) and showed a progressive change from fDPC-P0 to tDPC-P3. This partitioning illustrates the progressive changes in the molecular characteristics of DPCs in a 2D culture environment. Some of the DP-specific expression markers such as ALP, β-catenin, versican, NCAM1, and NCAM2 were also found to be progressively downregulated from fDPC-P0 to tDPC-P3, which appeared to account, at least in part, for this partitioning of gene expression in fDPC and tDPC. The RT-qPCR and western blotting results also suggested that the characteristics of DPCs were better maintained in fDPC than in 2D-cultured tDPC-P0 and tDPC-P3, demonstrating that FDGS combined with CEDT helps improve the implications of DPCs in tissue engineering and cell subset research.

Conclusions

This article describes a novel FDGS method which requires only an ordinary centrifugal force and density gradient to isolate and purify dermal papilla (DP) spheres. A simple cocktail enzyme digestion treatment (CEDT) was used to obtain single DP cells (DPCs) from purified DP spheres. Simultaneously, dermal sheath cells and epithelial hair follicle stem cells can be obtained using traditional methods. This modification not only provides the possibility to obtain freshly isolated DPCs, but also improves their research potential.

Availability of data and materials

Data used to support the findings of this study are available from the corresponding author upon request. The original sequencing data was deposited in the Gene Expression Omnibus (GEO) database, and the related data was archived in supplementary files Table S2-S4.

Abbreviations

FDGS:

Ficoll density gradient sedimentation

CEDG:

Cocktail enzyme digestion treatment

DPC:

Dermal papilla cell

HFSC:

Hair follicle stem cell

DSC:

Dermal sheath cell

tDPC:

Traditionally isolated dermal papilla cell

fDPC:

Freshly isolated dermal papilla cell

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Acknowledgements

The authors declare that they have not used Artificial Intelligence in this study. The authors thank the Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering for providing experimental instruments.

Funding

This work was supported by the Guangdong Basic and Applied Basic Research Foundation (Grant Nos. 2024A1515011392, 2023A1515110686, and 2022A1515111070) and National Natural Science Foundation of China (Grant Nos. 82172235, 82300763, and 82304054).

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Authors and Affiliations

  1. Department of Plastic and Aesthetic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong, China

    Junfei Huang, Jian Chen, Zhexiang Fan, Yuyang Gan & Lijuan Du

  2. Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, China

    Haoyuan Li

  3. Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong, China

    Yangpeng Chen

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Contributions

JFH and LJD designed the experiments and performed the statistical analyses. JFH and JC wrote the manuscript and performed cell biology experiments. YPC produced the animal model. ZXF and YYG prepared tissues for histological evaluation. HYL provided suggestions during manuscript preparation. All the authors have read and approved the final version of the manuscript.

Corresponding author

Correspondence to Lijuan Du.

Ethics declarations

Ethics approval and consent to participate

All the excess HF units were extracted by FUE from the discarded scalp tissue of patients undergoing plastic surgery. All patients were aware of and agreed to use some of their HF units for research. This study was approved by the ethics committee of Nanfang Hospital (Approval number: NFYY-2021–1062; Title of the approved project: Force-triggered density gradient sedimentation and cocktail enzyme digestion treatment for isolation of single dermal papilla cells from follicular unit extraction harvesting human hair follicles; Date: 15 Nov 2021), and followed the Principles of the Declaration of Helsinki. All methods were carried out in accordance with relevant guidelines and regulations. This study was conducted in compliance with the ARRIVE guidelines.

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Huang, J., Chen, J., Li, H. et al. Force-triggered density gradient sedimentation and cocktail enzyme digestion treatment for isolation of single dermal papilla cells from follicular unit extraction harvesting human hair follicles. Stem Cell Res Ther 15, 416 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04026-2

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512