Isolation and Characterization of Multipotent Skin-Derived Precursors from Human Skin
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《干细胞学杂志》
a Departments of Developmental Biology and
b Urology, Hospital for Sick Children, Toronto, Ontario, Canada;
c Departments of Physiology and
d Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada;
e Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada
Key Words. Neural stem cells ? Neural crest ? Neurons ? Schwann cells ? Smooth muscle cells ? Foreskin ? Stem cells ? Dermis
Correspondence: Freda D. Miller, Ph.D., Senior Scientist and Professor, Department of Developmental Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Telephone: 416-813-7654, ext. 1434; Fax: 416-813-2212; e-mail: fredam@sickkids.ca
ABSTRACT
A significant amount of recent interest has focused on the possibility that adult human stem cells are a realistic therapeutic alternative to embryonic stem cells. This interest has arisen largely as a consequence of recent work demonstrating that several adult stem cells show a surprisingly diverse differentiation repertoire . Although in some of these cases this multipotency was due to unanticipated cellular fusion events that occurred in vivo , compelling evidence still exists for the multipotency of several cultured adult stem cell populations, including multipotent adult progenitor cells isolated after long-term culture of bone marrow cells and neural stem cells from the central nervous system (CNS) .
The most obvious therapeutic use of such multipotent adult human precursors is for cell transplantation and replacement. However, perhaps equally important is the possibility that expandable adult stem cell populations could be used to generate human cell types on an individual basis for screening or discovery research. In either case, the ideal human precursor cell population would be one that could be derived in an autologous fashion from small amounts of accessible human tissue biopsies. With this in mind, we previously isolated and characterized a multi-potent precursor cell population from a highly accessible tissue source, adult mammalian dermis , using an approach similar to that originally described by Reynolds and Weiss to isolate adult stem cells from the CNS. These cells, termed skin-derived precursors (SKPs), were isolated and expanded from rodent skin and would differentiate into both neural and mesodermal progeny, including cell types that are never found in skin, such as neurons. One endogenous embryonic stem cell population that has a similar broad potential and that contributes to the dermis is neural crest stem cells , and, in this regard, our recent work indicates that SKPs represent an embryonic neural crest–related precursor cell that arises in skin during embryogenesis and that persists in lower numbers into adulthood .
We have previously provided evidence that a similar SKP-like precursor may reside in adult human skin . Specifically, we demonstrated that small punch biopsies of human scalp contained cells that would proliferate in response to fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF), that a subpopulation of these cells expressed the SKP markers nestin and fibronectin, and that they could differentiate into ?III-tubulin–positive cells with the morphology of newly born neurons. In this study, we have built upon these initial findings and have isolated and expanded SKPs from human foreskin tissue. We demonstrate that these human SKPs are multipotent adult precursor cells that are capable of generating neural and mesodermal progeny even after long-term expansion and that they may well represent an endogenous neural crest–related precursor present in adult human dermis.
MATERIALS AND METHODS
SKPs Can Be Routinely Isolated and Expanded from Neonatal Human Foreskin
Because rodent SKPs are more abundant in neonatal than adult skin , we used neonatal human foreskin tissue in our attempts to isolate and characterize human SKPs. Foreskin samples from circumcisions of children ranging from 4 weeks to 3 years of age were used for this study, with over 30 samples in all being analyzed. These samples were indistinguishable in terms of SKP isolation and properties. One sample was also obtained from a 12-year-old, and no overt differences in SKP growth or differentiation properties were noted relative to SKPs derived from younger children. Nonetheless, all data presented in this report derive from SKPs cultured from foreskins of children aged 3 years or younger.
To isolate SKPs, epidermis and dermis were dissociated from skin samples of approximately 1–2 cm2, and dissociated dermal cells were cultured in defined medium containing EGF, FGF2, and B27. Over the first 2–3 weeks under these culture conditions, most cells adhered to the tissue culture plastic and/or died, but a small population of proliferating spheres of cells formed, with a morphology similar to that previously seen with rodent SKPs . At approximately 3 weeks, the spheres were isolated, dissociated, and then split into medium containing the same growth factors and filtered SKP-conditioned medium. SKP cultures were then passaged and split every 2–3 weeks, so that at the end of 3 months, a 1-cm2 piece of developing foreskin gave rise to approximately 1 to 2 x 107 cells present in culture as floating spheres (Fig. 1A).
Figure 1. Isolation and characterization of human foreskin SKPs. (A): Phase photomicrograph of human foreskin SKP spheres immediately before passaging. Note the characteristic morphology, which is very similar to rodent SKP spheres. (B): Phase photomicrographs of human SKP spheres that were grown in EGF, FGF2, or both EGF and FGF2 for 2 weeks. (C): Fluorescence photomicrograph of human SKP cells that were adhered to a poly-D-lysine/laminin substratum overnight and then double-labeled with antibodies to nestin (left panel) and fibronectin (right panel). (D): Fluorescence photomicrograph of adherent SKP cells immunostained for vimentin. (C, D): The blue is Hoechst staining of nuclei to show all of the cells in the field. (E): Reverse transcription–polymerase chain reaction analysis for the expression of mRNAs for Pax3, Snail, Slug, p75NTR, and, as a control, GAPDH, in two human SKP preparations (SKPs-1 and SKPs-2) that had been passaged five and six times. Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth factor 2; SKP, skin-derived precursor.
To ask whether these proliferating spheres shared the same growth factor requirements as rodent SKPs, cells in spheres were triturated to single cells and were then passaged into defined medium containing B27, with or without EGF and FGF2. This experiment revealed that the human SKPs absolutely required EGF and FGF2 to proliferate, because no spheres were generated in their absence. Visual examination of the cultures also revealed that, although spheres could grow in EGF, their number and size were reduced relative to FGF2 alone or EGF plus FGF2 (Fig. 1B). Measurements of sphere number and size confirmed this conclusion; after 2.5 weeks of growth in EGF or FGF2 alone, the sphere numbers were approximately 20% and 80%, respectively, of those obtained with both growth factors, and spheres were approximately 50% and 90%, respectively, of the size of those generated in EGF plus FGF2 (mean sphere diameter of 58 ± 3.4 μm for EGF, 124 ± 4.9 μm for FGF2 versus 134 ± 5.9 μm for EGF plus FGF2 in one representative experiment; p < .0001 for the comparison between EGF and EGF plus FGF2; p < .05 for the comparison between FGF2 and EGF plus FGF2). The diameter of individual SKP cells was also measured, and cell size was in the range of 10–15 μm in diameter, thereby distinguishing SKPs from the very small spore stem cells previously isolated from skin .
To confirm that these human spheres were similar to rodent SKPs, we initially examined the cell-surface markers that they expressed. Human spheres of three passages or less were either plated onto poly-D-lysine and laminin overnight or cytospun as spheres onto slides and then were immunostained for nestin, fibronectin (Fig. 1C), and vimentin (Fig. 1D), all of which are expressed by rodent SKP cells . This analysis revealed that the human SKPs expressed all of these proteins but that they did not express tyrosinase or trp1, markers for melanoblasts/melanocytes (data not shown). Similar results were obtained with all samples whether the cells were analyzed as single cells, attached cells, or spheres.
We have recently found that rodent SKPs express several transcription factors that are associated with embryonic neural crest stem cells and some of their developing embryonic derivatives . We therefore used RT-PCR to analyze two independently isolated human SKP populations for their expression of a subset of these transcription factors, Pax3 , snail , and slug . This analysis (Fig. 1E) revealed that, like rodent SKPs, human SKPs express these three transcription factors. In addition, human SKPs expressed low levels of the mRNA for p75 neurotrophin receptor (p75NTR) (Fig. 1E), another marker for embryonic neural crest stem cells. However, immunocytochemical analysis indicated that the p75NTR protein was expressed only at low or undetectable levels in SKP spheres (Fig. 2B). Thus, human SKPs share the same growth factor requirements as rodent SKPs and express similar genes.
Figure 2. Single human SKP cells are multipotent. (A): Phase photomicrographs of a human SKP clone generated initially from a single, isolated cell on the day of plating (day 0, top panel) and after 4 (middle panel) and 8 (bottom panel) weeks of proliferation. Note that at 8 weeks, a nascent sphere is forming that will ultimately detach from the dish and grow in suspension. (B): Immunocytochemical analysis of single clonal SKP spheres that were double-labeled for nestin and fibronectin (top panels), nestin and p75NTR (middle panels), or vimentin and fibronectin (bottom panels). Nuclei of all the cells are blue as a consequence of the Hoechst counterstain. (C): Fluorescence photomicrograph of cells from a single SKP clone that were differentiated for 3 weeks in the presence of 5% FBS and then immunostained for NF-M. (D): Fluorescence photomicrographs of cells from a single SKP clone that were differentiated for 3 weeks in the presence of 5% FBS and then double-labeled for CNPase (top panels) and GFAP (bottom panels). The arrow indicates the same cell in both panels. (E): Fluorescence photomicrograph of cells from a single SKP clone differentiated for 3 weeks and then immunostained for SMA. (F): Phase photomicrograph of cells from a single SKP clone differentiated for 3 weeks in the presence of retinoic acid, showing cells with the characteristic morphology and lipid droplet inclusions of adipocytes. (G): G-banding of a metaphase chromosome spread obtained from one of the three SKP clones that were analyzed as shown in panels (A–F) after passaging for 15 months. The left panel shows the metaphase spread, and the right panel shows the ordered chromosomal pairs. Abbreviations: FBS, fetal bovine serum; NF-M, neurofilament; SKP, skin-derived precursor; SMA, smooth muscle actin.
SKPs Differentiate into Neurons, Glia, and Smooth Muscle Cells, Including Cells with Peripheral Neural Phenotypes
We have previously demonstrated that rodent SKPs will differentiate into both neural and mesodermal cell types, including neurons, glia, smooth muscle cells, and adipocytes . Moreover, our recent studies indicate that the neural cell types are largely peripheral cells that derive from the neural crest during development, including catecholaminergic neurons and Schwann cells . To ask whether human SKPs could generate the same differentiated cells, spheres at passages 3 through 9 were plated down on poly-D-lysine/laminin for 2–4 weeks in the presence of defined medium plus 5% FBS. Immunocytochemical analysis of these cultures revealed that the human SKPs generated many morphologically complex neurons that were positive for ?III-tubulin and/or NF-M (Figs. 3A, 3B). These cells always differentiated on top of a layer of nonneuronal cells. Almost all of the ?III-tubulin–positive neurons coexpressed p75NTR (Fig. 3C), a hallmark of peripheral neurons, and something that is also seen for the rodent SKP-derived neurons . When cells were differentiated in the presence of neurotrophins to enhance the survival and differentiation of peripheral neurons (neuronal conditions), then these ?III-tubulin–positive cells also coexpressed two other neuron-specific proteins, GAP-43 (Fig. 3E) and MAP2 (Fig. 3D). These putative neurons did not express SMA, nor did they express markers of glial cells, including CNPase, GFAP, or S100?. To estimate the percentage of cells that differentiated into neurons, random fields of cells from three different experiments were quantitated to determine the total number of cells (as determined by counting Hoechst-positive nuclei) versus the total number of ?III-tubulin–positive, morphologically complex cells. This analysis revealed that 9.4% ± 0.2% of the cells in these experiments were differentiated neurons.
Figure 3. Human SKPs differentiate into neurons. (A, B): Fluorescence photomicrographs of two different human SKP preparations that were differentiated for 2–3 weeks in the presence of 5% fetal bovine serum and immunostained for (A) ?III-tubulin or (B) NFM. The blue is from the Hoechst staining of nuclei. Note that the positive cells are of typical neuronal morphology and have differentiated on top of a layer of nonneuronal cells. (C–E): Fluorescence photomicrographs of SKPs differentiated for 3 weeks in the presence of neurotrophins and then double-labeled for (C) ?III-tubulin (left panel) and p75NTR (right panel), (D) ?III-tubulin (left panel) and all isoforms of MAP2 (right panel), or (E) ?III-tubulin (left panel) and GAP43. In all cases, the arrowheads denote cells that are double-labeled for both markers, whereas in (C) the arrows denote cells that are positive for p75NTR but not for ?III-tubulin. Coexpression of these markers is characteristic of peripheral neurons, whereas the cells that express p75NTR but not ?III-tubulin are potentially p75NTR-positive Schwann cells. Note that the MAP2 antibody used in (D) detects MAP2a, b, c and is therefore not specific to dendrites in developing neurons. Abbreviations: NFM, neurofilament; SKP, skin-derived precursor.
In addition to neurons, under the same differentiation conditions, human SKPs generated glial cells and potential smooth muscle cells (Fig. 4). With regard to glial cells, a subpopulation of bipolar cells coexpressed S100? and p75NTR (Fig. 4A), an expression profile typical of peripheral Schwann cells . A similar subpopulation of bipolar cells also coexpressed CNPase, a marker of myelinating glia, including oligodendrocytes and Schwann cells, and GFAP, which is expressed in Schwann cells and astrocytes (data not shown; Figs. 2D, 5D). The coexpression of these four proteins and this bipolar morphology indicate that these cells are likely to be Schwann cells. Quantitation of the total number of CNPase-positive cells versus the total number of cells in three different experiments revealed that 4.3% ± 0.3% of the cells were differentiated glial cells. A subpopulation of SMA-positive cells with the morphology of smooth muscle cells or myofibroblasts was also observed in these experiments (Fig. 4B). These latter cells did not express any glial or neuronal proteins. These three different cell types, neurons, glial cells, and smooth muscle cells, were consistently seen upon differentiation of human SKP cultures.
Figure 4. Human SKPs differentiate into glial and smooth muscle cells. (A): Fluorescence photomicrograph of human SKPs that were differentiated for 3 weeks in the presence of 1% serum, forskolin, and heregulin-? and then double-labeled for S100? (left panel) and p75NTR (right panel), a morphological and antigenic profile characteristic of Schwann cells. (B): Fluorescence photomicrograph of human SKPs differentiated in 5% fetal bovine serum for 3 weeks and then immunostained for SMA. Note the characteristic morphology of these cells, indicating that they are likely to be smooth muscle cells or myofibroblasts. Abbreviations: SKP, skin-derived precursor; SMA, smooth muscle actin.
Figure 5. After 1 year in culture, human SKPs differentiate appropriately. (A): Fluorescence photomicrographs of human SKPs that had been expanded for 1 year in culture and that were double-labeled for nestin and versican (left panels), nestin and p75NTR (middle panels), or nestin and fibronectin (right panels). (B–E): Fluorescence photomicrographs of human SKPs that were expanded for 1 year in culture, differentiated for 3 weeks, and then immunostained for (B) ?III-tubulin and (C) NF-M to detect neurons, (D) CNPase and GFAP to detect Schwann cells, or (E) SMA to detect smooth muscle cells and myofibroblasts. In all panels, the nuclei are blue due to Hoechst counterstaining. Note that the morphology of the differentiated cell types is indistinguishable from that observed with shorter-term SKP cultures. (F): G-banding of a metaphase chromosome spread obtained from SKPs after passaging for 15 months. The left panel shows the metaphase spread, and the right panel shows the ordered chromosomal pairs. Abbreviations: NF-M, neurofilament; SKP, skin-derived precursor; SMA, smooth muscle actin.
To determine whether SKPs could be expanded long-term and still maintain their potential to generate these three cell types, we maintained and analyzed two human SKP cultures for 1 year, at the end of which they had been passaged 16 times. Over this entire time period, the SKPs maintained the same relatively slow growth rate, with the cells doubling in number every 2–3 weeks. In addition, G-banding of one of these lines revealed that their chromosomal karyotype was normal after 15 months of passaging (Fig. 5F). Immunocytochemical analysis of these long-passage SKP spheres demonstrated that they expressed the same markers as the lower-passage SKP spheres, including nestin, fibronectin, versican (Fig. 5A), and vimentin (data not shown). These cells were then differentiated for 3–4 weeks and examined immunocytochemically. This analysis revealed that these highly expanded SKPs were able to generate morphologically complex, ?III-tubulin and NF-M–positive neurons (Figs. 5B, 5C), as well as bipolar glial cells coexpressing CNPase and GFAP (Fig. 5D) and SMA-positive smooth muscle cells or myofibroblasts (Fig. 5E). Thus, SKPs retain their ability to differentiate appropriately, even when maintained and expanded for long periods of time in culture.
Single SKPs Clonally Generate Neural and Mesodermal Progeny
To ask whether a single SKP cell was able to generate these different cell types, we performed clonal analysis. Human SKPs that had been passaged three times were dissociated to single cells; these single cells were isolated by limiting dilution into 48-well plates (Fig. 2A) and then cultured in medium containing EGF, FGF2, B27, and 50% of the filtered medium conditioned by more densely growing SKP cultures. Initially, these cells were quiescent, but they slowly started to proliferate, so that by 4 weeks, small clones of dispersed cells were formed, whereas by 8 weeks, spheres were generated that would ultimately dissociate from the tissue culture plastic and grow in suspension (Fig. 2A). After 10 weeks, the clones were dissociated and moved to 24-well plates and then 12-well plates over a period of 3 weeks and in this manner were ultimately expanded into small flasks. Of the 92 wells containing single cells at the start of the experiment, 39 of these proliferated to generate clones, and 37 of those grew as spheres. The other two clones only ever grew adherently, even after expansion, and were not analyzed further. Thus, approximately 40% of the SKP cells were able to self-renew when isolated as clones.
Immunocytochemical analysis of these clonal spheres revealed that they were similar to the starting culture of SKPs with regard to marker protein expression. Immunocytochemical analysis of six of these clones revealed that all of the spheres coexpressed nestin, fibronectin, and vimentin, whereas only the occasional cell was detectably positive for p75NTR (Fig. 2B). We then asked whether these clonal SKP lines were able to generate both neural and mesodermal progeny by plating cells from three clones on poly-D-lysine/laminin, withdrawing their proliferation factors, and differentiating them either in the presence of 5% FBS plus or minus neurotrophins (neuronal differentiation conditions) or in 5 μm retinoic acid and 1% FBS to ask if they could generate adipocytes. G-banding of one of these clones demonstrated that the chromosomal karyotype was normal (Fig. 2G). Immunocytochemical analysis of these three clones revealed that all of them were able to generate neurons, glia, smooth muscle cells, and adipocytes. Under neuronal differentiation conditions, the clones generated a relatively dense network of ?III-tubulin and NF-M–positive, morphologically complex neurons on top of a bed of nonneuronal cells (Fig. 2C). Many of these ?III-tubulin–positive cells also coexpressed p75NTR (data not shown). Under these same conditions, a smaller subpopulation of potential bipolar Schwann cells was also observed, as indicated by their coexpression of CNPase and GFAP (Fig. 2D). Neuronal and glial proteins were never expressed in the same cells in these cultures. With regard to mesodermal cell types, in the neural differentiation conditions, we observed a subpopulation of SMA-positive cells with the morphology of smooth muscle cells or myofibroblasts (Fig. 2E) but never saw cells with the morphology and characteristic lipid droplet inclusions of adipocytes. In contrast, when the same clones were differentiated in the presence of retinoic acid, a small population of adipocytes was observed (Fig. 2F). Similar results were obtained with three independent clones, indicating that human SKPs are multipotent.
Human SKPs May Represent an Endogenous Dermal Precursor
We have recently demonstrated that rodent SKPs derive from an endogenous SKP-like precursor that is first present in skin during embryogenesis and that is then maintained at lower levels into adulthood . We therefore asked whether human foreskin contained an endogenous SKP-like precursor that could generate neurons, a cell type that is never present in skin. To ask this question, four different human foreskin samples were dissociated into single cells, and these cells were plated adherently in the presence of 5% FBS. Immunocytochemical analysis 2 weeks later revealed the presence, in all four samples, of a subpopulation of morphologically complex cells that expressed ?III-tubulin (Fig. 6A) and, in some cases, NF-M (data not shown), properties of newly born neurons. In addition, cells were observed that expressed CNPase and GFAP, consistent with the presence of Schwann cells in skin, and a separate population was observed that expressed SMA (data not shown). Because ?III-tubulin–positive neurons were never observed in freshly dissociated skin preparations (data not shown) and have never been reported in skin, we conclude that the neurons observed in these experiments were generated by differentiation of a precursor present in developing foreskin.
Figure 6. Primary human SKPs have the same properties as passaged SKPs. (A): Fluorescence photomicrograph of primary human foreskin cells that were differentiated for 2 weeks and then immunostained for ?III-tubulin (red). (B): Fluorescence micrographs of primary human SKP spheres that were double-labeled for either nestin and fibronectin (left panels) or nestin and vimentin (right panels). (C): Fluorescence micrographs of primary human SKPs that were differentiated for 3 weeks in the presence of 5% fetal bovine serum and then double-labeled for ?III-tubulin (top panel) and p75NTR (bottom panel). (D): Fluorescence micrographs of primary human SKPs that were differentiated for 3 weeks in the presence of neurotrophins and then double-labeled for ?III-tubulin and p75NTR (left panels) or ?III-tubulin and NF-M (right panels). Note that although all ?III-tubulin–positive neurons are p75NTR-positive, not all p75NTR-positive cells express ?III-tubulin. These latter p75NTR-positive cells may be Schwann cells. (E): Primary human SKPs that were differentiated for 3 weeks and then double-labeled for CNPase (top panel) and GFAP (bottom panel). Note the colocalization of these proteins in elongated, bipolar cells. (F): Primary human SKPs that were differentiated for 3 weeks and immunostained for SMA. In all panels, the blue nuclei are Hoechst stained to show all of the cells in the field. Abbreviations: NF-M, neurofilament; SKP, skin-derived precursor; SMA, smooth muscle actin.
We then asked whether the very first SKP spheres that were formed from skin (primary spheres) had the properties of the more long-term passaged human SKPs. Immunocytochemical analysis of nine different preparations of primary SKP spheres revealed that, like the passaged SKPs, they expressed nestin, fibronectin, and vimentin (Fig. 6B) but did not contain cells expressing tyrosinase or trp1 (data not shown), indicating that they did not contain melanoblasts/melanocytes. Similar results were obtained when these primary SKP spheres were plated down for 24 hours. We then differentiated primary SKP spheres under neuronal differentiation conditions to ask whether they could generate neurons. Immunocytochemical analysis demonstrated that all of the human samples analyzed in this way generated morphologically complex cells that coexpressed either ?III-tubulin and p75NTR (Figs. 6C, 6D) or ?III-tubulin and NF-M (Fig. 6D), a profile typical of peripheral neurons. In these same cultures, a significantly smaller population of cells had the bipolar morphology typical of Schwann cells and coexpressed CNPase and GFAP (Fig. 6E) or S100? and p75NTR (data not shown). Finally, some of the cells expressed SMA and had a morphology typical of smooth muscle cells or myofibroblasts (Fig. 6F). Thus, the initial, unpassaged spheres that are generated from dissociated skin cells are similar, if not identical, to passaged SKP spheres, supporting the idea that SKPs may arise from an endogenous precursor in human skin, as they do in rodent skin.
DISCUSSION
This work was supported by grants from the Canadian Stem Cell Network, the CIHR, and the Canadian Heart and Stroke Foundation. I.A.M. is supported by a Canadian Stem Cell Network training award and a studentship award from the Hospital for Sick Children Foundation. F.D.M. is a CIHR Senior Investigator and a Senior Canada Research Chair. We would like to thank Mahnaz Akhavan and Anne Aumont for excellent technical support throughout the course of this work, David Kaplan for many valuable ideas, Dr. Roman Jednak for his help with the initial tissue samples, and the Miller and Kaplan laboratories for ongoing discussions.
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b Urology, Hospital for Sick Children, Toronto, Ontario, Canada;
c Departments of Physiology and
d Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada;
e Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada
Key Words. Neural stem cells ? Neural crest ? Neurons ? Schwann cells ? Smooth muscle cells ? Foreskin ? Stem cells ? Dermis
Correspondence: Freda D. Miller, Ph.D., Senior Scientist and Professor, Department of Developmental Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Telephone: 416-813-7654, ext. 1434; Fax: 416-813-2212; e-mail: fredam@sickkids.ca
ABSTRACT
A significant amount of recent interest has focused on the possibility that adult human stem cells are a realistic therapeutic alternative to embryonic stem cells. This interest has arisen largely as a consequence of recent work demonstrating that several adult stem cells show a surprisingly diverse differentiation repertoire . Although in some of these cases this multipotency was due to unanticipated cellular fusion events that occurred in vivo , compelling evidence still exists for the multipotency of several cultured adult stem cell populations, including multipotent adult progenitor cells isolated after long-term culture of bone marrow cells and neural stem cells from the central nervous system (CNS) .
The most obvious therapeutic use of such multipotent adult human precursors is for cell transplantation and replacement. However, perhaps equally important is the possibility that expandable adult stem cell populations could be used to generate human cell types on an individual basis for screening or discovery research. In either case, the ideal human precursor cell population would be one that could be derived in an autologous fashion from small amounts of accessible human tissue biopsies. With this in mind, we previously isolated and characterized a multi-potent precursor cell population from a highly accessible tissue source, adult mammalian dermis , using an approach similar to that originally described by Reynolds and Weiss to isolate adult stem cells from the CNS. These cells, termed skin-derived precursors (SKPs), were isolated and expanded from rodent skin and would differentiate into both neural and mesodermal progeny, including cell types that are never found in skin, such as neurons. One endogenous embryonic stem cell population that has a similar broad potential and that contributes to the dermis is neural crest stem cells , and, in this regard, our recent work indicates that SKPs represent an embryonic neural crest–related precursor cell that arises in skin during embryogenesis and that persists in lower numbers into adulthood .
We have previously provided evidence that a similar SKP-like precursor may reside in adult human skin . Specifically, we demonstrated that small punch biopsies of human scalp contained cells that would proliferate in response to fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF), that a subpopulation of these cells expressed the SKP markers nestin and fibronectin, and that they could differentiate into ?III-tubulin–positive cells with the morphology of newly born neurons. In this study, we have built upon these initial findings and have isolated and expanded SKPs from human foreskin tissue. We demonstrate that these human SKPs are multipotent adult precursor cells that are capable of generating neural and mesodermal progeny even after long-term expansion and that they may well represent an endogenous neural crest–related precursor present in adult human dermis.
MATERIALS AND METHODS
SKPs Can Be Routinely Isolated and Expanded from Neonatal Human Foreskin
Because rodent SKPs are more abundant in neonatal than adult skin , we used neonatal human foreskin tissue in our attempts to isolate and characterize human SKPs. Foreskin samples from circumcisions of children ranging from 4 weeks to 3 years of age were used for this study, with over 30 samples in all being analyzed. These samples were indistinguishable in terms of SKP isolation and properties. One sample was also obtained from a 12-year-old, and no overt differences in SKP growth or differentiation properties were noted relative to SKPs derived from younger children. Nonetheless, all data presented in this report derive from SKPs cultured from foreskins of children aged 3 years or younger.
To isolate SKPs, epidermis and dermis were dissociated from skin samples of approximately 1–2 cm2, and dissociated dermal cells were cultured in defined medium containing EGF, FGF2, and B27. Over the first 2–3 weeks under these culture conditions, most cells adhered to the tissue culture plastic and/or died, but a small population of proliferating spheres of cells formed, with a morphology similar to that previously seen with rodent SKPs . At approximately 3 weeks, the spheres were isolated, dissociated, and then split into medium containing the same growth factors and filtered SKP-conditioned medium. SKP cultures were then passaged and split every 2–3 weeks, so that at the end of 3 months, a 1-cm2 piece of developing foreskin gave rise to approximately 1 to 2 x 107 cells present in culture as floating spheres (Fig. 1A).
Figure 1. Isolation and characterization of human foreskin SKPs. (A): Phase photomicrograph of human foreskin SKP spheres immediately before passaging. Note the characteristic morphology, which is very similar to rodent SKP spheres. (B): Phase photomicrographs of human SKP spheres that were grown in EGF, FGF2, or both EGF and FGF2 for 2 weeks. (C): Fluorescence photomicrograph of human SKP cells that were adhered to a poly-D-lysine/laminin substratum overnight and then double-labeled with antibodies to nestin (left panel) and fibronectin (right panel). (D): Fluorescence photomicrograph of adherent SKP cells immunostained for vimentin. (C, D): The blue is Hoechst staining of nuclei to show all of the cells in the field. (E): Reverse transcription–polymerase chain reaction analysis for the expression of mRNAs for Pax3, Snail, Slug, p75NTR, and, as a control, GAPDH, in two human SKP preparations (SKPs-1 and SKPs-2) that had been passaged five and six times. Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth factor 2; SKP, skin-derived precursor.
To ask whether these proliferating spheres shared the same growth factor requirements as rodent SKPs, cells in spheres were triturated to single cells and were then passaged into defined medium containing B27, with or without EGF and FGF2. This experiment revealed that the human SKPs absolutely required EGF and FGF2 to proliferate, because no spheres were generated in their absence. Visual examination of the cultures also revealed that, although spheres could grow in EGF, their number and size were reduced relative to FGF2 alone or EGF plus FGF2 (Fig. 1B). Measurements of sphere number and size confirmed this conclusion; after 2.5 weeks of growth in EGF or FGF2 alone, the sphere numbers were approximately 20% and 80%, respectively, of those obtained with both growth factors, and spheres were approximately 50% and 90%, respectively, of the size of those generated in EGF plus FGF2 (mean sphere diameter of 58 ± 3.4 μm for EGF, 124 ± 4.9 μm for FGF2 versus 134 ± 5.9 μm for EGF plus FGF2 in one representative experiment; p < .0001 for the comparison between EGF and EGF plus FGF2; p < .05 for the comparison between FGF2 and EGF plus FGF2). The diameter of individual SKP cells was also measured, and cell size was in the range of 10–15 μm in diameter, thereby distinguishing SKPs from the very small spore stem cells previously isolated from skin .
To confirm that these human spheres were similar to rodent SKPs, we initially examined the cell-surface markers that they expressed. Human spheres of three passages or less were either plated onto poly-D-lysine and laminin overnight or cytospun as spheres onto slides and then were immunostained for nestin, fibronectin (Fig. 1C), and vimentin (Fig. 1D), all of which are expressed by rodent SKP cells . This analysis revealed that the human SKPs expressed all of these proteins but that they did not express tyrosinase or trp1, markers for melanoblasts/melanocytes (data not shown). Similar results were obtained with all samples whether the cells were analyzed as single cells, attached cells, or spheres.
We have recently found that rodent SKPs express several transcription factors that are associated with embryonic neural crest stem cells and some of their developing embryonic derivatives . We therefore used RT-PCR to analyze two independently isolated human SKP populations for their expression of a subset of these transcription factors, Pax3 , snail , and slug . This analysis (Fig. 1E) revealed that, like rodent SKPs, human SKPs express these three transcription factors. In addition, human SKPs expressed low levels of the mRNA for p75 neurotrophin receptor (p75NTR) (Fig. 1E), another marker for embryonic neural crest stem cells. However, immunocytochemical analysis indicated that the p75NTR protein was expressed only at low or undetectable levels in SKP spheres (Fig. 2B). Thus, human SKPs share the same growth factor requirements as rodent SKPs and express similar genes.
Figure 2. Single human SKP cells are multipotent. (A): Phase photomicrographs of a human SKP clone generated initially from a single, isolated cell on the day of plating (day 0, top panel) and after 4 (middle panel) and 8 (bottom panel) weeks of proliferation. Note that at 8 weeks, a nascent sphere is forming that will ultimately detach from the dish and grow in suspension. (B): Immunocytochemical analysis of single clonal SKP spheres that were double-labeled for nestin and fibronectin (top panels), nestin and p75NTR (middle panels), or vimentin and fibronectin (bottom panels). Nuclei of all the cells are blue as a consequence of the Hoechst counterstain. (C): Fluorescence photomicrograph of cells from a single SKP clone that were differentiated for 3 weeks in the presence of 5% FBS and then immunostained for NF-M. (D): Fluorescence photomicrographs of cells from a single SKP clone that were differentiated for 3 weeks in the presence of 5% FBS and then double-labeled for CNPase (top panels) and GFAP (bottom panels). The arrow indicates the same cell in both panels. (E): Fluorescence photomicrograph of cells from a single SKP clone differentiated for 3 weeks and then immunostained for SMA. (F): Phase photomicrograph of cells from a single SKP clone differentiated for 3 weeks in the presence of retinoic acid, showing cells with the characteristic morphology and lipid droplet inclusions of adipocytes. (G): G-banding of a metaphase chromosome spread obtained from one of the three SKP clones that were analyzed as shown in panels (A–F) after passaging for 15 months. The left panel shows the metaphase spread, and the right panel shows the ordered chromosomal pairs. Abbreviations: FBS, fetal bovine serum; NF-M, neurofilament; SKP, skin-derived precursor; SMA, smooth muscle actin.
SKPs Differentiate into Neurons, Glia, and Smooth Muscle Cells, Including Cells with Peripheral Neural Phenotypes
We have previously demonstrated that rodent SKPs will differentiate into both neural and mesodermal cell types, including neurons, glia, smooth muscle cells, and adipocytes . Moreover, our recent studies indicate that the neural cell types are largely peripheral cells that derive from the neural crest during development, including catecholaminergic neurons and Schwann cells . To ask whether human SKPs could generate the same differentiated cells, spheres at passages 3 through 9 were plated down on poly-D-lysine/laminin for 2–4 weeks in the presence of defined medium plus 5% FBS. Immunocytochemical analysis of these cultures revealed that the human SKPs generated many morphologically complex neurons that were positive for ?III-tubulin and/or NF-M (Figs. 3A, 3B). These cells always differentiated on top of a layer of nonneuronal cells. Almost all of the ?III-tubulin–positive neurons coexpressed p75NTR (Fig. 3C), a hallmark of peripheral neurons, and something that is also seen for the rodent SKP-derived neurons . When cells were differentiated in the presence of neurotrophins to enhance the survival and differentiation of peripheral neurons (neuronal conditions), then these ?III-tubulin–positive cells also coexpressed two other neuron-specific proteins, GAP-43 (Fig. 3E) and MAP2 (Fig. 3D). These putative neurons did not express SMA, nor did they express markers of glial cells, including CNPase, GFAP, or S100?. To estimate the percentage of cells that differentiated into neurons, random fields of cells from three different experiments were quantitated to determine the total number of cells (as determined by counting Hoechst-positive nuclei) versus the total number of ?III-tubulin–positive, morphologically complex cells. This analysis revealed that 9.4% ± 0.2% of the cells in these experiments were differentiated neurons.
Figure 3. Human SKPs differentiate into neurons. (A, B): Fluorescence photomicrographs of two different human SKP preparations that were differentiated for 2–3 weeks in the presence of 5% fetal bovine serum and immunostained for (A) ?III-tubulin or (B) NFM. The blue is from the Hoechst staining of nuclei. Note that the positive cells are of typical neuronal morphology and have differentiated on top of a layer of nonneuronal cells. (C–E): Fluorescence photomicrographs of SKPs differentiated for 3 weeks in the presence of neurotrophins and then double-labeled for (C) ?III-tubulin (left panel) and p75NTR (right panel), (D) ?III-tubulin (left panel) and all isoforms of MAP2 (right panel), or (E) ?III-tubulin (left panel) and GAP43. In all cases, the arrowheads denote cells that are double-labeled for both markers, whereas in (C) the arrows denote cells that are positive for p75NTR but not for ?III-tubulin. Coexpression of these markers is characteristic of peripheral neurons, whereas the cells that express p75NTR but not ?III-tubulin are potentially p75NTR-positive Schwann cells. Note that the MAP2 antibody used in (D) detects MAP2a, b, c and is therefore not specific to dendrites in developing neurons. Abbreviations: NFM, neurofilament; SKP, skin-derived precursor.
In addition to neurons, under the same differentiation conditions, human SKPs generated glial cells and potential smooth muscle cells (Fig. 4). With regard to glial cells, a subpopulation of bipolar cells coexpressed S100? and p75NTR (Fig. 4A), an expression profile typical of peripheral Schwann cells . A similar subpopulation of bipolar cells also coexpressed CNPase, a marker of myelinating glia, including oligodendrocytes and Schwann cells, and GFAP, which is expressed in Schwann cells and astrocytes (data not shown; Figs. 2D, 5D). The coexpression of these four proteins and this bipolar morphology indicate that these cells are likely to be Schwann cells. Quantitation of the total number of CNPase-positive cells versus the total number of cells in three different experiments revealed that 4.3% ± 0.3% of the cells were differentiated glial cells. A subpopulation of SMA-positive cells with the morphology of smooth muscle cells or myofibroblasts was also observed in these experiments (Fig. 4B). These latter cells did not express any glial or neuronal proteins. These three different cell types, neurons, glial cells, and smooth muscle cells, were consistently seen upon differentiation of human SKP cultures.
Figure 4. Human SKPs differentiate into glial and smooth muscle cells. (A): Fluorescence photomicrograph of human SKPs that were differentiated for 3 weeks in the presence of 1% serum, forskolin, and heregulin-? and then double-labeled for S100? (left panel) and p75NTR (right panel), a morphological and antigenic profile characteristic of Schwann cells. (B): Fluorescence photomicrograph of human SKPs differentiated in 5% fetal bovine serum for 3 weeks and then immunostained for SMA. Note the characteristic morphology of these cells, indicating that they are likely to be smooth muscle cells or myofibroblasts. Abbreviations: SKP, skin-derived precursor; SMA, smooth muscle actin.
Figure 5. After 1 year in culture, human SKPs differentiate appropriately. (A): Fluorescence photomicrographs of human SKPs that had been expanded for 1 year in culture and that were double-labeled for nestin and versican (left panels), nestin and p75NTR (middle panels), or nestin and fibronectin (right panels). (B–E): Fluorescence photomicrographs of human SKPs that were expanded for 1 year in culture, differentiated for 3 weeks, and then immunostained for (B) ?III-tubulin and (C) NF-M to detect neurons, (D) CNPase and GFAP to detect Schwann cells, or (E) SMA to detect smooth muscle cells and myofibroblasts. In all panels, the nuclei are blue due to Hoechst counterstaining. Note that the morphology of the differentiated cell types is indistinguishable from that observed with shorter-term SKP cultures. (F): G-banding of a metaphase chromosome spread obtained from SKPs after passaging for 15 months. The left panel shows the metaphase spread, and the right panel shows the ordered chromosomal pairs. Abbreviations: NF-M, neurofilament; SKP, skin-derived precursor; SMA, smooth muscle actin.
To determine whether SKPs could be expanded long-term and still maintain their potential to generate these three cell types, we maintained and analyzed two human SKP cultures for 1 year, at the end of which they had been passaged 16 times. Over this entire time period, the SKPs maintained the same relatively slow growth rate, with the cells doubling in number every 2–3 weeks. In addition, G-banding of one of these lines revealed that their chromosomal karyotype was normal after 15 months of passaging (Fig. 5F). Immunocytochemical analysis of these long-passage SKP spheres demonstrated that they expressed the same markers as the lower-passage SKP spheres, including nestin, fibronectin, versican (Fig. 5A), and vimentin (data not shown). These cells were then differentiated for 3–4 weeks and examined immunocytochemically. This analysis revealed that these highly expanded SKPs were able to generate morphologically complex, ?III-tubulin and NF-M–positive neurons (Figs. 5B, 5C), as well as bipolar glial cells coexpressing CNPase and GFAP (Fig. 5D) and SMA-positive smooth muscle cells or myofibroblasts (Fig. 5E). Thus, SKPs retain their ability to differentiate appropriately, even when maintained and expanded for long periods of time in culture.
Single SKPs Clonally Generate Neural and Mesodermal Progeny
To ask whether a single SKP cell was able to generate these different cell types, we performed clonal analysis. Human SKPs that had been passaged three times were dissociated to single cells; these single cells were isolated by limiting dilution into 48-well plates (Fig. 2A) and then cultured in medium containing EGF, FGF2, B27, and 50% of the filtered medium conditioned by more densely growing SKP cultures. Initially, these cells were quiescent, but they slowly started to proliferate, so that by 4 weeks, small clones of dispersed cells were formed, whereas by 8 weeks, spheres were generated that would ultimately dissociate from the tissue culture plastic and grow in suspension (Fig. 2A). After 10 weeks, the clones were dissociated and moved to 24-well plates and then 12-well plates over a period of 3 weeks and in this manner were ultimately expanded into small flasks. Of the 92 wells containing single cells at the start of the experiment, 39 of these proliferated to generate clones, and 37 of those grew as spheres. The other two clones only ever grew adherently, even after expansion, and were not analyzed further. Thus, approximately 40% of the SKP cells were able to self-renew when isolated as clones.
Immunocytochemical analysis of these clonal spheres revealed that they were similar to the starting culture of SKPs with regard to marker protein expression. Immunocytochemical analysis of six of these clones revealed that all of the spheres coexpressed nestin, fibronectin, and vimentin, whereas only the occasional cell was detectably positive for p75NTR (Fig. 2B). We then asked whether these clonal SKP lines were able to generate both neural and mesodermal progeny by plating cells from three clones on poly-D-lysine/laminin, withdrawing their proliferation factors, and differentiating them either in the presence of 5% FBS plus or minus neurotrophins (neuronal differentiation conditions) or in 5 μm retinoic acid and 1% FBS to ask if they could generate adipocytes. G-banding of one of these clones demonstrated that the chromosomal karyotype was normal (Fig. 2G). Immunocytochemical analysis of these three clones revealed that all of them were able to generate neurons, glia, smooth muscle cells, and adipocytes. Under neuronal differentiation conditions, the clones generated a relatively dense network of ?III-tubulin and NF-M–positive, morphologically complex neurons on top of a bed of nonneuronal cells (Fig. 2C). Many of these ?III-tubulin–positive cells also coexpressed p75NTR (data not shown). Under these same conditions, a smaller subpopulation of potential bipolar Schwann cells was also observed, as indicated by their coexpression of CNPase and GFAP (Fig. 2D). Neuronal and glial proteins were never expressed in the same cells in these cultures. With regard to mesodermal cell types, in the neural differentiation conditions, we observed a subpopulation of SMA-positive cells with the morphology of smooth muscle cells or myofibroblasts (Fig. 2E) but never saw cells with the morphology and characteristic lipid droplet inclusions of adipocytes. In contrast, when the same clones were differentiated in the presence of retinoic acid, a small population of adipocytes was observed (Fig. 2F). Similar results were obtained with three independent clones, indicating that human SKPs are multipotent.
Human SKPs May Represent an Endogenous Dermal Precursor
We have recently demonstrated that rodent SKPs derive from an endogenous SKP-like precursor that is first present in skin during embryogenesis and that is then maintained at lower levels into adulthood . We therefore asked whether human foreskin contained an endogenous SKP-like precursor that could generate neurons, a cell type that is never present in skin. To ask this question, four different human foreskin samples were dissociated into single cells, and these cells were plated adherently in the presence of 5% FBS. Immunocytochemical analysis 2 weeks later revealed the presence, in all four samples, of a subpopulation of morphologically complex cells that expressed ?III-tubulin (Fig. 6A) and, in some cases, NF-M (data not shown), properties of newly born neurons. In addition, cells were observed that expressed CNPase and GFAP, consistent with the presence of Schwann cells in skin, and a separate population was observed that expressed SMA (data not shown). Because ?III-tubulin–positive neurons were never observed in freshly dissociated skin preparations (data not shown) and have never been reported in skin, we conclude that the neurons observed in these experiments were generated by differentiation of a precursor present in developing foreskin.
Figure 6. Primary human SKPs have the same properties as passaged SKPs. (A): Fluorescence photomicrograph of primary human foreskin cells that were differentiated for 2 weeks and then immunostained for ?III-tubulin (red). (B): Fluorescence micrographs of primary human SKP spheres that were double-labeled for either nestin and fibronectin (left panels) or nestin and vimentin (right panels). (C): Fluorescence micrographs of primary human SKPs that were differentiated for 3 weeks in the presence of 5% fetal bovine serum and then double-labeled for ?III-tubulin (top panel) and p75NTR (bottom panel). (D): Fluorescence micrographs of primary human SKPs that were differentiated for 3 weeks in the presence of neurotrophins and then double-labeled for ?III-tubulin and p75NTR (left panels) or ?III-tubulin and NF-M (right panels). Note that although all ?III-tubulin–positive neurons are p75NTR-positive, not all p75NTR-positive cells express ?III-tubulin. These latter p75NTR-positive cells may be Schwann cells. (E): Primary human SKPs that were differentiated for 3 weeks and then double-labeled for CNPase (top panel) and GFAP (bottom panel). Note the colocalization of these proteins in elongated, bipolar cells. (F): Primary human SKPs that were differentiated for 3 weeks and immunostained for SMA. In all panels, the blue nuclei are Hoechst stained to show all of the cells in the field. Abbreviations: NF-M, neurofilament; SKP, skin-derived precursor; SMA, smooth muscle actin.
We then asked whether the very first SKP spheres that were formed from skin (primary spheres) had the properties of the more long-term passaged human SKPs. Immunocytochemical analysis of nine different preparations of primary SKP spheres revealed that, like the passaged SKPs, they expressed nestin, fibronectin, and vimentin (Fig. 6B) but did not contain cells expressing tyrosinase or trp1 (data not shown), indicating that they did not contain melanoblasts/melanocytes. Similar results were obtained when these primary SKP spheres were plated down for 24 hours. We then differentiated primary SKP spheres under neuronal differentiation conditions to ask whether they could generate neurons. Immunocytochemical analysis demonstrated that all of the human samples analyzed in this way generated morphologically complex cells that coexpressed either ?III-tubulin and p75NTR (Figs. 6C, 6D) or ?III-tubulin and NF-M (Fig. 6D), a profile typical of peripheral neurons. In these same cultures, a significantly smaller population of cells had the bipolar morphology typical of Schwann cells and coexpressed CNPase and GFAP (Fig. 6E) or S100? and p75NTR (data not shown). Finally, some of the cells expressed SMA and had a morphology typical of smooth muscle cells or myofibroblasts (Fig. 6F). Thus, the initial, unpassaged spheres that are generated from dissociated skin cells are similar, if not identical, to passaged SKP spheres, supporting the idea that SKPs may arise from an endogenous precursor in human skin, as they do in rodent skin.
DISCUSSION
This work was supported by grants from the Canadian Stem Cell Network, the CIHR, and the Canadian Heart and Stroke Foundation. I.A.M. is supported by a Canadian Stem Cell Network training award and a studentship award from the Hospital for Sick Children Foundation. F.D.M. is a CIHR Senior Investigator and a Senior Canada Research Chair. We would like to thank Mahnaz Akhavan and Anne Aumont for excellent technical support throughout the course of this work, David Kaplan for many valuable ideas, Dr. Roman Jednak for his help with the initial tissue samples, and the Miller and Kaplan laboratories for ongoing discussions.
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