Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34

Carol S. Trempus; Rebecca J. Morris; Carl D. Bortner; George Cotsarelis; Randall S. Faircloth; Jeffrey M. Reece; Raymond W. Tennant

doi:10.1046/j.1523-1747.2003.12088.x

Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34

Carol S. Trempus, Rebecca J. Morris, Carl D. Bortner, George Cotsarelis, Randall S. Faircloth, Jeffrey M. Reece, Raymond W. Tennant

Research output: Contribution to journal › Article › peer-review

504 Scopus citations

Abstract

It is widely believed that epithelial stem cells reside in the hair follicle bulge region. We investigated the hematopoietic stem and progenitor cell marker, CD34, as a potential marker of hair follicle bulge keratinocytes. Using a CD34-specific antibody, we identified intense membrane staining on keratinocytes in the bulge region of the mouse hair follicle. CD34 expression colocalized with both slowly cycling (label retaining) cells and keratin 15 expression. Live CD34⁺ keratinocytes were positively selected using antibodies to CD34 and α6 integrin in combination with fluorescent activated cell sorting. Sorted cells were analyzed for DNA content, and a staining profile was generated to confirm these cells as keratinocytes. CD34⁺ keratinocytes were predominantly in G_o/G₁, in contrast to CD34^- cells, which had well defined G₂/M and S phases. In addition, CD34⁺ keratinocytes were found to express α6 integrin more intensely than CD34^- cells (p < 0.05), identifying this population as an α6 integrin bright subset. When seeded at clonal density, CD34⁺ keratinocytes formed larger colonies than CD34^- cells (p < 0.05), indicating a higher proliferative potential. All flow-sorted cells were positive for keratin 14 expression, and negative for keratin 1, loricrin, vimentin, and CD31. The majority of CD34⁺ cells (98%) were positive for keratin 6, establishing this population as basal keratinocytes of follicular origin. CD34 message was detected by reverse transcription polymerase chain reaction predominantly in the CD34⁺ keratinocytes, confirming specificity of the antibody. This work is the first to demonstrate that CD34 is a specific marker of bulge cell keratinocytes in the cutaneous epithelium. Furthermore, the use of this marker facilitates isolation of live epithelial cells with stem and progenitor cell characteristics, potentially providing a tool for the study of carcinogen target cells, gene therapy, and tissue engineering applications.

Original language	English (US)
Pages (from-to)	501-511
Number of pages	11
Journal	Journal of Investigative Dermatology
Volume	120
Issue number	4
DOIs	https://doi.org/10.1046/j.1523-1747.2003.12088.x
State	Published - Apr 1 2003
Externally published	Yes

Bibliographical note

Funding Information:
The authors wish to extend their thanks to Norris Flagler and Pat Stockton of the Laboratory of Experimental Pathology at NIEHS for aid in preparing photomicrographs (N.F.) and frozen sections (P.S.) for histology, and Dr. Joseph Haseman (Biostatistics, NIEHS) for statistical analysis. We also wish to thank Drs Jackie Akunda and Anton Jetten for their review of this manuscript. This work was supported in part by NIH grant CA45293 and CA87780 (R.J.M.).

Keywords

Epidermal
Keratinocyte
Stem cell

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title = "Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34",

abstract = "It is widely believed that epithelial stem cells reside in the hair follicle bulge region. We investigated the hematopoietic stem and progenitor cell marker, CD34, as a potential marker of hair follicle bulge keratinocytes. Using a CD34-specific antibody, we identified intense membrane staining on keratinocytes in the bulge region of the mouse hair follicle. CD34 expression colocalized with both slowly cycling (label retaining) cells and keratin 15 expression. Live CD34+ keratinocytes were positively selected using antibodies to CD34 and α6 integrin in combination with fluorescent activated cell sorting. Sorted cells were analyzed for DNA content, and a staining profile was generated to confirm these cells as keratinocytes. CD34+ keratinocytes were predominantly in Go/G1, in contrast to CD34- cells, which had well defined G2/M and S phases. In addition, CD34+ keratinocytes were found to express α6 integrin more intensely than CD34- cells (p < 0.05), identifying this population as an α6 integrin bright subset. When seeded at clonal density, CD34+ keratinocytes formed larger colonies than CD34- cells (p < 0.05), indicating a higher proliferative potential. All flow-sorted cells were positive for keratin 14 expression, and negative for keratin 1, loricrin, vimentin, and CD31. The majority of CD34+ cells (98%) were positive for keratin 6, establishing this population as basal keratinocytes of follicular origin. CD34 message was detected by reverse transcription polymerase chain reaction predominantly in the CD34+ keratinocytes, confirming specificity of the antibody. This work is the first to demonstrate that CD34 is a specific marker of bulge cell keratinocytes in the cutaneous epithelium. Furthermore, the use of this marker facilitates isolation of live epithelial cells with stem and progenitor cell characteristics, potentially providing a tool for the study of carcinogen target cells, gene therapy, and tissue engineering applications.",

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note = "Funding Information: The authors wish to extend their thanks to Norris Flagler and Pat Stockton of the Laboratory of Experimental Pathology at NIEHS for aid in preparing photomicrographs (N.F.) and frozen sections (P.S.) for histology, and Dr. Joseph Haseman (Biostatistics, NIEHS) for statistical analysis. We also wish to thank Drs Jackie Akunda and Anton Jetten for their review of this manuscript. This work was supported in part by NIH grant CA45293 and CA87780 (R.J.M.).",

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AU - Cotsarelis, George

AU - Faircloth, Randall S.

AU - Reece, Jeffrey M.

AU - Tennant, Raymond W.

N1 - Funding Information: The authors wish to extend their thanks to Norris Flagler and Pat Stockton of the Laboratory of Experimental Pathology at NIEHS for aid in preparing photomicrographs (N.F.) and frozen sections (P.S.) for histology, and Dr. Joseph Haseman (Biostatistics, NIEHS) for statistical analysis. We also wish to thank Drs Jackie Akunda and Anton Jetten for their review of this manuscript. This work was supported in part by NIH grant CA45293 and CA87780 (R.J.M.).

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N2 - It is widely believed that epithelial stem cells reside in the hair follicle bulge region. We investigated the hematopoietic stem and progenitor cell marker, CD34, as a potential marker of hair follicle bulge keratinocytes. Using a CD34-specific antibody, we identified intense membrane staining on keratinocytes in the bulge region of the mouse hair follicle. CD34 expression colocalized with both slowly cycling (label retaining) cells and keratin 15 expression. Live CD34+ keratinocytes were positively selected using antibodies to CD34 and α6 integrin in combination with fluorescent activated cell sorting. Sorted cells were analyzed for DNA content, and a staining profile was generated to confirm these cells as keratinocytes. CD34+ keratinocytes were predominantly in Go/G1, in contrast to CD34- cells, which had well defined G2/M and S phases. In addition, CD34+ keratinocytes were found to express α6 integrin more intensely than CD34- cells (p < 0.05), identifying this population as an α6 integrin bright subset. When seeded at clonal density, CD34+ keratinocytes formed larger colonies than CD34- cells (p < 0.05), indicating a higher proliferative potential. All flow-sorted cells were positive for keratin 14 expression, and negative for keratin 1, loricrin, vimentin, and CD31. The majority of CD34+ cells (98%) were positive for keratin 6, establishing this population as basal keratinocytes of follicular origin. CD34 message was detected by reverse transcription polymerase chain reaction predominantly in the CD34+ keratinocytes, confirming specificity of the antibody. This work is the first to demonstrate that CD34 is a specific marker of bulge cell keratinocytes in the cutaneous epithelium. Furthermore, the use of this marker facilitates isolation of live epithelial cells with stem and progenitor cell characteristics, potentially providing a tool for the study of carcinogen target cells, gene therapy, and tissue engineering applications.

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Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34

Abstract

Bibliographical note

Keywords

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