Characterization of Rat Hair Follicle Stem Cells Selected by Vario Magnetic Activated Cell Sorting System

En-yi, Huang; Lian, Xiaohua; Chen, Wei; Yang, Tian; Yang, Li

doi:10.1267/ahc.09016

Cited by 9 publications

(5 citation statements)

References 33 publications

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“…1,2 This method provides purities ‡ 90% but requires expensive instrumentation typically found only in core facilities and a minimum run time of over 2 h, from incubation of cells with fluorescent tags to running through the FACS instrument. MACS, which is another well-developed cell isolation technique utilized in epidermal stem cell isolation, 18 requires similar run times. By contrast, our microfluidic technique is capable of providing an enriched suspension of epidermal stem cells in less than 30 min and does not require preprocessing labeling of the sample with fluorescent or magnetic tags or the associated incubation and centrifugation steps that may contribute to cell loss.…”

Section: Discussionmentioning

confidence: 99%

“…17 The standard methods of isolating bulge stem cells are fluorescence-and magnet-activated cell sorting (FACS and MACS, respectively). 1,2,18 Both methods require preprocessing labeling of cells with antibody tags followed by centrifugation steps before cell separation. While both FACS and MACS are well established and reliable, the sample processing steps and time required present challenges when considering translational regenerative applications of resident stem cells, such as skin stem cells.…”

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confidence: 99%

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Microfluidic Enrichment of Mouse Epidermal Stem Cells and Validation of Stem Cell Proliferation In Vitro

Zhu

Smith

Yarmush

et al. 2013

Tissue Engineering Part C: Methods

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Bulge stem cells reside in the lowest permanent portion of hair follicles and are responsible for the renewal of these follicles along with the repair of the epidermis during wound healing. These cells are identified by surface expression of CD34 and the a6-integrin. When CD34 and a6 double-positive cells are isolated and implanted into murine skin, they give rise to epidermis and hair follicle structures. The current gold standard for isolation of these stem cells is fluorescence-activated cell sorting (FACS) based on cell surface markers. Here, we describe an alternative method for CD34 bulge stem cell isolation: a microfluidic platform that captures stem cells based on cell surface markers. This method is relatively fast, requiring 30 min of time from direct introduction of murine skin tissue digestate into a two-stage microfluidic device to one-pass elution of CD34+ enriched cells with a purity of 55.8% -5.1%. The recovered cells remain viable and formed colonies with characteristic morphologies. When grown in culture, enriched cells contain a larger a6 + population than un-enriched cells.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Microfluidic Enrichment of Mouse Epidermal Stem Cells and Validation of Stem Cell Proliferation In Vitro

Zhu

Smith

Yarmush

et al. 2013

Tissue Engineering Part C: Methods

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“…The rats were sacrificed by decollation and subsequently sterilized using 75% ethyl alcohol. HFSCs were isolated and cultured as previously reported, with some modifications ( 29 ). Briefly, hair follicles were separated from the vibrissae and subsequently digested with 1 g/l collagenase type I (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 30 min at 37°C, followed by 2.5 g/l trypsin (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

miR‑339‑5p negatively regulates loureirin A‑induced hair follicle stem cell differentiation by targeting DLX5

Xie

et al. 2018

Mol Med Report

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Our previous study indicated that loureirin A induces hair follicle stem cell (HFSC) differentiation through Wnt/β-catenin signaling pathway activation. However, if and how microRNAs (miRNAs/miRs) modulate loureirin A-induced differentiation remains to be elucidated. In the present study, HFSCs were separated from the vibrissae of rats and identified by CD34 and keratin, type 1 cytoskeletal (K)15 expression. Microarray-based miRNA profiling analysis revealed that miR-339-5p was downregulated in loureirin A-induced HFSC differentiation. miR-339-5p overexpression by transfection with miR-339-5p mimics markedly inhibited the expression of K10 and involucrin, which are markers of epidermal differentiation, whereas inhibition of miR-339-5p by miR-339-5p inhibitor transfection promoted the expression of K10 and involucrin. These results suggest that miR-339-5p is a negative regulator of HFSC differentiation following induction by loureirin A. These findings were confirmed by a luciferase assay. Homeobox protein DLX-5 (DLX5) was identified as a direct target of miR-339-5p. Furthermore, it was demonstrated that miR-339-5p inhibited DLX5. Overexpression of miR-339-5p by mimic transfection significantly inhibited protein Wnt-3a (Wnt3a) expression, while inhibition of miR-339-5p by inhibitor transfection significantly increased the expression of Wnt3a. Furthermore, small interfering RNA targeting DLX5 was transfected into HFSCs, and western blot analysis revealed that Wnt3a, involucrin and K10 expression was significantly downregulated. Taken together, these results suggest that miR-339-5p negatively regulated loureirin A-induced HFSC differentiation by targeting DLX5, resulting in Wnt/β-catenin signaling pathway inhibition. This may provide a possible therapeutic target for skin repair and regeneration.

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“…Using our method, unknown animal pathogens can be avoided and it supports xeno-free human cell isolation. Our HFSCs showed high viability and were easily enriched from disaggregated dermis, suggesting an alternative technique to cell-sorting systems [17]. Although many types of cells reside in the dermis, for example, sweat gland [18], dermal sheath and dermal papilla [19], nestin [20], endothelial cells [21], melanocytes [22], Merkel cells [23] and fibroblasts [24], serum-free and defined media do not support the growth of unspecific cells and causes these cells to degrade during cultivation.…”

Section: Isolation and Viability Of Hfscs In Cnt07mentioning

confidence: 95%

A simple culture method for epithelial stem cells derived from human hair follicle

et al. 2013

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The challenge arises among researchers when hair follicle stem cells (HFSCs) derived from a human hair follicle remain poorly expanded in defined culture medium. In this study, we isolated the HFSCs and they became confluent after 10 days of cultivation. Comparing the viability of HFSCs cultured in defined keratinocytes serum free medium (KSFM) in a coated plate and CnT07 medium in an uncoated plate, the number of HFSCs cultured in CnT07 was significantly higher at days 2, 4, 6 and 8 (P=0.004). The population doubling time of HFSCs was 21.48±0.44 hours in non-coated plates with CnT07 and 30.73±0.75 hours in coated plates with KSFM. Our primary HFSC cultures were positive for CD200 and K15 with brownish color. Flow cytometry analysis showed that the percentage of HFSCs expressing CD200 and K15 were 65.20±3.16 and 72.07±6.62 respectively. After reaching 100% confluence, the HFSCs were differentiated into an epidermal layer in vitro using CnT02-3D defined media. HFSCs were differentiated into an epidermal layer after 2 weeks of induction. Involucrin- and K6-positive cells were detected in the differentiated epidermal layer. This method is a simple technique for HFSC isolation and has a lower cost of processing and labor, and it represents a promising tool for skin tissue engineering.

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Characterization of Rat Hair Follicle Stem Cells Selected by Vario Magnetic Activated Cell Sorting System

Cited by 9 publications

References 33 publications

Microfluidic Enrichment of Mouse Epidermal Stem Cells and Validation of Stem Cell Proliferation In Vitro

Microfluidic Enrichment of Mouse Epidermal Stem Cells and Validation of Stem Cell Proliferation In Vitro

miR‑339‑5p negatively regulates loureirin A‑induced hair follicle stem cell differentiation by targeting DLX5

A simple culture method for epithelial stem cells derived from human hair follicle

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