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Keywords:

  • Stem cells;
  • Aging;
  • Human skin-derived precursors

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

A major unanswered question in autologous cell therapy is the appropriate timing for cell isolation. Many of the putative target diseases arise with old age and previous evidence, mainly from animal models, suggests that the stem/progenitor cell pool decreases steadily with age. Studies with human cells have been generally hampered to date by poor sample availability. In recent years, several laboratories have reported on the existence, both in rodents and humans, of skin-derived precursor (SKP) cells with the capacity to generate neural and mesodermal progenies. This easily obtainable multipotent cell population has raised expectations for their potential use in cell therapy of neurodegeneration. However, we still lack a clear understanding of the spatiotemporal abundance and phenotype of human SKPs. Here we show an analysis of human SKP abundance and in vitro differentiation potential, by using SKPs isolated from four distinct anatomic sites (abdomen, breast, foreskin, and scalp) from 102 healthy subjects aged 8 months to 85 years. Human SKP abundance and differentiation potential decrease sharply with age, being extremely difficult to isolate, expand, and differentiate when obtained from the elderly. Our data suggest preserving human SKP cell banks early in life would be desirable for use in clinical protocols in the aging population. Stem Cells 2009;27:1164–1172


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

The relative contribution of adult stem cells (as compared with more committed, differentiated cells) to organ homeostasis and its putative relationship to human body aging are fundamental questions in biology that remain poorly understood [1–3]. Data generated in animal models such as rodents are often difficult to extrapolate to human beings because the animal, in most cases, is only a few months old at the time of analysis. Besides, meaningful developmental data from human beings such as lineage tracing are difficult to obtain for obvious ethical reasons. However, there are some published studies of human stem/progenitor cell fate that avoid recurrence to grafting to animal models. Through postmortem analyses of patients who had been treated with bromodeoxyuridine (BrdU) and neurosurgical resections, we have learned that new neurons are generated from dividing progenitor cells in the dentate gyrus, but not in the neocortex of the adult human brain [4–6]. Moreover, retrospective 14C-dating can determine the average age of cells in postmortem samples of the human intestine, intercostal skeletal muscle, and brain, thus providing a new tool to measure cell turnover in human beings [4, 7].

However, the question of functional availability of the stem/progenitor pool of a given human tissue and its relationship to age is basically unanswered. To our knowledge, evidence for stem/progenitor cell pool depletion in the human body with aging includes peripheral blood stem cells [8, 9], satellite cells of skeletal muscle [10], and putative stem cells of the epidermal compartment of the skin [11]. Interestingly, in a retrospective analysis of >6,900 bone marrow transplantations conducted by the National Marrow Donor Program, age was the only donor trait significantly associated with recipient survival [12]. On the other hand, donor age does not apparently affect phenotype of human corneal epithelial cells expanded by limbal culture, differentiation potential of mesenchymal stromal cells from bone marrow, or the number and phenotype of pluripotent hepatic progenitors [13–16]. Although some of the observed discrepancies will undoubtedly be accounted for by tissue-specific differences [17], ill-defined cell populations of origin and unreliable assays used for phenotypic readout have also contributed to blur the general picture [18]. Besides, little is known as to whether age-related changes in stem/progenitor cell functionality are due to intrinsic (cell autonomous) factors or otherwise induced by extrinsic factors such as aging of the somatic environment (stem cell niche) [19].

Human skin is an easily accessible reservoir of adult stem/progenitor cells with a potential use in cell therapy. Among many others, multipotent progenitor cells (dubbed skin-derived precursors – SKPs) with potentiality to generate neural and mesodermal progeny have been described in both rodents and humans (reviewed by Fernandes et al. [20]). These progenitor cells have been reported to reside in the dermal papillae (DP) of human hair follicles [21, 22], their physiological role being unclear at the moment. This easily obtainable multipotent cell population has raised expectations because of their potential use in cell therapy of neurodegeneration [23–29]. However, we still lack a clear understanding of the spatiotemporal abundance and phenotype of human SKPs. To address these issues, we have taken advantage of availability of skin from healthy subjects undergoing surgical procedures in four distinct anatomic sites (abdomen, breast, foreskin, and scalp). We present an analysis of human SKP abundance and differentiation potential in vitro, by using SKPs isolated from 102 subjects aged 8 months to 85 years. We show an age-dependent, functional depletion of the SKP pool in adult human skin, as seen either in progenitor cell number and/or differentiation potential.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Derivation and Passage of SKP Cell Suspension from Human Skin Biopsies

A total of 104 human biopsies were obtained from 102 donors who gave informed consent, after protocol approval by the Clinical Research Ethical Committees of Hospital Donostia and Policlínica Gipuzkoa (supporting information Table 1). Informed consent was obtained from parents when donors were under age 12. Biopsies were stored in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% P/S and processed at a maximum 4 hours postsurgery. We used the protocol of Toma et al. [30] with minor modifications depending on the tissue origin, as detailed in supporting information. For methylcellulose passaging experiments, methylcellulose stock solution (2.8%, R&D Systems, Minneapolis, MN, http://www.rndsystems.com HSC001) was diluted to a final concentration of 0.8% in proliferation medium.

Derivation and Passage of Human Dermal Fibroblasts

Foreskin of 1–2 cm2 pieces derived from voluntary circumcisions of men were cut into small pieces and incubated with Type I Collagenase at 37°C for 2–4 hours, until full tissue disaggregation was achieved. Cells were resuspended in DMEM supplemented with 10% fetal bovine serum (PAA, Laboratories GmbH, Linz, Austria; http://www.paa.com), 1% P/S, and 1% L-glutamine. Human dermal fibroblasts (FBS) (HDF) cells were initially plated at a density of 90,000 cell/cm2 in tissue culture-treated flasks (BD Biosciences “Falcon”, Franklin Lakes, NJ; http://www.bdbiosciences.com). If passaged further, HDFs were seeded at a density of 1,000 cells/cm2 in the same culture medium.

Adherent Culture of Human SKPs

We followed the protocol of Joannides et al. [31]. Primary dermospheres (day 7) were centrifuged at 1,000 rpm for 5 minutes. The dermosphere pellet was resuspended in DMEM/F12 (3:1) supplemented with 20% FBS (Cambrex, Walkersville, MD, http://www.cambrex.com), B27, 1% P/S, and 1% L-glutamine (adhesion medium). Twenty to forty dermospheres were seeded per 25 cm2 flask in 5 ml medium. In these conditions, dermospheres adhered to the flasks with cells continuously migrating out of the sphere. When confluent, adherent SKP cultures were expanded at a density of 1,000 cells/cm2 supplemented with 20% FBS.

Cell Proliferation Analyses

Cells were seeded at a density of 1,000 cells/cm2 in six-well plates, trypsinized and counted by trypan blue exclusion at day 7. As a proliferation control, HDFs were used. Cumulative cell doublings were calculated as follows: number of cell doublings = log2 (no. of cells at subculture/no. of cells seeded).

Histological, Histochemical, and Immunofluorescence Staining of Skin Sections

Pieces of human skin of 0.5 cm2 were embedded in OCT resin (Tissue-Tek, Sakura Finetechnical Co., Ltd., Tokyo, Japan, http://www.sakuraus.com) and snap-frozen in liquid nitrogen. Five micrometer thick, consecutive skin sections were cut at a Tissue-Tek II Cryostat (Miles, Diagnostic/Bayer, Inc., Pittsburg, PA) and maintained at −80°C for at least 24 hours. Consecutive tissue sections were either stained with hematoxylin and eosin or processed for immunofluorescence. To this end, sections were fixed with cold acetone for 10 minutes at 4°C and washed three times with phosphate buffered saline (PBS) for 10 minutes. Primary antibodies specific for human Nestin (Abcam, Cambridge, U.K., http://www.abcam.com; Chemicon, Temecula, CA, http://www.chemicon.com), Fibronectin (Sigma-Aldrich Co., St. Louis, MO; http://www.sigmaaldrich.com), p75NTR (Alomone Labs, Jerusalem, Israel; http://www.alomone.com), and Vimentin (Sigma-Aldrich) were incubated for 1 hour at room temperature (RT). Sections were then washed twice with PBS and incubated with secondary antibodies conjugated to the appropriate fluorophores (Alexa Fluor 488 and Alexa Fluor 546; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) for 1 hour at RT. Sections were then washed twice with PBS, once with distilled water, and mounted with Gel Mount (Biomeda, Foster City, CA; http://www.biomeda.com) to be analyzed by confocal microscopy (LSM 510 META, Carl Zeiss Inc., Jena, Germany; http://www.zeiss.com). In situ histochemical semiquantitative detection of alkaline phosphatase activity was achieved by using leukocyte alkaline phosphatase kit (Sigma-Aldrich 85L3R), following manufacturer's instructions.

Differentiation of Human SKPs

Primary dermospheres (2–5 units) were seeded in 12-mm diameter coverslips (Menzel-Gläser, Braunschweig, Germany, http://www.menzel.de) previously treated to improve adherence with poly-L-Lysine (Fluka, Sigma-Aldrich) and Laminin (Sigma-Aldrich) or with extracellular cell matrix rich in Laminin 5 from 804G cells [32]. To this end, the 804G cells grown confluent on coverslips were removed with 20 mM NH4OH for 5–10 minutes, followed by extensive washing of the coverslip, previous to dermosphere seeding. Once the dermospheres were attached, and depending on the experiment, three differentiation media were used: (a) basal medium: same as proliferation medium with no growth factors and supplemented with 1%–5% FBS (Cambrex); (b) neurogenic medium (low): dermospheres were resuspended in basal medium for 3 days and then in Neural Stem Cell medium (Chemicon) supplemented with 1% FBS (Cambrex), 1% N2 (Gibco, Grand Island, NY, http://www.invitrogen.com), 4 μM Forskolin (Sigma-Aldrich), and 10 ng/ml Heregulin-β (Peprotech, Rocky Hill, NJ, http://www.peprotech.com); or (c) neurogenic medium (high): DMEM-F12 3:1 supplemented with 1% N2, 5 μM Forskolin, and 40 ng/ml Heregulin-β. All differentiation experiments were performed for 2 weeks, replacing differentiation media every 3 days.

Immunofluorescence of Dermospheres and Adherent SKPs

Primary day 7 dermospheres (20–40 units) were seeded in 12-mm diameter; coverslips or chamber slides (Nunc, Rochester, NY, http://www.nuncbrand.com) previously treated to improve adherence as above. Two hours later, dermospheres were rinsed with PBS and fixed with 4% paraformaldehyde for 45 minutes, PBS-washed three times and processed for immunofluorescence (see later). Cells attached to coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 15 minutes, PBS-washed three times, and processed for immunofluorescence. Briefly, cells were permeabilized with 0.5% Triton X-100 for 10 minutes, washed twice with PBS, and blocked with 10% FBS (in PBS) for 20 minutes at RT. Primary and secondary antibodies were incubated with 10% FBS (in PBS) for 1 hour at RT, with three 10 minutes PBS washes in between. Cells were washed again with PBS and nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich), Topro III (Molecular Probes Inc.), or Propidium iodide (Sigma-Aldrich) depending on the experiment. Primary antibodies used include human βIII-tubulin (Abcam), smooth muscle actin-SMA (Sigma-Aldrich), and Versican (Affinity Bioreagent, Thermo Fisher Scientific, Rockford, IL; http://www.bioreagents.com). Secondary antibodies were used as mentioned earlier.

Cell Differentiation Analyses

To estimate the percent cells differentiated to a given phenotype, the central 9-mm of the diameter of the coverslips were fully counted. Total cells were estimated through nuclear counts and cells in a given phenotype through counting of cells positive to the aforementioned markers.

Dermosphere Quantification

Primary day 7 dermospheres were attached to 24-well plates previously treated with 804G cell matrix, washed with PBS, fixed with ice cold Methanol for 10 minutes, and stained with Crystal Violet dye (Sigma-Aldrich) for 15 minutes at RT. Excess dye was washed away with water. Dermospheres obtained for each 25,000 cells were estimated by counting the actual spheres present in five fields covering 0.03 cm2 each. Dermosphere areas were quantified by a protocol adapted from Lehr et al. [33] by imaging with a ×10 objective and Photoshop 7.0 software (Adobe Systems, San Jose, CA; http://www.adobe.com). Five independent fields were chosen so as to best reflect the overall staining of the dermospheres contained on the entire well. The tolerance level of the Magic Wand tool was adjusted to 32 so that well-stained dermospheres were clearly separated from background. The mean staining intensity (in arbitrary units, AU) of both dermospheres and background was recorded. Subsequently, the apparent dermosphere size (mm2) was calculated as follows: [Mean staining intensity × total area (mm2)]/total staining intensity.

Statistical Analyses

Pearson correlation coefficients and p values were calculated by using the Microsoft Excel and Graphpad software packages (www.graphpad.com/quickcalcs/), respectively. Correlation was deemed significant at the p = .05 level (two-tailed). Exponential regression trendlines shown in Figure 6 and supporting information Figure 8 were calculated by Microsoft Excel software package. R-squared values are displayed on charts.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Derivation of Skin-Derived Precursor (SKP) Cell Suspensions from Human Foreskin Biopsies

To quantify the SKP cell pool in human samples, we set to analyze two characteristics that define a SKP as such: (a) the ability to form spheres when expanded in proliferation medium as first published by Miller and coworkers [20, 34] and (b) the potential to differentiate in vitro to at least two different lineages (neural and smooth muscle) when put in differentiation media, as assessed by immunofluorescence and confocal microscopy (for relevant lineage expression markers). Human biopsies were obtained from healthy subjects after informed consent, by making use of skin tissue remnants of medical procedures such as aesthetic surgery or circumcisions (supporting information Table 1). Human SKPs were derived as described [20, 30]. Primary spheres derived from human foreskin appeared as round floating structures with a regular size at day 7 in proliferation medium, as expected (Fig. 1A). We dubbed the floating spheres as “dermospheres” to emphasize their dermal origin. To confirm that SKPs were being obtained, dermospheres were analyzed by immunofluorescence with the neural precursor marker Nestin and the mature, peripheral neuron marker p75-NTR (Fig. 1B, upper panels). As expected, human foreskin dermospheres were nestin-positive and p75-NTR-negative. Spheres were also positive for dermal markers Vimentin and Fibronectin and negative for glial marker glial fibrillary acidic protein, neural marker βIII-tubulin, and the proteoglycan Versican, as expected (Fig. 4 and data not shown). When allowed to differentiate for 14 days in basal medium, spheres attached to the tissue culture surface and started to express differentiation markers characteristic of the neural (βIII-tubulin) and smooth muscle (smooth muscle actin-SMA) lineages (Fig. 1B, lower panels).

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Figure 1. Isolation and characterization of dermospheres derived from adult human skin. (A): Skin-derived precursor (SKP) cells derived from adult human foreskin biopsies grow as floating dermospheres in proliferation medium. Scale bar, 100 μm. (B): Characterization of primary dermospheres and differentiated cells. Primary dermospheres were attached to coverslips and analyzed by immunofluorescence and confocal microscopy with anti-Nestin and anti-p75-NTR antibodies (upper left and middle panels, respectively) or by phase contrast (right upper panel). The dermospheres show an undifferentiated phenotype, as expected. Cells explanted from the dermospheres and grown for 2 weeks in basal differentiation medium show morphological changes and immune reactivity consistent with neural precursor cells (βIII tubulin, left lower panel) and smooth muscle cells (anti-SMA, central lower panel), respectively. Merged image and cell nuclei counterstained with Hoechst (blue) are shown on the right lower panel, demonstrating specificity of the antisera. Scale bars = 50 μm. Abbreviation: SMA, smooth muscle actin.

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In Vitro Passage of Human Skin-Derived Precursor (SKP) Cells is Dependent on Expansion Protocol and Donor Age

In our hands, human SKP isolation protocol was extremely reproducible, with 75/78 foreskin biopsies analyzed (∼96%) being “spherogenic” in culture (supporting information Fig. 1 and Table 1). When standard seeding conditions were used, dermosphere number at P0 was directly dependent on cell seeding density (supporting information Fig. 2). In contrast, when media containing methylcellulose were used for P0 dermosphere formation, largest number of dermospheres was obtained at 90,000 cells/cm2, with higher cell seeding densities being detrimental for sphere formation. However, the number of cells dissociated from the spheres at each condition was still directly dependent on cell concentration, indicating that the cellularity of dermospheres formed at higher cell concentrations in media containing methylcellulose was also bigger.

On the other hand, in vitro expansion of adult human SKPs in liquid dermosphere culture was hampered by a clear loss of spherogenic precursor cells at P4, following an apparent enrichment within the first two passages in proliferation medium (Fig. 2A, left panel). In contrast, when media containing methylcellulose were used, adult human SKPs were maintained in dermosphere culture through at least four passages although with high variability and slower turnover (Fig. 2A, right panel; data not shown). As a control, both human infant and newborn rat SKPs were maintained for several passages in liquid media dermosphere culture (Fig. 2B), as expected [21, 22, 24, 25, 27, 35].

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Figure 2. In vitro passage of human skin-derived precursor (SKP) cells is dependent on expansion protocol and donor age. (A): Adult human dermospheres were passaged in liquid proliferation medium (left panel) or semisolid, methylcellulose-containing medium (right panel) for four passages. Total dermosphere number at each passage was calculated for a seeding cell density of 10,000 cells per square centimeter. Two independent donor biopsies were used for each experiment. Adult human SKPs were only expanded initially in liquid proliferation medium and in a more prolonged manner when semisolid medium was used. (B): Expansion of human infant and newborn (postnatal day 3) rat dermospheres in liquid proliferation medium. Two independent human or rat SKP cultures were passaged at a constant density of 10,000 cells per square centimeter and the resulting dermospheres quantified at day 7 in proliferation medium. Both infant human cells and rat SKPs were serially passaged as dermosphere cultures.

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When allowed to proliferate in attached culture (as described by Joannides et al. [31]), cells were easily expandable for up to 70–80 cell doublings, when they presumably reached senescence (supporting information Fig. 3). However, differentiation potential of the expanded cells was gradually lost (at least for the neural lineage), both after sphere and attached culture (supporting information Fig. 4). Taken together, our data suggest that adult human SKPs, in contrast to those obtained from the newborn and children up to 12 years of age [30, 36], can be easily isolated but are not expandable under previously published culture conditions. Expansion of adult human SKPs in semisolid but not in liquid media might be a reflection of aggregate formation in the liquid dermosphere cultures.

Derivation and Phenotypic Characterization of Human SKPs from Other Skin Areas

Human SKPs have been previously derived from scalp [22, 34, 37, 38], foreskin [30, 36], arm [39], and the facial area [22]. However, there has been little or no phenotypic characterization of the SKPs isolated from these areas. To phenotypically characterize human SKPs from other areas than foreskin, tissue remnants from aesthetic surgery operations were used to derive SKPs from human scalp (n = 12), breast (n = 7), and abdomen (n = 7; Fig. 3). All areas produced SKPs in a reproducible manner, although in much lower numbers than foreskin (Fig. 3D). Primary dermospheres derived from human foreskin, scalp, breast, and abdomen were attached to the cell culture substrate and analyzed by immunofluorescence with SKP markers Nestin, Fibronectin, Vimentin, p75-NTR, and Versican (Fig. 4A). Dermospheres from all areas stained positive for Nestin, Fibronectin, and Vimentin, as expected. However, some tissue-specific patterns were also found: scalp dermospheres were positive for p75-NTR, in contrast to all others, and versican was undetectable in dermospheres derived from foreskin. To assess multipotentiality of SKPs derived from different donor areas, dermospheres from foreskin, scalp, breast, and abdomen were put into basal differentiation media for 14 days (Fig. 4B). SKPs from all areas retained the ability to differentiate into βIII-tubulin-positive and SMA-positive cells. Similar results were also obtained with two different neurogenic media (not shown) indicating that multipotent precursor cells were present in all skin areas analyzed.

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Figure 3. Comparison of human skin-derived precursors (SKPs) derived from other areas. Primary SKPs derived from human scalp (A), breast (B), and abdomen (C) show dermosphere growth in proliferation medium, as observed with foreskin. Scale bars = 200 μm. (D): Reproducibility of the isolation protocol in the different areas. The graph shows the cells isolated per gram of tissue in the 104 biopsies processed of human foreskin (For), scalp (Sca), breast (Bre), and abdomen (Abd). Each circle represents one biopsy. Horizontal lines represent mean values.

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Figure 4. Phenotype comparison of dermospheres derived from different human skin areas. (A): Primary dermospheres derived from human foreskin (a1–j1), scalp (a2–j2), breast (a3–j3), and abdomen (a4–j4) were analyzed by immunofluorescence with anti-Nestin (a,b), Fibronectin (c,d), Vimentin (e,f), p75-NTR (g,h), and Versican (i,j) antibodies. Phase contrast images are shown to the right of the corresponding immunofluorescence picture. Scale bars = 50 μm. (B): Cells explanted from the dermospheres obtained of human foreskin, scalp, breast, and abdomen and grown for 2 weeks in basal differentiation medium showed morphological changes and immune reactivity consistent with neural precursor cells (βIII tubulin, green) and smooth muscle cells (anti-SMA, red), respectively. Cell nuclei, counterstained with Hoechst, are shown in blue. Scale bars = 50 μm.

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Dermal Niche of Human SKPs Within Glabrous Skin Areas

Hair follicle DPs have been proposed as a niche for SKPs both in rodents and human. Foreskin is a glabrous tissue with no hair follicles, a fact that led these authors to speculate about the existence of a different niche for foreskin SKPs [21, 22]. To contribute on the localization pattern of SKPs in the human dermis, we analyzed consecutive sections of human foreskin, scalp, breast, and abdomen by hematoxilin-eosin staining and immunofluorescence detection of SKP markers Fibronectin, Nestin, and Vimentin, as well as p75-NTR for peripheral neurons (Fig. 5 and supporting information Figs. 5 and 6). Cells consistent with the SKP phenotype (Fibronectin+ Nestin+ Vimentin+ p75-NTR−) were observed in discrete locations of every donor tissue, even when interfollicular epidermis was analyzed. Nestin was apparently more abundant in foreskin as compared with other areas. To further characterize these nonpapillary niches for human SKPs, we performed a histochemical assay to detect alkaline phosphatase activity, a known DP marker, in human scalp sections. The assay detected functional alkaline phosphatase activity in discrete locations within the scalp dermis, in regions unrelated to the hair follicle DPs (supporting information Fig. 7). These data indicate that SKP distribution might be ubiquitous in the human dermis, with no restriction to the DP niche as previously assumed.

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Figure 5. Localization of cell markers associated with skin-derived precursors in human skin sections. Skin biopsies from human foreskin (A), scalp (B), breast (C), and abdomen (D) were included in optimal cutting temperature (OCT) resin, sectioned and stained with hematoxylin-eosin (a). Sections of human foreskin (A), scalp (B), breast (C), and abdomen (D) were stained with anti-Fibronectin (b), Nestin (c), p75-NTR (e), and Vimentin (f) antibodies. Merged images are shown overlapped with Phase (d; g). Scale bar = 100 μm.

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Quantification of Actual Spherogenic Precursors (SKPs) in the Pool of Human Cells Obtained at Processing Reveals an Age-Dependent Depletion of Human SKPs

Cells obtained at the biopsy processing stage are in fact a complex mixture of stem, precursor, and terminally differentiated cells that might largely not contribute to dermosphere formation. There is currently no bona fide marker to distinguish true dermosphere forming cells from other dermal cells. For this reason, we quantified the dermospheres formed by a set number of primary cells as obtained in foreskin biopsy processing (n = 21; Fig. 6A). The resulting analyses showed a negative correlation of dermosphere forming cells with age (r = −0.624; p = .0019) and depletion of the spherogenic cell pool by age 50. Because of the fact that floating spheres may adhere to each other in standard liquid cultures [40], we controlled this variable by analyzing the apparent dermosphere area for each sample (Fig. 6B), showing a correlation of larger dermospheres with younger age (r = −0.610; p = .0025). These data indicated that the SKP cell pool in a given area of human skin decrease with age. To test whether the observed difference in the SKP pool size was also detectable in situ, human foreskin biopsy sections (n = 7) from subjects aged 8 months to 80 years were analyzed by immunofluorescence detection of SKP cell marker Nestin and Hoechst staining for total cell counts. Nestin-positive cells were quantified as a percent of total cells and the percentage plotted against donor age (supporting information Fig. 8). The results showed a negative correlation (r = −0.610) between Nestin-positive cells and age, which was not deemed significant (p = .1081). To strengthen the argument we compared the number and/or differentiation potential of SKPs present in the foreskins of nine subjects of young (3, 9, and 10 years), middle (23, 24, and 36 years), and old age (61, 84, and 85 years), respectively. Primary dermospheres obtained from these biopsies (n = 9) were put into three differentiation media as described in Materials and Methods section, and comparative differentiation potentials after 14-day culture were analyzed by immunofluorescence and confocal microscopy with anti-βIII-tubulin and anti-SMA antibodies (Fig. 6C and Table 1). Major differences were observed between young–middle and older sample groups, independently of the differentiation protocol. The younger subjects (mean ages of 7.3 and 27.7 years) had 8–29X more βIII-tubulin+ cells when compared with older men (mean age 76.7 years, Table 1). Overall, our data indicate that the pool size and/or differentiation potential of SKPs decreases with donor age in healthy human subjects.

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Figure 6. Age-dependent depletion of dermosphere-forming skin-derived precursors. (A): Dermosphere number decrease with donor age. The graph shows the number of dermospheres generated per 25,000 cells after 7 days in dermosphere proliferation medium, as obtained from 21 independent biopsies of human foreskin with donor ages shown on the x-axis. (B): Dermosphere size decrease with donor age. The graph shows the mean dermosphere area (in squared millimeters) apparent after 7 days in dermosphere proliferation medium, same biopsies as before. (C): SKP abundance and/or differentiation potential decrease with donor age. Three foreskin biopsies from men aged 24 (BP91, a–c), 61 (BP96, d–f), and 84 years (BP98, g–i) were processed and the primary, day 7 dermospheres put into three differentiation media [basal (a, d, g), neurogenic-low (b, e, h), and neurogenic-high (c, f, i)] for 14 days. They were then analyzed by immunofluorescence and confocal microscopy with anti-βIII tubulin (green) and anti-SMA antibodies (red). Merged images are shown with cell nuclei counterstained with Hoechst (blue). Scale bar = 50 μm.

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Table 1. Differentiation potential of SKPs and donor age
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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

An as yet unresolved, fundamental problem in biology is how tissues regulate their size. Cell replenishment in homeostasis may occur by stem cell differentiation or by duplication of pre-existing differentiated cells. Our main goal in this work was to analyze a well-characterized human progenitor cell pool and correlate its size in relation to body aging. Several laboratories have now reported the existence of a multipotent dermal precursor cell population, the skin-derived precursor (SKP) cells (reviewed by Miller and coworkers [20, 41]). Initially derived from rodents, now several groups reported isolation of SKPs from human scalp, foreskin, arm, beard and chin, in subjects ranging from fetal to old age [22, 23, 30, 31, 34, 36–39]. Transplantation of SKPs to animal models of disease has raised expectations for their potential use in human cell therapy [23–29]. However, translation of results from animal models to a human setting has often resulted in the past in inconsistencies or plain lack of success.

With the aim to translate basic results to the clinical practice, we processed >100 human biopsies to address several basic questions that were still unanswered, such as: (a) how reproducible is the SKP isolation protocol? (b) which of the published protocols works best for adult human SKP expansion? (c) is there an optimal donor area for SKP derivation? and (d) does donor age influence SKP number and/or differentiation potential? In the course of this work, we had several unexpected findings.

We found the SKP isolation protocol was extremely robust, with >96% foreskin biopsies being “spherogenic” (supporting information Fig. 1). Of note, SKPs obtained from foreskin and breast appeared to be more often spherogenic than those from scalp and abdomen (supporting information Table 1), although greater numbers of biopsies of areas other than foreskin are needed to support this conclusion.

With regard to cell expansion, widely used culture conditions for dermospheres cause a selective loss of adult human precursors, that others and we have not seen in rodents nor in human children-derived SKPs (Fig. 2). After an apparent enrichment within the first passages, adult human SKPs were gradually lost in liquid sphere culture, disappearing in P4. In contrast, use of semisolid medium permitted continuous expansion of adult human SKPs. Our data support the use of semisolid media both for the initial seeding (supporting information Fig. 2) and for further expansion of adult human SKPs in proliferation media, because better sphere-to-cell ratios are obtained (Fig. 2). Although use of methylcellulose does not warrant clonality of dermospheres [42], expansion of adult human SKPs in semisolid but not in liquid media might reflect aggregate formation in the liquid dermosphere cultures, that is somehow deleterious for cells.

Adherent passage of younger human SKPs with no apparent loss of potentiality has been previously reported [22, 31]. In our hands, adult human SKPs disaggregated from primary dermospheres can be attached and expanded for many passages until they reach senescence, but multipotentiality is gradually lost (supporting information Figs. 3 and 4). Of interest, neural precursor cells were specifically depleted from cultures obtained both after dermosphere passaging and after attached cell passaging. This was not observed for smooth muscle, where differentiation potential was maintained in sphere cultures and greatly diminished, but not completely lost, within attached cultures (supporting information Fig. 4). These data suggest that human SKPs are either lost with passage or lose potentiality, but also indicate that a more committed progenitor with “smooth muscle only” specification is preferentially grown under present culture conditions. Clearly further work is needed to elucidate the mechanisms involved in this apparent lack of in vitro expansion of adult human SKPs, as well as to characterize and improve their expansion in semisolid media.

As for the donor area, probably our most unexpected finding was the lower cell numbers obtained from areas other than foreskin (Fig. 3). Although we do not have a definitive explanation, we believe it might be due to a technical issue. Foreskin might be more easily dissociated than skin from other areas, because of the fact that the foreskin epidermis is either mono-stratified or bi-stratified, its dermis being often thinner than that of scalp, breast, and abdomen. The fact that dermospheres coming from different areas do not show the same staining patterns (Fig. 4) is not surprising in the light of developmental biology. Recent work has elucidated anatomic patterning of dermal fibroblasts that reflects the fibroblast position relative to the three developmental axes [43]. The fact that different skin areas will provide progenitor cells with different specifications deserves further investigation that we are currently undertaking. Recent work has characterized a highly enriched niche of human and rodent SKPs within the DP of the hair follicle [21, 22]. Although DP has the obvious advantage of being accessible in a noninvasive manner, it would also restrict donor skin areas to those rich in hair follicles. In this work, we have convincingly described a dermal SKP niche outside of the DP of the hair follicle (Fig. 5 and supporting information Figs. 5–7) that might explain previous derivation of SKPs from glabrous skin areas. Clearly further work is needed to assess the ontogenic relationship, if any, among these otherwise phenotypically similar cells, as well as to clarify which of the dermal niches will be most useful to extract SKPs for therapy purposes.

Finally, we have demonstrated through several lines of evidence that the SKP cell pool and/or its differentiation potential diminishes sharply with aging (Fig. 6, Table 1 and supporting information Fig. 8), for unknown reasons, in healthy human subjects. We do not believe this is due to more difficult extraction of cells in old skin (as seen by data distribution in supporting information Fig. 1). Although we cannot formally rule out the alternative explanation that the skin-derived progenitor pool remains intact but increasingly resistant to dissociation with age, we believe our data support the need to cryopreserve these cells early in life for putative future use in cell therapy protocols.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Depletion of human stem/progenitor pool with aging of tissues has long been hypothesized. However, experiments involving use of human tissue have many regulatory hurdles that prevent steady advancement in this area. The data we present here suggest that adult human SKPs are depleted in an age-dependent manner. Regardless of the underlying mechanism, our data indicate the importance of establishing master cell banks of younger subjects that might be of use later in life when degenerative disease arises.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

We thank Carlos Vicario and an anonymous reviewer of the article for helpful experimental suggestions, Adrian Perez and Karmele Valencia for technical assistance, and Angel G. Martin and Rosario Sanchez-Pernaute for critical reading of the manuscript. This work was supported by grants from Diputación Foral de Gipuzkoa (OF0184/2007, OF0148/2006 and OF808/2004), the Department of Industry at the Basque Government (Saiotek Program) and Instituto de Salud Carlos III (FIS PI052614) from Spain. A.I. was supported by a “Miguel Servet” contract from the Spanish Ministry of Science and Innovation (CP03/00056). N.G. was supported by a Fellowship from the Department of Industry at the Basque Government (Spain).

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Additional supporting information available online.

FilenameFormatSizeDescription
STEM_00027_sm_SupLegend1.doc23KSupporting Information Figure Legend 1
STEM_00027_sm_SupFigure1.tif6182KSupporting Information Figure 1. Reproducibility of the isolation protocol for human SKPs.
STEM_00027_sm_SupLegend2.doc24KSupporting Information Figure Legend 2
STEM_00027_sm_SupFigure2.tif6981KSupporting Information Figure 2. Optimal cell seeding density for primary dermosphere cultures.
STEM_00027_sm_SupLegend3.doc31KSupporting Information Figure Legend 3
STEM_00027_sm_SupFigure3.tif15407KSupporting Information Figure 3. Serial passaging of human SKPs and dermal fibroblasts in attached cultures.
STEM_00027_sm_SupLegend4.doc22KSupporting Information Figure Legend 4
STEM_00027_sm_SupFigure4.tif6298KSupporting Information Figure 4. Adult human SKPs are gradually lost in both sphere and attached cultures under previously published culture conditions.
STEM_00027_sm_SupLegend5.doc26KSupporting Information Figure Legend 5
STEM_00027_sm_SupFigure5.tif13640KSupporting Information Figure 5. Localization of cell markers associated with SKPs in human skin sections (II).
STEM_00027_sm_SupLegend6.doc31KSupporting Information Figure Legend 6
STEM_00027_sm_SupFigure6.tif8244KSupporting Information Figure 6. Localization of cell markers associated with SKPs in human skin sections (III).
STEM_00027_sm_SupLegend7.doc26KSupporting Information Figure Legend 7
STEM_00027_sm_SupFigure7.tif14678KSupporting Information Figure 7. Histochemical detection of alkaline phosphatase activity in human skin sections.
STEM_00027_sm_SupLegend8.doc27KSupporting Information Figure Legend 8
STEM_00027_sm_SupFigure8.tif12035KSupporting Information Figure 8. Quantification of markers associated with SKPs in human skin sections.
STEM_00027_sm_SupMat.doc25KSupporting Information Materials and Methods.
STEM_00027_sm_SupTable1.doc259KSupporting Information Table 1. Biopsies processed in this study.

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