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Keywords:

  • Stem cell;
  • Multipotent;
  • Oral mucosa;
  • Neural crest;
  • Neuroectoderm;
  • Definitive endoderm;
  • Tumor

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

The highly regenerative capacity of the human adult oral mucosa suggests the existence of a robust stem cell (SC) population in its lamina propria (OMLP). The purpose of this study was to characterize the availability, growth, immunophenotype, and potency of this presumable SC population. Cells positive for the embryonic stem cell transcription factors Oct4 and Sox2 and for p75 formed distinct cord-like structure in the OMLP. Regardless of donor age, trillions of cells, termed human oral mucosa stem cells (hOMSC), 95% of which express mesenchymal stromal cell markers, were simply, and reproducibly produced from a biopsy of 3–4 × 2 × 1 mm3. A total of 40–60% of these cells was positive for Oct4, Sox2, and Nanog and 60–80% expressed constitutively neural and neural crest SC markers. hOMSC differentiated in culture into mesodermal (osteoblastic, chondroblastic, and adipocytic), definitive endoderm and ectodermal (neuronal) lineages. Unexpectedly, hOMSC treated with dexamethasone formed tumors consisting of two germ layer-derived tissues when transplanted in severe combined immune deficiency mice. The tumors consisted of tissues produced by neural crest cells during embryogenesis—cartilage, bone, fat, striated muscle, and neural tissue. These results show that the adult OMLP harbors a primitive SC population with a distinct primitive neural-crest like phenotype and identifies the in vivo localization of putative ancestors for this population. This is the first report on ectodermal- and mesodermal-derived mixed tumors formation by a SC population derived from a nonmalignant somatic adult human tissue. STEM CELLS 2010;28:984–995


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

Embryonic stem cells (ESCs) are pluripotent and endowed with therapeutic potential [1, 2]. A myriad of multipotent stem cells (SC) populations have been isolated from adult tissues and characterized in vitro [3–10] and in vivo [11–14]. Mesenchymal SC or according to the preferred terminology of International Society for Stem Cell Research (ISSCR), mesenchymal stromal cells (MSCs) derived from adult tissues of mesodermal origin exhibit various degrees of plasticity in vitro and in vivo, which have been attributed to fusion [15], culture conditions, and mesenchymal-to-epithelial transition [16]. An alternative proposed explanation is the preservation in the adult organism of rare populations of primitive cells characterized by an embryonic-like immunophenotype capable to differentiate into lineages of two or three germ layers [4–7, 14, 17]. However, these populations do not develop to sizable proportions under normal culture conditions and their isolation and expansion require enriched substrates and culture media. In spite of their presumed existence in vivo, accumulating evidence indicates that ageing decreases the frequency, growth, and differentiation potential of adult SC [18–22].

Oral mucosa lines the oral cavity. Similarly to the ectomesenchymal origin of connective tissues in the oral cavity, cells of the oral mucosa lamina propria (OMLP) originate from the embryonic neural crest [23]. Wounds in human oral mucosa heal mainly by regeneration. The rate of healing is faster than that in the skin or other connective tissues and seems to be affected negligibly by age and gender [24, 25]. Human oral mucosa-derived fibroblasts behave in some respects similarly to fetal-derived fibroblasts [26]. Approximately 44% of rodent oral mucosa-derived cells cycle in vivo and in vitro and on explantation additional ∼39% start cycling in vitro [27]. Recently, we have provided first preliminary evidence that the OMLP gives rise to a robust multipotent SC population and suggested the human oral mucosa as a novel source for therapeutic adult SC (data presented at the sixth ISSCR Annual Meeting [28]). Widera et al. [29] recently reported the isolation of a neural population from the rat palate and identified in the human palate the message for transcription factors used for the generation of induced pluripotent stem (iPS) cells. Taken together, these data suggest that a resident primitive population is preserved in the adult human OMLP. However, to date, this population has never been isolated and its in vivo potency has not been characterized.

In this study, we report that explantation of the adult human OMLP reproducibly generates trillions of SC that we human oral mucosa stem cells (hOMSC). Immunophenotyping reveal a primitive neural crest stem cells (NCSC) phenotype, which is not affected by donor age. We show for the first time that hOMSC differentiate in vitro into lineages of the three germ layers and after stimulation with dexamethasone, their implantation in vivo resulted in the formation of bilineage mixed tumors consisting of tissues that develop from cranial neural crest cells during embryogenesis.

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

The sources of the materials, including antibodies, used in the study are detailed in supporting information.

Donors

Gingival and alveolar mucosa biopsies were obtained from donors following the study approval by the Institutional Helsinki Committee of Sheba Medical Center, Ramat-Gan, Israel.

Animal Experimentation

Severe combined immune deficiency (SCID) mice (C3H.CSCID/IcrSmnHsd) were obtained from Harlan (Jerusalem, Israel), (www.harlan.com). Animal experiments were approved by the Animal Ethic Committee, Tel Aviv University and the Israeli Ministry of Health.

Tissue Immunofluorescence

Processing and immunostaining of biopsies procedures are described in detail in supporting information. Immunofluorescent stained sections were visualized with epiflourescent light. To identify the histological nature of the fluorescent stained structures the microscope table was stabilized, the slides were carefully removed from the microscope, the coverslips were removed, the mounting solution was washed in phosphate-buffered saline (PBS), the sections were stained with hematoxylin and eosin (H&E), the slides were returned to the microscope at the same position as before their removal and the same field previously visualized with epifluorescence was captured with light microscopy.

Cell Culture

Oral mucosa biopsies particularly of gingival origin were obtained from donors aged 25–80 years. hOMSC were generated by explantation as described by us elsewhere [30] and expanded in low-glucose Dulbecco's modified Eagle's medium (LG-DMEM) and 10% fetal calf serum (FCS); (expansion medium). Human embryonic stem cells (hESCs) (HES-1 cell line [31]) with a stable normal karyotype (46XX) were maintained as described in [32] and served as positive controls in assays described below. Human foreskin fibroblasts (HSF) (ATCC#SCRC-1041) were cultured in high-glucose DMEM and 10% FCS.

Cumulative Population Doublings

Primary cultures (P0) were generated and passaged as described above. At each harvesting point, the total number of cells in each of three separate flasks was counted and the average number of cells was calculated. The number of doublings between two subsequent passages was calculated using the formula (log (10)H − log (10)I)/log (10)2, where H and I are the harvested and plated cells number, respectively.

Cloning Efficiency

To assess the number of clonogenic cells cultures at P9 to P11 obtained from two donors (aged 21 and 61 years) were plated by limited dilution in 96-well plates in expansion medium [30] at a density of 0.33, 1, 2, and 4 cells per well. Ninety-six wells were tested for each cell density. The experiment was repeated three times for each donor. Fourteen days thereafter, the number of empty wells was determined and their average fraction plotted against the cell density. Data were analyzed according to the Poisson distribution as previously described [27] (supporting information Fig. S6).

Immunophenotyping

Flow cytometry, immunofluorescence, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression of cell surface markers and transcription factors as described in detail in supporting information.

Growth Factors Secretion

Cultures at passages 9–11 obtained from three young (20, 20, and 23 years old) and two elderly (61 and 63 years old) donors were plated in expansion medium in T-75 culture flasks at 50% confluent density and cultured for 48 hours. Thereafter, the medium was removed, the cultures were washed 3× with PBS and cultured in serum-free expansion medium. After 48 hours, the conditioned medium of each donor was collected, concentrated eightfold, and analyzed with enzyme-linked immunosorbent assay kits for the secretion of epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), nerve growth factor (NGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), and vascular endothelial growth factor (VEGF) according to the manufacturer's instructions.

In Vitro Differentiation

hOMSC cultures at passages 4–8 were used for the differentiation experiments. Unless mentioned otherwise, the differentiation experiments were repeated for three donors in each of the young and elderly donors groups.

Mesodermal Differentiation

hOMSC cultures were subjected to osteoblastic, chondroblastic, and adipocytic differentiation regimens and analyzed for the expression of lineage specific differentiation markers by histochemistry, immunohistochemistry, and RT-PCR (see supporting information for details).

Definitive Endoderm Differentiation

hOMSC were plated in 35-mm culture dishes or in Labtek 8-well plates and maintained in LG-DMEM supplemented with 100 ng/ml of activin A and 0.5% FCS. The expression of Sox17, FoxA2, and CXCR4 markers were assessed by flow cytometry and indirect immunofluorescence in cultures treated with activin A for 24 and 72 hours. Untreated cultures served as controls.

Neuronal Differentiation

Cells derived from cultures of two young (20 and 24 years old) and two elderly (60 and 63 years old) donors at passages 9–11 were plated in Labtek 8-well plates (500 or 1,000 cells per well) or in 6-well plates (2 × 105 cells per well) and maintained in expansion medium for 48 hours. Then, the medium was replaced with serum-free expansion medium supplemented with 10 ng/ml neurotrophin-3 (NT-3), 10 ng/ml NGF-β, 50 ng/ml brain-derived neurotrophic factor, and N2. For determining expression of neural markers, cultures were harvested for immunofluorescence and real-time RT-PCR at 0 (72 hours for immunofluorescence), 7, and 14 days thereafter.

Glial Differentiation

Cells derived from cultures of two young (20 and 24 years old) and two elderly (60 and 63 years old) donors at passages 9–11 were plated on 18-mm glass coverslips (10,000 cells per slide) and cultured in 12-well plates in expansion medium. After 24 hours, the expansion medium was replaced with induction medium consisting of expansion medium supplemented with 35 ng/ml all-trans-retinoic acid for 72 hours and then with differentiation medium consisting of expansion medium supplemented with 5 ng/ml PDGF, 10 ng/ml basic fibroblast growth factor, 14 μM forskolin, and glial growth factor-2. This medium was replaced every 72 hours for 2 weeks. PC12 cells plated on 18-mm glass coverslips in 12 wells were cultured for 24 hours in LG-DMEM supplemented with 10% horse serum and 5% FBS, 1% glutamine, and 1% penicillin-streptomycin. Then, the coverslips bearing each of the two cell types were rinsed 3× in PBS and cocultured without coming into contact in 50-mm tissue bacterial culture dishes in serum-free LG-DMEM. To test the effect of hOMSC putatively differentiated into Schwann cells (ShOMSC) on PC12 cells survival and neuritogenesis, eight randomly selected fields on each of two coverslips bearing PC12 cells were photographed at a magnification of 200×, 24 hours after the beginning of the coculture. The total number of cells and the number of cells exhibiting neurite outgrowth were counted in each field. The results were pooled and expressed as mean ± SD per donor. Cultures of hOMSC maintained for 2 weeks only in expansion medium and then cocultured with PC12 as described above and PC12 cells cultured alone in serum-free medium served as controls.

Clones Differentiation

For clones differentiation, see supporting information.

In Vivo Differentiation

Thin fibrin matrices were produced by mixing a fibrinogen solution of 50 mg/ml with five units of thrombin for each milliliter of fibrinogen solution and pouring the mixed solution into 24-well plates. After 24 hours, the plates were washed in PBS and 500,000 hOMSC at passage seven derived from a young donor (20 years old) were plated in each well. The cultures were maintained in expansion medium for 48 hours and then in osteogenic medium comprising 10−7 M dexamethasone for additional 10 days at which time the fibrin matrices were detached from the wells and implanted subcutaneously in six SCID mice. The experiment was repeated with hOMSC derived from a second young donor (20 years old) that were implanted in five animals. Control dexamethasone-untreated hOMSC cultured at the same density on fibrin matrices, only in expansion medium, were implanted subcutaneously in five SCID mice. hOMSC cultured in expansion medium in culture dishes were harvested, suspended in PBS, labeled with the fluorescent dye DiI, and injected (2 × 106 per animal) in the quadriceps of four controls SCID mice. Animals were sacrificed 8 weeks thereafter, the implantation site was excised, fixed in 10% buffered formalin, and processed for histological examination as described elsewhere [33]. Sections were stained with H&E and Masson trichrome. To demonstrate hOMSC differentiation into neuroectoderm, sections were stained by immunochemistry with an anti-human S100B antibodies kit according to the manufacturer's instructions. To confirm the human origin of the transplants section were tested for the expression of mitochondrial antigen using an anti-human mitochondrial kit. Mouse-derived muscular and brain tissues served as negative controls for the anti-human mitochondrial antigen.

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

Expression of p75, Sox2, and Oct4 in the Gingival Lamina Propria

The low-affinity neurotrophin (p75) is a marker of NCSC and was used to isolate these cells from fetal and adult tissues [34, 35]. p75 was localized in groups of cells organized in cord-like structures within the OMLP (Fig. 1Ac–e). Similar cord-like structures were stained positively for the ESC transcription factors Oct4 and Sox2 (Fig. 1Ah–k,n–p). Double staining for Oct4 and p75 demonstrate their colocalization in part of p75 positive cells (Fig. 1B). Staining with anti-Nanog, nestin, and β-III tubulin antibodies was negative (data not shown). Sections in which the primary antibody was replaced with PBS stained negatively (data not shown).

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Figure 1. (A): In vitro and in vivo expression of primitive stem cell markers. The majority of hOMSC stain positively for p75 [(a, b), ×200]. Groups of human oral mucosa stem cells (hOMSC) stain positively for Oct4 and Sox2 [(f, g, l, m), ×400]. See also Figure 2 and Table 1. Immunostaining of oral mucosa lamina propria (OMLP)-derived sections localizes p75 to cord-like structure of high-cellular density as indicated by the DAPI and H&E staining [arrows in (c–e)]. Oct4 [arrows in (h–k)] and Sox2 [arrows in (n–p)] are localized to similar structures (×200). (B): Colocalization of Oct4 and p75 in a cord-like structure in the OMLP (×400). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin.

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Yield and Growth of hOMSC

Cells outgrew from the explants 7–10 days after explantation. On an average, a biopsy of 3–4 × 2 × 1 mm3 yielded 8 × 106 ± 1.1 × 105 cells at P0 during a period of 2–3 weeks. No differences were found between the yields of primary cultures obtained from young (YhOMSC) and elderly (EhOMSC) donors. To date, we have expanded YhOMSC between P1 and P27 for 70 cumulative population doublings (CPDs) (supporting information Fig. 1S) with an average doubling time of 49.2 ± 7.2 hours. EhOMSC were expanded between P1 and P27 for 50 CPD with an average doubling time of 87.2 ± 3.8. Thus, if the entire primary population were expanded, it would be feasible to generate trillions of YhOMSC or EhOMSC during 5–6 weeks of culture (7–8 passages) in expansion medium only.

Considering that the number of population doublings during the development of the primary cultures could not be assessed because of the explantation method used for their generation, the CPD of YhOMSC and EhOMSC is beyond the Hayflick limit [36]. Real-time RT-PCR did not reveal any telomerase transcripts indicating that the increased self-renewal capacity of these cells is telomerase-independent (see Discussion).

Characterization of hOMSC Cell Markers

Immunofluorescence indicated that the large majority of hOMSC at passages 1–8 were positive for p75 (Fig. 1Aa,b). Flow cytometry analysis of cultures at passages 4–8 (Table 1 and Fig. 2A) revealed that >95% of the cells in each of the tested populations expanded only in standard culture conditions expressed the consensus profile of MSC surface markers—CD29, CD73, CD90, CD105, and CD166. The interdonor standard deviations ranged between 0.62 and 3.91 pointing to the high reproducibility of the MSC immunophenotype in these populations. Approximately 33% of these populations were positive for Stro1 and CD106, which are mainly detected in MSC located in the paravascular niche in several adult tissues [37]. The pericytes marker CD146 was identified only in 16% of the cells indicating that the majority of hOMSC did not originate from pericyte-like cells as recently suggested to be the case for other adult tissue-derived MSC [38]. hOMSC were negative for CD34 and CD45 indicating nonhematopoietic origin. hOMSC were negative for human leukocyte antigen-DR (HLA-DR), but 95% were positive for human leukocyte antigen-DR (HLA-ABC) eliminating the possibility for their allogeneic utilization.

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Figure 2. Immunophenotype of human oral mucosa stem cells (hOMSC). (A): Representative flow cytometry histograms of the markers shown in Table 1. (B): Mean quantitative relative expression (y-axis) of Oct4, Nanog, and Sox2 observed in cultures of 33 doublings obtained from three young (Y-red bars) and three elderly (E-green bars) donors. ESC (blue bars) served as positive controls (C): Immunofluorescent illustrations of the ESC markers Nanog, and Oct4 in a young donor-derived hOMSC culture 48 hours after plating in expansion medium (original photograph, ×400). Nonreduced photograph (two right panels) illustrates nuclear localization of Oct4 and Nanog (original photograph, ×400). Control cultures in which the primary antibody was replaced with phosphate-buffered saline were negative (data not shown). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; ESC, embryonic stem cell.

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Table 1. Flow cytometry analysis of human oral mucosa stem cells (hOMSC)
  1. Averaged percentage of positive cells for the cell surface, cytoplasmatic and nuclear markers are shown in the table. Each marker was tested in cultures derived from three young (18–25 years old) and three elderly (over 60 years old) donors at passages 4–8.

  2. Abbreviations: ESC, embryonic stem cell; HLA-ABC, human leukocyte antigen-ABC; HLA-DR, human leukocyte antigen-DR; MSC, mesenchymal stromal cell.

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Next, we assessed whether hOMSC are positive for cell surface and nuclear markers typical to hESC [39]. Surprisingly, a relatively high percentage of hOMSC expressed pluripotency-related hESC markers constitutively. SSEA4, Oct4, and Sox2 were immunolocalized in >65% of the hOMSC and Nanog in 40% (Table 1 and Fig. 2A). Unlike most of ESC lineages, hOMSC were negative for SSEA3, SSEA1, Tra1-60, and Tra1-81, but 22–26% of these cells expressed Tra2-54 and Tra2-49 similar to some ESC lineages [39]. Alkaline phosphatase (ALP) was identified qualitatively at the protein level in a fraction of the cells and quantified by real-time RT-PCR to be 40 ± 26 and twofold higher than the reference human skin fibroblasts and hESCs, respectively (supporting information Fig. 2S). The expression levels of Oct4, Sox2, and Nanog were quantified at the message levels against the levels of HSF and found to be 250- to 350-fold higher than the baseline but by one order of magnitude lower than that of hESC (Fig. 2B; supporting information Fig. 3S), suggesting an intermediate level of Oct4 and Nanog in hOMSC. (Since the real expression of Oct4 in adult SC has been recently questioned by Liedtke et al. [40], we have used the primers suggested by these authors to confirm the expression of the Oct4 gene and not its pseudogenes.) Protein expression of these transcription factors was demonstrated at the single cell level (Figs. 1, 2). Nanog and Oct4 were specifically localized to the nucleus in hOMSC and in hESC (Fig. 2C and supporting information Fig. 4S for ESC).

The Effect of Ageing on the hOMSC Immunophenotype

In Vivo Ageing.

Comparison between the immunophenotypes of YhOMSC and EhOMSC revealed that donor ageing did not affect the pattern and expression levels of MSC and ESC markers at the protein levels (Table 1) and neither the message levels of Oct4, Nanog, and Sox2 (Fig. 2B).

In Vitro Ageing.

The levels of expression of SSEA4, Oct4, Nanog, Sox2, and nestin in three cultures derived from two young and one elderly donors at P27 and at 62 CPD were statistically equal to those of cultures at P4 with an average of eight CPD, demonstrating that in vitro ageing did not affect the ESC-like aspect of the hOMSC immunophenotype (supporting information Table 3).

Karyotype analysis of two cultures at 74 and 55 CPD derived from a young (23 years old) and elderly (63 years old) donor, respectively, did not reveal chromosomal aberrations (supporting information Fig. 5S).

Growth Factors

Analysis of growth factors in hOMSC conditioned medium obtained from three young and two old donors revealed that ∼7.5 × 105 cells secrete on an average (pooled data for YhOMSC and EhOMSC) 121 ± 47 pgr/ml/24 hours of FGF-2, 80 ± 45.4 pgr/ml/24 hours of VEGF, 17 ± 14 pgr/ml/24 hours of EGF, and 11 ± 5 pgr/ml/24 hours of NGF. PDGF-BB and SCF were not detected.

Differentiation Capacity of hOMSC

In Vitro Differentiation.

To test the multipotency of hOMSC, whole unsorted hOMSC cultures were induced with appropriate differentiation regimens toward specification to cell lineages derived from the three germ layers.

Mesodermal Differentiation.

Osteogenic, chondrogenic, and adipocytic lineages represented the mesodermal germ layer. Nodular mineralized deposits characteristic to human osteoblastic cultures derived from bone marrow stromal progenitor cells [30, 41] were identified with Alizarin Red S staining in 5 weeks old cultures as described elsewhere [41] (Fig. 3A). The early osteogenic differentiation markers Runx2, osterix, collagen type I, and ALP were expressed in cultures maintained for 1 week in osteogenic differentiation medium (Fig. 3B). Differentiation into the adipocytic lineage was shown by demonstrating the expression of the specific adipocytic factors lipoprotein lipase and peroxisome proliferator-activated receptor γ-2 in hOMSC cultures maintained for 5 weeks in adipocytic medium and by the positive oil red O staining of 5–10% of the cells in these cultures (Fig. 3C, 3D). Chondrogenic differentiation was evidenced in micromasses maintained for 5 weeks in chondrogenic medium by positive staining of proteoglycans with Alcian Blue and by immunolocalization of aggrecan and collagen type II (Fig. 3E–3H). Cultures maintained only in expansion medium were negative for the aforementioned late mesodermal differentiation markers (data not shown).

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Figure 3. Mesodermal and endodermal differentiation of hOMSC. (A–H): Mesodermal differentiation. (A): Mineralized deposits identified by Alizarin Red S in hOMSC grown in osteogenic medium for 5 weeks. (B): Expression of early osteoblastic differentiation markers Runx2, osterix, alkaline phosphatase, and type I collagen (lanes 2–5, respectively) at 1 week of osteogenic induction; lane 1: glyceraldehyde 3-phosphate dehydrogenase (GAPDH); arrow indicates 500 bp. (C): hOMSC cultured in adipocytic media for 5 weeks expressed the adipocytic markers proliferator-activated receptor-γ2 and lipoprotein lipase (lanes 2–3, respectively); lane 1: GAPDH; arrow indicates 500 bp. (D): Triglycerides vacuoles that stained positively with oil red O staining (bar 50 μm). (E): Cartilage proteoglycans identified by Alcian Blue staining of histologic sections obtained from micromasses of hOMSC grown in suspension for 5 weeks in chondrogenic medium (bar 160 μm). (F): Alcian Blue and red fast counterstaining illustrates chondroblast-like cells within proteoglycans reach matrix lacunae (bar 80 μm). (G, H): Immunohistochemical identification of aggrecan (G) and collagen type II (H) (bar 160 μm). Sections treated only with the second antibody were negative (data not shown). (I–K): Endodermal differentiation. Expression of the definitive endoderm differentiation markers Sox17 (I), Foxa2 (J), and CRCX4 (K) in the large majority of hOMSC cultured for 72 hours in 100 ng/ml of activin A and 0.5% fetal calf serum (bars 180 μm). Cultures incubated only with second antibodies were negative (data not shown). Abbreviation: DAPI, 4,6-diamidino-2-phenylindole.

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Endodermal Differentiation.

To demonstrate the capacity of hOMSC to differentiate into a putative definitive endoderm (DE) lineage, the expression of the DE markers Sox17, Foxa2, and CRCX4 [42] was assessed by flow cytometry and immunofluorescence 24 and 72 hours after activin A induction. At 24 hours, 91%, 93%, and 70% of the cells in unsorted hOMSC cultures were positive for the DE markers Sox17, Foxa2, and CRCX4, respectively. At 72 hours (Fig. 3I–3K), the expression of Sox17 and Foxa2 remained stable and that of CXCR4 increased to 85% of the cells indicating that the majority of the hOMSC exhibited DE markers and pointing to their putative differentiation into DE.

Neuronal Differentiation.

hOMSC cultures at passages 4–11 maintained only in expansion medium were positive for p75, nestin, βIII-tubulin, MAP2, and GFAP at the message level (Fig. 4A). Flow cytometry analysis showed that 60–70% of the cells in these cultures were positive for nestin, βIII-tubulin, and GFAP pointing to the constitutive expression of neuronal and glial markers in these cultures.

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Figure 4. Neural differentiation of hOMSC. (A): Real-time reverse transcription-polymerase chain reaction quantification of neuronal marker expression by hOMSC subjected for 2 weeks to neural differentiation. Values on y-axis are normalized to human foreskin fibroblasts. (B): Immunolocalization of βIII-tubulin (bar 150 μm), Neu-N (bar 40 μm), and O4 (bar 40 μm) in hOMSC cultures maintained for 2 weeks in neural differentiation medium. (C): hOMSC grown for 2 weeks in glial differentiation medium support the survival and neuritogenesis of PC12 cells. ShOMSC + PC12: PC12 cells cocultured with hOMSC subjected to glial differentiation; hOMSC + PC12: PC12 cells cocultured with undifferentiated hOMSC. (D): D1–D3 illustrates general views of the number of PC12 cells in cultures of ShOMSC + PC12, hOMSC + PC12, and PC12 only, respectively, cultured in serum-free medium (×40). D4 is a higher magnification of D1 demonstrating neurites outgrowth (×200). Abbreviations: %CN, average percentage of cell number/field/donor exhibiting neuritogenesis; DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary protein; hOMSC, human oral mucosa stem cells; MAP2, microtubule associated protein-2; PC12, pheochromocytoma cell line-12; TCN, average of total cell number/field/donor; ShOMSC, hOMSC induced to glial differentiation.

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Neural Differentiation.

Treatment with neural differentiation medium for 2 weeks decreased the message for Nanog and Oct4 by one order of magnitude, the message of βIII-tubulin by threefold (p < .05), increased the message of MAP2 by 3.3-fold (p < .05) (Fig. 4A) and induced the expression of and Neu-N at the protein level (Fig. 4B). It also decreased the levels of p75 and GFAP, even though not statistically significant. Nestin expression was not affected. The results indicate a differentiation process of hOMSC toward a neural lineage, albeit incomplete as nestin, p75 and O4 were still expressed in the differentiated cultures.

Glial Differentiation.

To test glial differentiation a functional assay was developed: The capacity of hOMSC subjected to glial differentiation regimen (ShOMSC) to induce neuritogenesis and support survival of PC12 cells was assessed. It was found that 24 hours after coculturing ShOMSC with PC12 cells in serum-free medium, the percentage of PC12 cells exhibiting neurite outgrowth was 3- and 1.8-fold higher (p < .05) than that of PC12 grown only in serum-free expansion medium and PC12 cocultured with control undifferentiated hOMSC, respectively (Fig. 4C, 4D). The average number of PC12 cells per field was reduced by 60% in PC12 cultures grown only in serum-free medium (p < .05) compared with that of PC12 cells cocultured with ShOMSC. Coculturing with undifferentiated hOMSC supported PC12 survival similarly to ShOMSC. The results indicate that ShOMSC supported neuritogenesis and cell survival of PC12 cells in serum-free medium.

Cloning and Clone Differentiation.

Using limited dilution and Poisson distribution analysis (supporting information Fig. S6), it was found that 1 of 1.88 cells in the hOMSC cultures was a clonogenic cell that formed a colony of 50 cells or more. YhOMSC and EhOMSC slightly differ in this respect (1/1.78 and 1/1.99, respectively). Cloned colonies obtained by limiting dilution in 96 wells were subjected to differentiation regimens. A large fraction of clones subjected to specific differentiation regimens expressed characteristic lineage specific markers of mesodermal (50%), neural (60%), and DE lineages (30%) (see supporting information and Fig. 7S for detailed information).

In Vivo Differentiation.

The original purpose of the in vivo experiment was to test the capacity of hOMSC induced for 10 days with dexamethasone to form mesodermal tissues and particularly bone, ectopically in vivo. To do this, hOMSC were seeded on fibrin membranes at high density to obtain cell aggregates. These were treated with vitamin C to induce extracellular matrix formation and dexamethasone to induct cell differentiation. Phase contrast microscopic examination revealed that at the implantation time multilayered structures developed on the fibrin membranes. At 6 weeks after implantation, a swelling (tumor) of 4–5 mm in diameter and 2–3 mm in height (Fig. 5A) was observed at the supracalvarial implantation site in each animal. During the remaining 2 weeks, two experimental animals of the first experiment died and the implants could not be retrieved for analysis. Surprisingly, the histological examination of these swellings in the remaining four animals revealed the formation of bilineage (mesodermal and ectodermal) mixed tumors consisting of fetal fat, striated muscle, cartilage, bone, epithelial, and neural tissues (Fig. 5B–5M). The distribution of the various tissues within the tumor was reminiscent of a teratoma-like structure. Endodermal derivatives were not detected. Striated muscle and fetal fat formed a considerable part of these tumors. The muscular bundles were often randomly oriented. Cartilage was observed in the vicinity of epithelial structures (Fig. 5B–5D), but mostly it was embedded within muscular bundles (Fig. 5F). Islands of bone formation were observed emerging from cartilage structures simulating enchondral osteogenesis (Fig. 5G). Prominent neural tissue that stained positively for the glial S100B marker was observed within the connective tissue (Fig. 5H–5M). Staining with anti-human mitochondria antibody stained positively the various tissues of the bilineage tumor, including endothelial cells pointing to their human origin (Fig. 6A, 6B). Mouse muscle and brain tissues that served as controls were negative (Fig. 6C, 6D). In the second experiment, two animals died, one in each of the control and experimental groups. Swellings as described above were also observed in the supracalvarial implantation sites in each of the remaining four experimental and four control animals (Fig. 6C). On dissection, tumor-like masses were found between the calvaria bones and skin in all animals. Soft x-ray radiography indicated mineralized tissue formation within these masses (Fig. 6E–6G). Histological examination of sections obtained from control animals implanted with untreated hOMSC revealed that these masses consisted of bony lacunae filled with an acellular basophilic material surrounded by a white hallo (Fig. 6H, 6I). Basophilic cells delineated these lacunae, but occasionally the basophilic material contacted directly the bony tissue. Masson trichrome staining showed that the basophilic material stained green-blue with red spots (Fig. 6K), pointing to its mineralized collagenous nature. Histological examination of sections of the four experimental animals of the second experiment uncovered the formation of similar bony structures. Notably, in one experimental animal, a bilineage tumor consisting of muscle, cartilage, and neural tissue was observed adjacent to the bony mass. In a second experimental animal, a neural primitive tissue, which stained positively for βIII-tubulin, was identified (data not shown). Fibrin membranes implanted without cells could not be identified 8 weeks postimplantation. Fluorescent microscopic examination of the muscles injected with cell suspensions in the control animals did not identify the DiI labeled cells. It should be mentioned that this examination was made difficult by the strong autofluorescence of the muscle tissue. Furthermore, thorough light microscopic examination of the injected muscles did not reveal any tumors or other ectopic tissue formation. The histological appearance of the injected muscles was normal and did not differ from that of contralateral control muscles.

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Figure 5. hOMSC form bilineage mixed tumors with a teratoma-like appearance in vivo. A representative swelling in the supracalvarial implantation site of a severe combined immune deficiency mice (A). Representative histological general view of sections obtained from tissues forming swellings as shown in (A) depicting: cartilage (C), neural tissue (N), muscle bundles (M), fetal fat (F), and epithelium (arrows) intermingled within the tumor [(B) ×40]. Low and high magnification of epithelium (arrows) and cartilage [(C), ×40; (D), ×200] and fetal fat [(C), ×40; (E), ×200] within a tumor. Cartilage developing within striated muscular tissue [(F), ×100]. Island of bone tissue emerging from cartilaginous tissue (asterisks) [(G), ×100]. General view [(H), ×40] and high magnifications [(I, L), ×100] of neural structures. An adjacent section stained positively with anti-S100 antibody confirm the neural nature of these structures [(K, M), ×100].

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Figure 6. (A): Immunohistochemical staining with anti-human mitochondrial antibodies of a specimen from a bilineage mixed tumor demonstrating positive staining for muscle (M), neural tissue (red arrow), and vascular cells (black arrows). (B): An adjacent control section that was not incubated with the primary antibody shows negative staining. (C, D): Control sections of mice muscle (C) and brain (D) tissues labeled with anti-human mitochondrial antibodies stain negatively. Panels (E)–(L) are representative of macroscopic (E), radiographic (F), and microscopic (H–L) illustrations of mineralized masses formed between the calvaria bones and the skin in control animals implanted with aggregates of untreated hOMSC on fibrin membranes. The arrows in (E) and (F) point to the mineralized mass. (G): A radiograph of a control animal that was implanted with the fibrin membrane only. (H): H&E staining at low magnification of a mineralized mass (arrow) showing that it is not connected to the calvaria bones (×40). (I): H&E staining of a mineralized mass depicting the basophilic material (arrow) within the bony tissue (×200). (K): Masson trichrome staining showing the green-blue with red spots staining of the material found in a bony lacunae pointing to mineralized matrix (arrow) (×200). (L): Control Masson trichrome staining of the parietal bone of a severe combined immune deficiency mice after demineralization demonstrating the greenish and red staining characteristic to collagenous mineralized matrix.

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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

The connective tissues of the head are of neural crest origin [23, 43]. The fact that similar to fetal tissues, wounds in the oral mucosa heal by regeneration and without scar formation suggested the existence of a primitive NCSC-derived population in the OMLP. To mimic wound healing in culture [44], minimize the “trauma” associated with cell culture generation by enzymatic digestion and preserve as much as possible the SC niche, hOMSC cultures were produced by explantation.

Immunophenotyping revealed the expression of markers consistent with MSC [45] and neural stem/progenitor cell [46] phenotypes. At the message level, hOMSC expressed constitutively transcription factors known to maintain the pluripotency of ESC (Oct4, Nanog, and Sox2) at levels lower than those of hESC, probably due to hOMSC population heterogeneity and/or consequent to lower number of transcripts per cell. Even though Sox2 is shared by both neural SCs and ESC, the expression of Oct4 and Nanog suggests that these factors maintain in an undifferentiated state a relatively large fraction of hOMSC culture. The expression of early neural crest stem/progenitor cell markers (Sox2 and p75) in vitro and in vivo points to the neural crest origin of this population. These data are in line with recent reports on the existence of neural crest-derived stem/progenitor cells in bone marrow, dermis, whisker pad and the gastrointestinal tract including oral mucosa albeit at lower frequencies [29, 34, 35, 47–49]. The identification of mRNA for Oct4, Sox2, Myc-c, and Kfl4, the four transcription factors used for iPS induction [50] in the human palate [29] suggests that OMLP harbors a primitive SC population. We show for the first time that p75, Sox2, and Oct4 are localized in cells constituting cord-like structures in the human OMLP and that Oct4 and p75 colocalize in some of these cells suggesting that these cords might constitute a niche for the preservation of neural crest-derived SCs in the adult organism. Since Nanog and nestin were not identified in the cells forming these cords, it is conceivable that their expression is initiated either in the explant or in cells that left the cord niche to migrate out to the culture dish.

hOMSC are endowed with a relatively high, telomerase-independent self-renewing capacity and cloning efficiency compared with MSC derived from other tissue [19, 22]. hOMSC produce and secrete FGF-2, EGF, VEGF, and NGF. These growth factors, particularly FGF-2 [51–53], are known to support cell proliferation. Thus, their activity may explain the high-renewal capacity of hOMSC. Recently, Enoch et al. [54] reported that OMLP-derived fibroblasts reach 80–115 population doublings before senescence, while donor matched skin fibroblasts only 40–65. This increased self-renewal capacity of OMLP-fibroblasts was telomerase-independent, but was related to increased telomeres length compared with skin fibroblasts suggesting that the OMPL population is endowed with inherent mechanisms that preserves telomere length. Endogenous FGF-2 has recently been proposed to contribute to maintaining pluripotency genes expression and reduce stress-induced apoptosis in hESC [55]. It might be possible that similar endogenous FGF-2 activity in hOMSC increases their lifespan and maintain them in a relatively undifferentiated state.

The NCSC-like immunophenotype of hOMSC and the expression of ESC-markers predicted that they are multipotent and possibly pluripotent. During development, cranial-derived NCSC are specified to differentiate into mesodermal and neuroectodermal lineages [43]. In vitro assays demonstrated that unsorted hOMSC subjected to neuronal differentiation regimens differentiated into neuroectoderm lineages as evidenced by the decrease in Oct4 and Nanog, increase in MAP2 expression (neural) and induction of neuritogenesis in PC12 cells (glial), the last being considered a functional assay for glial differentiation [56]. Undifferentiated hOMSC supported only PC12 cells survival, probably via the secretion of NGF and FGF-2. Differentiated ShOMSC supported both survival and neuritogenesis indicating that ShOMSC secreted additional trophic factors that remain to be determined in futures studies.

As described for hESC [42], when induced with activin A, hOMSC expressed markers of the DE lineage. Even though these markers are produced by different cell lineages, their concomitant expression suggests initial differentiation toward the endodermal lineage. However, we have been unsuccessful to promote the further differentiation of hOMSC toward pancreatic lineages (data not shown).

On the basis of the capacity of hOMSC to differentiate into mesodermal lineages in vitro, we first tested their capacity to do that in vivo. To do this, cultures were induced with dexamethasone for 10 days. Since dexamethasone is mainly an inducer of the osteoblastic lineage, we presumed that dexamethasone-treated hOMSC would mainly form ectopic bone tissue. The surprising formation of bilineage mixed tumors consisting of mesodermal and ectodermal tissues in this experimental model implies that: (a) The hOMSC population (or at least part of it) is multipotent and as this, it is capable of giving rise in vivo to tissues that develop from cranial neural crest cells during embryogenesis. Moreover, this finding confirm the capacity of hOMSC to differentiate into mesodermal and ectodermal lineages observed in vitro. (b) The lack of endodermal tissue formation in the tumor suggests that either similar to cranial NCSC, hOMSC are more restricted in their potency than ESC or iPS or that the endodermal pathway was blocked by dexamethasone treatment. (c) Despite the prolonged inductive treatment, the hOMSC population responded heterogeneously to dexamethasone.

Not less intriguing are the findings that implantation of control suspensions of untreated hOMSC from two donors did not result in any type of ectopic tissue formation. Control cultures of untreated hOMSC aggregates did not form bilineage mixed tumors, but rather developed into ectopic mineralized tissue consisting of bone and an acellular basophilic material reminiscent of dental cementum suggesting that cell–cell interactions are required for ectopic tissue development in this experimental setup. However, dexamethasone-treated hOMSC aggregates from the same donor formed bilineage mixed tumors in two of four animals. Since it was expected that dexamethasone untreated hOMSC would express a less restricted differentiation repertoire than the treated ones, these findings suggest that: (a) in the culture conditions used in this study dexamethasone activated in vitro various lineage specifications in addition to the osteoblastic one; (b) because untreated hOMSC did not form mineralized-like tissue in vitro, the final stages of osteoblastic lineage specification occurred in vivo; and (c) the propensity of dexamethasone-treated hOMSC to form bilineage mixed tumors might be donor dependent.

In contrast to reports on the negative effect of ageing on adult SC growth and functionality [18–22, 57], the growth during the first 10 population doublings, the fraction of cells expressing Nanog and Oct4, the level of transcription of these factors, and the in vitro differentiation capacity of hOMSC were not affected by the donor age, thus, providing a biological explanation for the negligible ageing effect on wound healing in the elderly oral mucosa [25]. However, the increase in the doubling time at higher population doublings in elderly derived cultures suggests that mechanisms active in preserving telomeres length in EhOMSC is less effective than in YhOMSC.

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

In summary, the results of this study indicate that a multipotent NCSC-like population can be reproducibly generated from the adult lamina propria of the adult human oral mucosa. The study provides first evidence that dexamethasone-treated hOMSC produce bilineage mixed tumors on its transplantation in vivo and that the putative ancestors of hOMSC within the adult human lamina propria are organized in specific cord-like structures. The wide repertoire of ectopic tissues formation points to the possible future therapeutic use of hOMSC.

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 Dr. K. Fuchs, Tel Aviv University for excellent statistical assistance, Dr. B. Reubinoff, Hebrew University for kindly providing the hESC line, Dr. E. Okon, Tel Aviv University for confirming the histological results, and H. Orenstein, H. Vered, and L. Mittelman for excellent technical assistance. This work was supported by the Israel Science Foundation of the Israeli Academy of Science, Binational United States - Israel Science Foundation, and the Chief Scientist of the Ministry of Health Foundation.

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_00425_sm_suppfigure1.tif174KSupporting Figure 1
STEM_00425_sm_suppfigure2.tif1054KSupporting Figure 2
STEM_00425_sm_suppfigure3.tif119KSupporting Figure 3
STEM_00425_sm_suppfigure4.tif198KSupporting Figure 4
STEM_00425_sm_suppfigure5.tif289KSupporting Figure 5
STEM_00425_sm_suppfigure6.tif164KSupporting Figure 6
STEM_00425_sm_suppfigure7.tif2332KSupporting Figure 7
STEM_00425_sm_suppinfo.doc95KSupporting Information

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