- Email: [email protected]
Identification of hematopoietic cell populations from the dermal papillae of human hair follicles
Identification of Hematopoietic Cell Populations From the Dermal Papillae of Human Hair Follicles C. Shi, Y. Mai, and T. Cheng ABSTRACT Hematopoietic stem cell (HSC) activity has been identified from the hair follicles (HFs) in mice; however, it has not been identified in human HFs. We used immunohistochemistry and flow cytometry to identify cultured dermal papilla (DP) cells expressing CD45 to test for hematopoietic activity in colony-forming assays of granulocyte/macrophage hematopoietic progenitors (CFU-GM). Occasional CD45-positive cells were detected in cultured DP cells. After in vitro stimulation with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) for 7 days, about 1% of the cells were CD45-positive by flow cytometry analysis, an fiftyfold expansion in cell numbers. We further examined whether mesenchymal stem/progenitor cells reside in human dermal papillae. Cultured DP papilla cells incubated with monoclonal antibodies to remove the CD45 positive cells were induced into multilineage differentiation with the formation of CFU-GM. Our findings preliminarily indicate that human dermal papilla contain at least a CD45-positive hematopoietic cell population and a mesenchymal stem/progenitor cell population.
R
ECENT STUDIES SUGGEST that several stem/progenitor cell populations reside in skin and hair follicles. Ectopic hematopietic tissue may be induced after subcutaneous implantation of demineralized tooth matrix with human bone morphogenetic protein 2.1,2 Several leukocyte cell populations reside within or around hair follicles (HFs) in skin.3 Hair follicle dermal cells have been found to have hematopoietic stem cells (HSCs) that can repopulate the hematopoietic system in mice,4 a finding that suggests therapeutic implications. However, whether cells from human HFs display hematopoietic cell activity is not clear. The dermal papillae (DP) of hair follicles are anatomically well-defined regions that can be dissected microscopically to examine whether hematopoietic stem/progenitor cells could be generated from human scalp tissue. MATERIALS AND METHODS Isolation and Culture of Human DP Cells Scalp tissue specimens were obtained from three male donors (age 21 to 30 years) after obtaining written informed consent, and approval of the protocol by the ethics committee of our university. Human scalp dermal papillae were isolated as previously described:5 In brief, dermal tissues were mechanically dissected, digested with collagenase, and sedimented. Single dermal papillae dissected microscopically were cultured in Iscove’s modified Dulbecco’s medium (IMDM) with 10% fetal bovine serum. The
papilla explants were incubated in a humidified incubator (5% CO2/37°C). When the papilla cells were more than 80% confluent, they were dissociated with 0.25% trypsin for serial passage.
Immunocytochemistry Immunocytochemistry study with the peroxidase-labeled streptavidin biotin method was used to detect CD45-positive cells among cultured human DP cells. After fixation with 4% buffered paraformaldehyde, blockage for 30 minutes in PBS with 10% BSA, the cells were incubated with primary antibodies (4°C/24 hour). Thereafter a 2-hour incubation with secondary antibodies and peroxidase complexes was performed at room temperature. A wash for 15 minutes in 0.01 mol/L PBS was used in all steps. The reaction product was developed with 0.05% DAB. After staining, the result was observed microscopically. From the Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined Injury, Department of Preventive Medicine, Third Military Medical University, Chongqing City, China. This work was supported by the state key project on basic science research (grant no. 1999054205). Address reprint requests to Dr Shi Chunmeng, Institute of Combined Injury, Department of Preventive Medicine, Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing City, 400038, China. E-mail: [email protected]
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.11.104
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3208
Transplantation Proceedings, 36, 3208 –3211 (2004)
HEMATOPOIETIC CELLS IN HUMAN HAIR FOLLICLES
Confirmation of Multilineage Differentiation To induce differentiation, DP cells in IMDM supplemented with 10% FBS (as a control) were cultured either nontreated or treated for 14 days with one of the two induction media: osteogenic medium: IMDM supplemented with 10% FBS, 0.1 mol/L dexamethasone, and 0.1 g/mL BMP-2; or adipogenic medium: IMDM supplemented with 10% FBS, 0.5 mmol/L isobutyl-methylxanthine (IMBX), and 1 mol/L dexamethasone. Differentiation was confirmed by histological staining and immunohistochemistry studies. Osteogenic changes were detected by alkaline phosphatase activity and alizarin red stain and adipogenic activity by oil red staining. The added agents such as dexamethasone, growth factors, or other supplements were changed every 3 days by replacing half the volume of the culture media. Cells maintained in control medium were examined as negative controls.
Preparation of Bone Marrow Cells and CFU-GM Assay Bone marrow specimens collected from three normal male donors were prepared as single nucleated cell suspensions. CD45-depleted DP cells were incubated with CD45 antibodies in 24-well dishes. When the cells become confluent, they were overlain with semisolid culture medium in the presence of bone marrow cells (2 ⫻ 105), methyl-cellulose (0.9%) in IMDM, L-glutamine (2.0 mmol/L), horse serum (30%), 2-mercaptoethanol (0.1 mmol/L), penicillin and streptomycin (100 U/mL and 100 ug/mL, respectively), and GM-CSF (10 ug/mL). Cells were incubated in a humidified incubator containing 5% CO2 at 37°C. After incubation for 7 days, colonies containing 50 or more cells were counted under an inverted microscope.
Flow Cytometry (FACS) Analysis Cultured DP cells first incubated with the primary unconjugated monoclonal antibody were washed twice and incubated with FITCconjugated mice anti-human antibody. Data on 10,000 cell fluorescence events were acquired and analyzed by flow cytometry using a
3209 FACSCalibur instrument (Becton Dickinson, San Jose, Calif, USA) using CellQuest software.
Statistical Analysis Data expressed as mean values ⫾ standard deviations were evaluated for statistical significance by Student t test. Statistical significance was assumed at P values less than .05.
RESULTS Identification of CD45-Positive Hematopoietic Cell Population From Human DPs
In culture, papilla cells spread slowly like fibroblasts (Fig 1A), eventually reaching confluency. To determine whether hematopoietic cells were present in the papilla, DP cells were incubated with anti-CD45 monoclonal antibody. Occasional positive cells bearing CD45, a marker for hematopoietic cells, were observed in confluent DP cells by immunohistochemistry (Fig 1B). However, rare positive cells could not be detected by flow cytometry and produced only a small number of hematopoietic colonies (approximately 1 in 5000 DP cells plated) as evidenced by colony-forming units in granulocyte/macrophage hematopoietic progenitors (CFU-GM) assay (Fig 1C). We next asked whether the CD45-positive DP cells could be expanded by hematopoietic growth factors in vitro. The papilla cells were further cultured in the presence of interleukin-3 (IL-3, 20 ng/mL), granulocytemacrophage colony-stimulating factor (GM-CSF, 10 ng/mL), and granulocyte colony-stimulating factor (G-CSF, 20 ng/mL) for 7 days to seek expansion of CD45-positive DP cells. Macroscopic colonies of CD45-positive cells were produced in culture (Fig 1D); about 1% of cytokine-treated DP cells were CD45-positive by flow cytometry analysis, suggesting a fiftyfold expansion in cell number (Fig 2). These data
Fig 1. Human dermal papilla cells in culture (⫻200). (A) Dermal papilla cells in culture. (B) One CD45-positive cell in cultured dermal papilla cells. (C) Hematopoietic colony formed in cultured dermal papilla cells subjected to CFU-GM assay. (D) CD45-positive colonies in cultured dermal papilla cells in the presence of IL-3, GM-CSF, and G-CSF.
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Fig 2. FACS analysis of CD45positive cells in cultured DP cells after treatment with IL-3, GM-CSF, and G-CSF.
preliminarily suggest the presence of a small population of CD45-positive hematopoietic cells in human hair papilla. In contrast, similar experiments performed on nonfollicular dermis and hair follicular epithelium, no CD45-positive hematopoietic cells or colonies were identified in the same assay. Identification of Mesenchymal Stem/Progenitor Cell Population From Human DP Cells
To test whether mesenchymal stem/progenitor cells reside in human dermal papillae, cultured DP cells were incubated with monoclonal antibodies against CD45 to remove CD45-positive cells (CD45-negative DP cells). After transfer into specific induction media for osteocyte or adipocyte differentiation for 2 weeks, CD45-negative DP cells in osteogenic medium showed increased calcium accumulation, as revealed by alizarin red staining, and adipocytes those in adipogenic medium, produced oil red-positive material (data not shown). Clonal analysis suggested that DP also contains a population of mesenchymal stem/progenitor cells. In contrast, hair follicular epithelium failed to generate mesenchymal stem/progenitor cell activities when tested using the same assay. To assess whether CD45-negative DP cell layers could support hematopoiesis, we established cocultures with marrow hematopoietic cells on DP cell layers in CFU-GM assay. Single nucleated cell suspensions of bone marrow specimens from three normal male donors were incubated for 14 days. About 48.5 ⫾ 4.1 colonies per 2 ⫻ 105 marrow nucleated cells were produced on the DP cell layer, with 14.2 ⫾ 1.3 colonies, in the controls without the DP cell layer. Thus DP cells support the growth of hematopoietic stem/ progenitor cells and may serve as a source of mesenchymal stem/progenitor cell activity in vitro. DISCUSSION
Hematopoiesis is a complex process requiring the collaborative efforts of hematopoietic cell lineages and supportive stromal cell lineages. Hematopoietic stem cell
(HSC) transplantation for treatment of hematological and solid malignancies is increasingly used in conjunction with radio and chemotherapy. However, the present source of hematopoietic stem/progenitor cells and mesenchymal stem/progenitor cells can not fulfill the clinical demand. Utilization of adult stem cells is a possible solution for replacement therapy. HSC activity was identified from remaining hair follicles. Our other studies have shown that populations of multipotent cells from rat dermis have the differentiation capacity to produce osteocytes and adipocyes and to support the growth of CFU-GM colonies in vitro as well as promote hematopoietic recovery in sublethally irradiated rats.5 In this study, our data suggested that human DP may contain at least a CD45-positive cell population with hematopoietic cell activity, and a mesenchymal stem/progenotor cell population supportive of hematopoiesis. Based on the present literature of hematopoietic stem cell populations in hair follicles and our data in this study, we hypothesized that DP may serve as a local reservior of hematopoietic stem/progenitor and mesenchymal stem/progenitor cells. Given their easy accessibility, DP may at least represent an alternative origin of hematopoietic stem/ progenitor cells to repopulate the hematopoietic system and of mesenchymal stem/progenitor cells to restore the marrow microenvironment. However, the functional activities of these cell populations in vivo still remain to be tested.6,7
REFERENCES 1. Gurevich OA, Samoilina NL, Medvinskii AL, et al: Induced hematopoietic foci in mice. I. Induction of extraskeletal hematopoietic areas using demineralized tooth matrix. Gematol Transfuziol 35:7, 1990 (in Russian) 2. An J, Rosen V, Cox K, et al: Recombinant human bone morphogenetic protein-2 induces a hematopoietic microenvironment in the rat that supports the growth of stem cells. Exp Hematol 24:768, 1996 3. Kumamoto T, Shalhevet D, Matsue H, et al: Hair follicles serve as local reservoirs of skin mast cell precursors. Blood 102:1654, 2003
HEMATOPOIETIC CELLS IN HUMAN HAIR FOLLICLES 4. Lako M, Armstrong L, Cairns PM, et al: Hair follicle dermal cells repopulate the mouse haematopoietic system. Journal of Cell Science 115:3967, 2002 5. Warren R, Chestnut MH, Wong TK, et al: Improved method for the isolation and cultivation of human scalp dermal papilla cells. J Invest Dermatol 5:693, 1992
3211 6. Devine SM, Hoffman R: Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Curr Opin Hematol 6:358, 2000 7. Shi C, Mai Y, Qu J, et al: Transplantation of dermal multipotent cells promotes the hematopoietic recovery in sublethally irradiated rats. Chin J Radiation Protection 23:435, 2003 (in Chinese)
R
ECENT STUDIES SUGGEST that several stem/progenitor cell populations reside in skin and hair follicles. Ectopic hematopietic tissue may be induced after subcutaneous implantation of demineralized tooth matrix with human bone morphogenetic protein 2.1,2 Several leukocyte cell populations reside within or around hair follicles (HFs) in skin.3 Hair follicle dermal cells have been found to have hematopoietic stem cells (HSCs) that can repopulate the hematopoietic system in mice,4 a finding that suggests therapeutic implications. However, whether cells from human HFs display hematopoietic cell activity is not clear. The dermal papillae (DP) of hair follicles are anatomically well-defined regions that can be dissected microscopically to examine whether hematopoietic stem/progenitor cells could be generated from human scalp tissue. MATERIALS AND METHODS Isolation and Culture of Human DP Cells Scalp tissue specimens were obtained from three male donors (age 21 to 30 years) after obtaining written informed consent, and approval of the protocol by the ethics committee of our university. Human scalp dermal papillae were isolated as previously described:5 In brief, dermal tissues were mechanically dissected, digested with collagenase, and sedimented. Single dermal papillae dissected microscopically were cultured in Iscove’s modified Dulbecco’s medium (IMDM) with 10% fetal bovine serum. The
papilla explants were incubated in a humidified incubator (5% CO2/37°C). When the papilla cells were more than 80% confluent, they were dissociated with 0.25% trypsin for serial passage.
Immunocytochemistry Immunocytochemistry study with the peroxidase-labeled streptavidin biotin method was used to detect CD45-positive cells among cultured human DP cells. After fixation with 4% buffered paraformaldehyde, blockage for 30 minutes in PBS with 10% BSA, the cells were incubated with primary antibodies (4°C/24 hour). Thereafter a 2-hour incubation with secondary antibodies and peroxidase complexes was performed at room temperature. A wash for 15 minutes in 0.01 mol/L PBS was used in all steps. The reaction product was developed with 0.05% DAB. After staining, the result was observed microscopically. From the Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined Injury, Department of Preventive Medicine, Third Military Medical University, Chongqing City, China. This work was supported by the state key project on basic science research (grant no. 1999054205). Address reprint requests to Dr Shi Chunmeng, Institute of Combined Injury, Department of Preventive Medicine, Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing City, 400038, China. E-mail: [email protected]
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.11.104
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3208
Transplantation Proceedings, 36, 3208 –3211 (2004)
HEMATOPOIETIC CELLS IN HUMAN HAIR FOLLICLES
Confirmation of Multilineage Differentiation To induce differentiation, DP cells in IMDM supplemented with 10% FBS (as a control) were cultured either nontreated or treated for 14 days with one of the two induction media: osteogenic medium: IMDM supplemented with 10% FBS, 0.1 mol/L dexamethasone, and 0.1 g/mL BMP-2; or adipogenic medium: IMDM supplemented with 10% FBS, 0.5 mmol/L isobutyl-methylxanthine (IMBX), and 1 mol/L dexamethasone. Differentiation was confirmed by histological staining and immunohistochemistry studies. Osteogenic changes were detected by alkaline phosphatase activity and alizarin red stain and adipogenic activity by oil red staining. The added agents such as dexamethasone, growth factors, or other supplements were changed every 3 days by replacing half the volume of the culture media. Cells maintained in control medium were examined as negative controls.
Preparation of Bone Marrow Cells and CFU-GM Assay Bone marrow specimens collected from three normal male donors were prepared as single nucleated cell suspensions. CD45-depleted DP cells were incubated with CD45 antibodies in 24-well dishes. When the cells become confluent, they were overlain with semisolid culture medium in the presence of bone marrow cells (2 ⫻ 105), methyl-cellulose (0.9%) in IMDM, L-glutamine (2.0 mmol/L), horse serum (30%), 2-mercaptoethanol (0.1 mmol/L), penicillin and streptomycin (100 U/mL and 100 ug/mL, respectively), and GM-CSF (10 ug/mL). Cells were incubated in a humidified incubator containing 5% CO2 at 37°C. After incubation for 7 days, colonies containing 50 or more cells were counted under an inverted microscope.
Flow Cytometry (FACS) Analysis Cultured DP cells first incubated with the primary unconjugated monoclonal antibody were washed twice and incubated with FITCconjugated mice anti-human antibody. Data on 10,000 cell fluorescence events were acquired and analyzed by flow cytometry using a
3209 FACSCalibur instrument (Becton Dickinson, San Jose, Calif, USA) using CellQuest software.
Statistical Analysis Data expressed as mean values ⫾ standard deviations were evaluated for statistical significance by Student t test. Statistical significance was assumed at P values less than .05.
RESULTS Identification of CD45-Positive Hematopoietic Cell Population From Human DPs
In culture, papilla cells spread slowly like fibroblasts (Fig 1A), eventually reaching confluency. To determine whether hematopoietic cells were present in the papilla, DP cells were incubated with anti-CD45 monoclonal antibody. Occasional positive cells bearing CD45, a marker for hematopoietic cells, were observed in confluent DP cells by immunohistochemistry (Fig 1B). However, rare positive cells could not be detected by flow cytometry and produced only a small number of hematopoietic colonies (approximately 1 in 5000 DP cells plated) as evidenced by colony-forming units in granulocyte/macrophage hematopoietic progenitors (CFU-GM) assay (Fig 1C). We next asked whether the CD45-positive DP cells could be expanded by hematopoietic growth factors in vitro. The papilla cells were further cultured in the presence of interleukin-3 (IL-3, 20 ng/mL), granulocytemacrophage colony-stimulating factor (GM-CSF, 10 ng/mL), and granulocyte colony-stimulating factor (G-CSF, 20 ng/mL) for 7 days to seek expansion of CD45-positive DP cells. Macroscopic colonies of CD45-positive cells were produced in culture (Fig 1D); about 1% of cytokine-treated DP cells were CD45-positive by flow cytometry analysis, suggesting a fiftyfold expansion in cell number (Fig 2). These data
Fig 1. Human dermal papilla cells in culture (⫻200). (A) Dermal papilla cells in culture. (B) One CD45-positive cell in cultured dermal papilla cells. (C) Hematopoietic colony formed in cultured dermal papilla cells subjected to CFU-GM assay. (D) CD45-positive colonies in cultured dermal papilla cells in the presence of IL-3, GM-CSF, and G-CSF.
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SHI, MAI, AND CHENG
Fig 2. FACS analysis of CD45positive cells in cultured DP cells after treatment with IL-3, GM-CSF, and G-CSF.
preliminarily suggest the presence of a small population of CD45-positive hematopoietic cells in human hair papilla. In contrast, similar experiments performed on nonfollicular dermis and hair follicular epithelium, no CD45-positive hematopoietic cells or colonies were identified in the same assay. Identification of Mesenchymal Stem/Progenitor Cell Population From Human DP Cells
To test whether mesenchymal stem/progenitor cells reside in human dermal papillae, cultured DP cells were incubated with monoclonal antibodies against CD45 to remove CD45-positive cells (CD45-negative DP cells). After transfer into specific induction media for osteocyte or adipocyte differentiation for 2 weeks, CD45-negative DP cells in osteogenic medium showed increased calcium accumulation, as revealed by alizarin red staining, and adipocytes those in adipogenic medium, produced oil red-positive material (data not shown). Clonal analysis suggested that DP also contains a population of mesenchymal stem/progenitor cells. In contrast, hair follicular epithelium failed to generate mesenchymal stem/progenitor cell activities when tested using the same assay. To assess whether CD45-negative DP cell layers could support hematopoiesis, we established cocultures with marrow hematopoietic cells on DP cell layers in CFU-GM assay. Single nucleated cell suspensions of bone marrow specimens from three normal male donors were incubated for 14 days. About 48.5 ⫾ 4.1 colonies per 2 ⫻ 105 marrow nucleated cells were produced on the DP cell layer, with 14.2 ⫾ 1.3 colonies, in the controls without the DP cell layer. Thus DP cells support the growth of hematopoietic stem/ progenitor cells and may serve as a source of mesenchymal stem/progenitor cell activity in vitro. DISCUSSION
Hematopoiesis is a complex process requiring the collaborative efforts of hematopoietic cell lineages and supportive stromal cell lineages. Hematopoietic stem cell
(HSC) transplantation for treatment of hematological and solid malignancies is increasingly used in conjunction with radio and chemotherapy. However, the present source of hematopoietic stem/progenitor cells and mesenchymal stem/progenitor cells can not fulfill the clinical demand. Utilization of adult stem cells is a possible solution for replacement therapy. HSC activity was identified from remaining hair follicles. Our other studies have shown that populations of multipotent cells from rat dermis have the differentiation capacity to produce osteocytes and adipocyes and to support the growth of CFU-GM colonies in vitro as well as promote hematopoietic recovery in sublethally irradiated rats.5 In this study, our data suggested that human DP may contain at least a CD45-positive cell population with hematopoietic cell activity, and a mesenchymal stem/progenotor cell population supportive of hematopoiesis. Based on the present literature of hematopoietic stem cell populations in hair follicles and our data in this study, we hypothesized that DP may serve as a local reservior of hematopoietic stem/progenitor and mesenchymal stem/progenitor cells. Given their easy accessibility, DP may at least represent an alternative origin of hematopoietic stem/ progenitor cells to repopulate the hematopoietic system and of mesenchymal stem/progenitor cells to restore the marrow microenvironment. However, the functional activities of these cell populations in vivo still remain to be tested.6,7
REFERENCES 1. Gurevich OA, Samoilina NL, Medvinskii AL, et al: Induced hematopoietic foci in mice. I. Induction of extraskeletal hematopoietic areas using demineralized tooth matrix. Gematol Transfuziol 35:7, 1990 (in Russian) 2. An J, Rosen V, Cox K, et al: Recombinant human bone morphogenetic protein-2 induces a hematopoietic microenvironment in the rat that supports the growth of stem cells. Exp Hematol 24:768, 1996 3. Kumamoto T, Shalhevet D, Matsue H, et al: Hair follicles serve as local reservoirs of skin mast cell precursors. Blood 102:1654, 2003
HEMATOPOIETIC CELLS IN HUMAN HAIR FOLLICLES 4. Lako M, Armstrong L, Cairns PM, et al: Hair follicle dermal cells repopulate the mouse haematopoietic system. Journal of Cell Science 115:3967, 2002 5. Warren R, Chestnut MH, Wong TK, et al: Improved method for the isolation and cultivation of human scalp dermal papilla cells. J Invest Dermatol 5:693, 1992
3211 6. Devine SM, Hoffman R: Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Curr Opin Hematol 6:358, 2000 7. Shi C, Mai Y, Qu J, et al: Transplantation of dermal multipotent cells promotes the hematopoietic recovery in sublethally irradiated rats. Chin J Radiation Protection 23:435, 2003 (in Chinese)