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TISSUE ENGINEERING: Part A Volume 17, Numbers 5 and 6, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2010.0385
The Potential of Stem Cells in Adult Tissues Representative of the Three Germ Layers
Haruko Obokata, M.S.,1–3 Koji Kojima, M.D., Ph.D.,1 Karen Westerman, Ph.D.,1 Masayuki Yamato, Ph.D.,3 Teruo Okano, Ph.D.,3 Satoshi Tsuneda, Ph.D.,2 and Charles A. Vacanti, M.D.1
1.Laboratory for Tissue Engineering and Regenerative Medicine, Department of Anesthesiology, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts. 2.Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan. 3.Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan.
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Abstract
Mature adult tissues contain stem cells that express many genes normally associated with the early stage of embryonic development, when maintained in appropriate environments. Cells procured from adult tissues representative of the three germ layers (spinal cord, muscle, and lung), each exhibiting the potential to mature into cells representative of all three germ layers. Cells isolated from adult tissues of different germ layer origin were propagated as nonadherent clusters or spheres that were composed of heterogeneous populations of cells. When the clusters or spheres were dissociated, the cells had the ability to reform new, nonadherent spheres for several generations. When implanted in vivo, in association with biodegradable scaffolds, into immunodeficient mice, tissue containing cells characteristic of the three germ layers was generated. These findings suggest the existence of a population of stem cells in adult tissues that is quite different and distinct from embryonic stem cells that demonstrate a greater potency for differentiation across germ lines than previously believed. Such cells could potentially be as useful as embryonic stem cells in tissue engineering and regenerative medicine.
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Introduction
The degree of potency of adult stem cells as compared to embryonic stem cells (ESCs) remains controversial. It is generally felt that while ESCs and induced pluripotent stem cells[1] demonstrate pluripotency, adult stems cells are limited to multipotency and are felt not to be able to cross the germ layer lineages as they develop. Although the existence of adult stem cells has been reported[2–5] for more than a decade, we have hypothesized the existence of adult stem cells, which are quite small and resistant to external stress, that reside in tissues representative of the three germ layers.[2] Adult stem cells have generally been felt to be limited to multipotency and unable to cross germ layer lineages as they develop. Cells derived from many adult tissues, including retina,[6] brain,[7–10] cornea,[11] olfactory neuroepithelium,[12,13] pancreas,[14] skin,[15,16] muscle,[17] and bone marrow (BM),[18,19] have now been propagated as nonadherent clusters or spheres,as have ESCs.[20] The cells contained within the spheres exhibit neural lineage markers and appear to possess varying degrees of stem cell potency. In addition, neurospheres have been shown to contain stem cells demonstrating gene express<Translation machine note:For 'express',read 'expression'. It's probably a typographical error.> that overlap that of ESCs.
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We believe that adult stem cells described in various reports represent the same adult stem cells at different stages of development, expressing different degrees of potency and that sphere-forming cells are much more immature than previously expected. We hypothesized that adult stem cells procured from any tissue, ectoderm, mesoderm, or endoderm cross germ layers as they develop, when maintained in appropriate environments in vitro and in vivo. In this study, we characterized cells isolated from three adult tissues (spinal cord, muscle, and lung) representative of the three different germ layers (ectoderm, mesoderm, and endoderm) and from BM.
Mature cells were disrupted by trituration and then propagated as nonadherent clusters or spheres in a serum-free culture medium. Cells procured from each source initially expressed many of the markers associated with ESCs and demonstrated the potential to differentiate into all three germ layers, in vitro and in vivo. The isolation initially contained a significant amount of floating debris, nonadherent cells, insoluble proteins or fibers, and other extraneous materials, all of which appeared to participate in the formation of nonadherent spherical clusters that contained the cells. The cellular make-up of individual spheres was not identical; that is, spheres were composed of heterogeneous populations of cells, even when the spheres were generated from cells procured from the same tissue at the same time. Similarities or differences seen in the cell content of different spheres were believed to be secondary to the environment in which they were cultured.
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Materials and Methods
Cell isolation and cell culture
BM, spinal cord (ectoderm), muscle (mesoderm), and lung (endoderm)were collected from 3-to4-week-old and 12-week-old C57BL/6J mice (Jackson Laboratory). C57BL/6J (B6) mice were anesthetized with Avertin(2,2,2-tribromoethanol)(SigmaAldrich). All tissues were obtained according to the guidelines from Harvard University IACUC. Mouse femurs and tibias were aseptically dissected and cut. The BM was extruded by flushing with culture media using an insulin syringe. The cell suspension obtained was centrifuged at 2000rpm for 5min and washed with F12/Dulbecco’s modified Eagle’s medium (DMEM; 1:1, v/v; Gibco). The cell suspension was passed through 100µm nylon filters (BD Falcon) and centrifuged at 2000rpm for 5min, washed once more, and centrifuged at 2000rpm for 5min. To remove the erythrocytes, the cell suspension was treated with RBC Lysis Buffer (eBiosciencel). Cells were triturated vigorously and passed through 70 and 40µm nylon filters. Spinal cord tissue was dissected and thoroughly minced. Cells were triturated vigorously and passed through 100, 70, and 40µm nylon filters.
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The cell suspension was collected and centrifuged at 2000rpm for 5min. Muscle from the thigh was excised and thoroughly minced. The tissue was treated with 0.2% collagenase D (Roche) for 1h at 37℃. Cells were triturated vigorously and passed through 100, 70, and 40µm nylon filters. Cell suspensions were collected and centrifuged at 2000rpm for 5min. Lung tissues were washed via intratracheal lumen with 10mL of cold phosphate buffered saline (PBS; Gibco) and were perfused with collagenase type P (Roche). Percoil gradient centrifugation was employed to remove contaminating erythrocytes, neutrophils, and cell debris. Cells were triturated vigorously and passed through 100, 70, and 40µm nylon filters. Cell suspensions were collected and centrifuged at 2000rpm for 5min.
Cells from each tissue type were plated at 1x10<to the power of 6> cells/㎠ in F12/DMEM (1:1, v/v) supplemented with 2% B27 (Invitrogen), 20ng/mL basic fibroblast growth factor (bFGF; R&D Systems), and 10ng/mL epidermal growth factor (EGF; R&D Systems). About 50% of the medium was replaced every 2–3 days for the duration of the culture; bFGF and EGF were added every other day. To calculate the doubling time of cells, the number of cells was counted daily as cells proliferated to form spheres. As the spheres grew in size, representative spheres were dissociated and the cell number were counted, doubling times were calculated.
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Cell size measurement
Marrowspheres from 4-week-old mice, collected after 5 days in the fresh medium, were dissociated into single cells. Dissociated cells were stained with propidium iodide filtered through a 40µm pore filter and measured on a DAKO Galaxy (DAKO) using FloMax software. Cytofluorimetric analysis was then performed to establish cell size. Microbeads of predefined sizes (Size Calibration Standards Kit; Bangs Laboratories, Inc.) were re-suspended in isotonic phosphate saline (pH 7.2) and used as a standard for which to compare size of cells contained in spheres using cytofluorimetric analysis.
Both cells and beads were analyzed using the same instrument setting (forward scatter, representing cell and bead size, and side scatter, representing cellular granularity). Cell size was calculated on a curve employing bead size on the x-axis and forward scatter values on the axis.
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Immunohistochemistry
The protein expression of spheres was assessed using immunohistochemical techniques. Spheres were collected using the cell strainer and rinsed with PBS twice. Then, spheres with the membrane were embedded and sliced at 4mm thickness. Each slide was fixed with 4% paraformaldehyde (Wako) for 15min at room temperature, washed with PBS, incubated with SuperBlock-R blocking buffer in PBS (P74370; Takara) for 30min to block nonspecific reactions, and incubated with anti-c-kit rat monoclonal antibody (sc-19619; Santa Cruz Biotechnology, Inc), anti-Sca-1 rat monoclonal antibody (ab25031; Abcam), or anti-E-cadherin rat monoclonal antibody (ab11512; Abcam) overnight at 48C. After washing with PBS, the cells were incubated with goat anti-rat IgG Texas Red-conjugated antibodies (112-076-062; Jackson ImmunoResearch) and goat anti-rat IgG Fluoresceinconjugated antibodies (112-096-062; Jackson ImmunoResearch) for 30min at room temperature. Stage-specific embryonic antigen-1 and alkaline phosphatase staining was performed using the ES cell detection Kit (Millipore) in accordance with the manufacturer’s protocol. In negative controls, the primary antibody was replaced with IgGnegative controls of the same isotype to ensure specificity.
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In vitro differentiation assay
Mesoderm lineage differentiation assay. The spheres were collected at 5 days and dissociated into single cells, and placed in DMEM supplemented 20%fetal calf serum(FCS).The medium was exchanged every 3 days. After 7–14 days, muscle cells were stained with anti-a-smooth muscle actin antibody (N1584; DAKO), anti-myosin antibody (ALX-805-503), and anti-desmin antibody (D1033; Sigma). In negative controls, the primary antibody was replaced with IgG-negative controls of the same isotype to ensure specificity. Chondrocytes were stained with Safranin-O (Fisher; S67025) and Fast Green (Fisher; F99). Osteocytes were stained with Alizarin Red S (Sigma; A5533). After 21 days, adipocytes were stained with Oil Red O (Sigma; O-0625).
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Neural lineage differentiation assay. The spheres were collected at 5 days and dissociated into single cells, which were plated on ortinin-coated chamber slides (Nalge Nunc International) in F12/DMEM (1:1, v/v) supplemented 2% B27 (Invitrogen), 10% FCS, 10ng/mL bFGF (R&D Systems), and 20ng/m EGF (R&D Systems). The medium was exchanged every 3 days. After 10–14 days, cells were fixed with 4% paraformaldehyde for 30min at 48C, washed with PBS containing 0.2% Triton X-100 (Sigma) for 15min at room temperature, incubated with PBS containing 2% FCS for 20min to block nonspecific reactions, and incubated with anti-bIII Tubuin mouse monoclonal antibody (G7121; Promega), anti-O4 mouse monoclonal antibody (MAB345; Millipore), and anti-glial fibrillary acidic protein (GFAP) mouse monoclonal antibody (AB5804; Chemicon). In negative controls, the primary antibody was replaced with IgG-negative controls of the same isotype to ensure specificity.
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Hepatic differentiation assay. The spheres were collected at day 5 and dissociated into single cells, which were plated on chamber two-well slide glass (Nalge Nunc International) in the hepatocyte culture medium composed of 500mL hepatocyte basal medium (Lonza), 0.5mL ascorbic acid, 10mL bovine serum albumin-fatty acid free, 0.5mL hydrocortisone, 0.5mL transferrin, 0.5mL insulin, 0.5mL EGF, and 0.5mL gentamycin-amphotericin (GA-1000; all from Lonza) supplemented with 10% FCS and 1% penicillin/streptomycin (Sigma). Differentiated cells were detected by immunohistochemistry using following antibodies: anti-a-fetoprotein (AFP) mouse monoclonal antibody (MAB1368; R&D System), anti-Albumin goat polyclonal antibody (sc-46293; Santa Cruz Biotechnology, Inc.), and anti-Cytokeratin 18 (CK18) mouse monoclonal antibody (ab668; Abcam). In negative controls, the primary antibody was replaced with IgGnegative controls of the same isotype to ensure specificity. The results from immunohistochemistry were confirmed by reverse transcription (RT)-polymerase chain reaction (PCR). The presence of microtubule-associated protein 2 (Map2) was evaluated for neural lineage, MyoD was evaluated for mesoderm lineage, and AFP was done for hepatic differentiation.
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Single sphere RT-PCR
Single spheres were individually collected at day 5 under a phase-contrast microscope. Total RNA was extracted using the RNeasy Micro Kit (Qiagen) in accordance with the manufacturer’s protocol. To eliminate the risk of false-positives, RNA was isolated fromES cells andE 6.5 fetalcells as positive controls, and RNA was also isolated from heart, muscle, liver, and adherent stromal cells as negative controls for embryonic gene marker expression in RT-PCR. RNA was treated with DnaseI before RT to remove any genomic DNA. Minus-RT controls were run for each cDNA preparation to rule out genomic contamination. Total RNA (10.0ng) was then subjected to oligo-dT-primed RT with SuperScriptIII First-Strand Synthesis SuperMix (Invitrogen). RT-PCR was performed using TaqDNA polymerase (Takara) on an iCycler (BioRad). After an initial incubation at 94℃ for 3min, PCR was carried out at 94℃, for 30s, at specific annealing temperature for 30s and at 72℃ for 30s with a final extension cycle at 72℃ for 1min. A total of 35 cycles were performed. PCR products were electrophoresed on 2% agarose TBE gels. Gene-specific primers for RT-PCR were designed based on published sequences (Supplementary Table S1; Supplementary Data are available online at www.liebertonline.com/ten).
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In vivo differentiation assay
Sphere cultures from representative tissues were washed twice with Hank’s balanced salt solution (Gibco). Under a microscope, 2000 spheres, each containing *1000 cells, were collected using a glass pipette and placed in a 50mL tube. Hank’s balanced salt solution (20mL) was added to each tube and subsequently centrifuged at 800rpm for 3min. The supernatant was discarded and the pellet was resuspended in 50mL of DMEM with 10% fetal bovine serum. This solution was seeded onto a sheet 3x3x1mm, composed of a nonwoven mesh of polyglycolic acid fibers, 200µm in diameter, and implanted subcutaneously into the dorsal flanks of a 4-week-old NOD/SCID mice (Jackson Laboratory).
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Four weeks later the implants were harvested, and analyzed using immunohistochemical techniques. The implants were fixed with 10% formaldehyde, embedded in paraffin, and routinely processed into 4µm thickness. Sections were stained with hematoxylin and eosin. Cartilage was confirmed using Safranin-O (Fisher; S670-25). Duct and gland-like tissues were identified using the endoderm marker, rabbit polyclonal FOXA2 (Abcam; ab40874). Epithelium-like structures were identified using mouse monoclonal [PCK-26] Pan cytokeratin antibody (Abcam; ab6401). Muscle-like structures were identified using mouse monoclonal desmin antibody (Sigma; D1033) and anti-a-smooth muscle actin antibody (N1584; Dako). Nerve-like structures were identified using mouse monoclonal beta III tubulin antibody (Promega; G7121). In negative controls, the primary antibody was replaced with IgG-negative controls of the same isotype to ensure specificity. All sections were then peroxidase stained using the LSAB 2 kit (DakoCytomation) according to the manufacturer’s protocol. The experiments were reviewed and approved by Harvard Medical Area Standing Committee in Animals.
4週間後、インプラントを採取し、免疫組織化学的手法を用いて分析した。移植細胞を10%ホルムアルデヒドで固定し、パラフィンに包埋し、手順通り4µmの厚さに加工した。切片をヘマトキシリンおよびエオシンで染色した。軟骨は、Safranin-O(Fisher; S670-25)を用いて確認した。樹状細胞および腺様組織は、内胚葉マーカーであるウサギポリクローナルFOXA2(Abcam; ab40874)を用いて同定した。上皮様構造は、マウスモノクローナル[PCK-26]汎サイトケラチン抗体(Abcam; ab6401)を用いて同定した。筋様構造は、マウスモノクローナルデスミン抗体(Sigma; D1033)および抗A平滑筋アクチン抗体(N1584; Dako)を用いて同定した。神経様構造は、マウスモノクローナルベータIIIチューブリン抗体(Promega; G7121)を用いて同定した。ネガティブコントロールとして、一次抗体を同じアイソタイプのIgG陰性対照と交換して特異性を確保した。次いで、全ての切片を、LSAB 2キット(DakoCytomation)を用いて製造業者のプロトコールに従ってペルオキシダーゼ染色した。実験はHarvard Medical Area Standing Committee in Animalsによって審査され承認された。
Sphere formation
Regardless of the tissue from which they had been isolated, cells triturated through reduced glass pipettes and grown in vitro formed nonadherent spheres. Spheres of up to 150mm in diameter, generated from marrow-derived cells, arose within 5 days (Fig. 1A). Cells from other adult tissue representative of the three germ layers, spinal cord (ectoderm), muscle (mesoderm), and lung (endoderm), formed spinal spheres, myospheres, and pneumospheres, respectively, within 5 days of isolation (Fig. 1B–D). Individual spheres possessed >2000 cells. The doubling time of cells within the spheres of 〜12±4h represented the average doubling time of mixed populations of cells within individual spheres.To assess the self-renewing potential of sphere-forming cells, spheres were dissociated into single cells and replated, resulting in the generation of secondary spheres (Fig. 1E, F, example marrowderived spheres). Disassociated cells were capable of forming spheres for at least five generations in this experiment.
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Immature cell marker expression
Expression of immature markers, which are widely recognized as stem cell markers using immunohistochemistry, was then assessed. Representative spheres from each tissue contained cells positive for stage-specific embryonic antigen1 (Fig. 1G), and expressed alkaline phosphatase activity (10% of the spheres) (Fig. 1H), c-kit (Fig. 1I), Sca-1 (Fig. 1J), and E-cadherin (Fig. 1K, example marrow-derived spheres). Expression of every surface marker was not identified in every sphere. Spheres generated from the same tissues were not identical and appeared to contain a heterogeneous population of primitive cells. The number of spheres formed was 116±5(n=5) per 10 million cells initially plated. When cells were procured from mice >12 weeks of age, the average number of spheres generated was less (Supplementary Fig. S1). Early in the formation of spheres, the contained cells were quite small in size, as demonstrated by the flow cytometry analysis, with the majority (〜60%–70% in all cases) being <8µm in diameter.
FIG. 1. Sphere generation from adult bone marrow (scale bar, A–H 20mm<Translation machine note:For 'mm',read 'µm'. It's probably a typographical error.>). Primary spheres formed from nonadherent bone marrow cells at day 5 (A). Sphere formation from cells representative of different adult tissue types. Muscle, lung, and spinal cord were harvested with trituration and cultured in the maintain media. Spheres from all type of tissues arose within 5 days. Spinal spheres (SS) (B), myospheres (MS) (C), and pneumospheres (PS) (D) at day 5. Secondary spheres formed from cells dissociated from primary marrowspheres (E) and tertiary spheres formed from cells dissociated from secondary marrowspheres (F). Stage-specific embryonic antigen-1 expression is shown in green (G), alkaline phosphatase expression is shown in red (H), C-kit expression is shown in red (I), Sca-1 expression is shown in green (J), and E-cadherin expression is shown in red (K) in primary bone marrow spheres.
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Expression of embryonic-like phenotypes
Expression of the following genes normally associated with the early stage of embryonic development was seen in cells procured from each tissue. The gene profiles that were expressed are recognized as ESC markers and suggestive of pluripotency. Oct4, Nanog, Zfp296, Cripto, Gdf3, Utf1, Ecat1, Esg1, Sox2, and Fgf5 (Figs. 2 and 3) were expressed in cells procured from each tissue source. In contrast, Rex1, Eras, and Cdx2 were not expressed at the mRNA level (Figs. 2 and 3; Supplementary Table S2), suggesting that although the cells analyzed in the study were very similar to ESCs, they were not identical. Smaller spheres, <100mm<Translation machine note:For 'mm',read 'µm'. It's probably a typographical error.> in diameter, contained relatively higher proportion of cells expressing markers associated with ESCs. About 5%–10% of the spheres expressed Oct4 (Supplementary Table S2). In the cells that expressed Oct4, the gene profiles were quite similar to those expressed in primitive ectoderm during the early stage of embryonic development (Supplementary Table S3). Spheres seemed to contain heterogeneous populations of cells, with some markers expressed in some spheres, and other markers expressed in different spheres generated from cells isolated from the same tissue, at the same time.
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The primitive ectoderm marker Fgf5 was frequently expressed, and pdgfa,a mesenchymal stem cell marker, was occasionally expressed (Figs. 2 and 3, Supplementary Table S2). Pdgfa-positive spheres were virtually negative for the mesoderm lineage marker Burachyury gene, which suggests that the spheres may contain precursors for mesenchymal stem cells.[21],[22] Expression of the neural stem cell marker, Nestin, was also documented after a few days in vitro, as well as the transcription genes Sox2, Pax6, and Eomes, critical in the development of the nervous system (Supplementary Table S2). During the early proliferation phase,especially in endodermal-and mesodermal-derived spheres, only 30% of spheres contained cells that expressed Nestin. Map2 and oligodendrocyte lineage transcription factor 2 (Olig2) were expressed only slightly, even when Nestin was also expressed (Supplementary Table S2). When cultured in the B27-supplemented serum-free medium used in this study, for >2 weeks without passaging, the gene expression profile demonstrated a reduction in ES marker gene expression and changed to a gene expression profile more similar to neural stem cells.
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Differentiation potential of cells in vitro
Regardless of the tissue from which they had been isolated, some spheres <7 days old contained cells that expressed gene markers that have been associated with pluripotency. A few cells slightly expressed primitive markers of the three germ layers (Map2, Brachyury, and Gata6) (Supplementary Table S2). Consequently, the potential to differentiate into cells representative of the three germ layers was examined.
FIG. 2. Gene expression profiles of cell contained in bone marrow spheres. Gene expression of each bone marrow sphere was analyzed by reverse transcription-polymerase chain reaction. Gene expression of cells in each bone marrow sphere was compared to those of embryonic stem cells.
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FIG. 3. Gene expression profiles of oct4-expressing cells in spheres from each tissue. Spinal spheres (SS), myospheres (MS), and pneumospheres (PS) at day 5 were compared to those of embryonic stem cells.
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Marrow-derived cells
When representative BM-derived spheres were dissociated into single cells and exposed to three different differentiation media, the cells differentiated to express specific genes of the three lineages, Map2 (ectoderm), MyoD (mesoderm), and AFP (endoderm). This is in contrast to undifferentiated spheres, which did not contain cells that expressed any of the specific genes associated with each lineage, that is, Map2, MyoD, and AFP (Fig. 4A). The addition of a neural differentiation medium to the in vitro environment of cells from BM spheres resulted in expression of bIII tubulin (a marker for neuron) (Fig. 4B), GFAP (for glia) (Fig. 4C), and O4 (representative of oligodendrocytes)(Fig.4D).
Alternatively,the addition of 20% fetal calf serum to the media resulted in expression of markers representative of mesoderm, that is, a-smooth muscle actin (Fig. 4E), myosin (Fig. 4F), and desmin (Fig. 4G) as well as the mesenchymal cells, chondrocytes, osteocytes, and adipocytes (Supplementary Fig. S2A–C). Thus, cells from spheres differentiated into all cell types of neural (neurons, oligodendrocytes, and glias) and mesenchymal stem cell lineage (chondrocytes, osteocytes, and adipocytes). When exposed to a hepatocyte differentiation media, expression of AFP (Fig. 4H),albumin(Fig.4I), andCK18(Fig.4J) wasseen, suggestive of differentiation into endodermal tissue.
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Cells derived from tissues representative of the three germ layers
Spinal spheres, myospheres, and pneumospheres were then exposed to the same three differentiation media. Neural cells (ectoderm lineage) expressing the neural cell marker, bIII tubulin (Fig. 5Ai–Ci), the glial cell marker, GFAP (Fig. 5Aii–Cii), and the oligodendrocyte marker, O4 (Fig. 5Aiii– Ciii), were identified when the spheres were exposed to a neural differentiating media. Muscle cells (mesodermal lineage) expressing a-smooth muscle actin (Fig. 5Aiv–Civ), myosin (Fig. 5Av–Cv), and desmin (Fig. 5Avi–Cvi) were identified when the spheres were exposed to a muscle differentiating media. Other mesenchymal lineage cells such as chondrocytes, osteocytes, and adipocytes (Supplementary Fig. S2D–I) were also observed. When spheres were exposed to a hepatocyte differentiating media, differentiated into hepatocyte (endoderm lineage), expression of AFP (Fig. 5Avii–Cvii), albumin (Fig. 5Aviii–Cviii), and CK18 (Fig. 5Aix–Cix) was seen.
The potential for multi-differentiation of cells contained in spheres that had been procured from tissues representative of the three germ layers seemed to possess the same differentiation potential as did cells procured from BM.
FIG. 4. In vitro differentiation assay of cells from three germ layers. Differentiation into cells representative of the three germ layers was confirmed by each specific gene expression of various lineages: Map2 (ectoderm), MyoD (mesoderm), and AFP (endoderm); however, nondifferentiated spheres did not express any of Map2, MyoD, and AFP (A). Marrowspheres were dissociated and plated in each appropriate medium. Cells from spheres differentiated into cells representative of the three germ layers: neural cells (B–D), muscle cells (E–G), and hepatocytes (H–J). Neurons stained with bIII tubulin (B), glias stained with GFAP (C), and oligodendrocytes were stained with O4 (D). Muscle cells stained with αsmooth muscle actin (E) and myosin (F) and desmin (G). Hepatocytes were stained with AFP (H), albumin (I), and CK18 (J). AFP, α-fetoprotein; Map2, microtubule-associated protein 2; GFAP, glial fibrillary acidic protein; CK18, cytokeratin 18.
FIG. 5. Differentiation assay of spheres derived from different adult tissue types into cells representative of the three germ layers. Differentiation assay from spinal spheres (A) into neural cells (i, ii, and iii), muscle cells (iv, v, and vi), and hepatocytes (vii, viii, and ix). bIII tubulin-expressing neurons are shown in green (i), GFAP-expressing glias are shown in red (ii), and O4-expressing oligodendrocytes are shown in red(iii).α-smooth muscle actin-expressing smooth muscle cells are shown in red(iv), myosin-expressing cells are shown in green (v), and desmin-expressing cells are shown in red (vi). AFP-expressing cells are shown in red(vii),albumin-expressing cells are shown in red(viii),and CK18 are shown in green(ix).
Differentiation assay from pneumospheres (B) into neural cells (i, ii, and iii), muscle cells (iv, v, and vi), and hepatocytes (vii, viii, and ix). bIII tubulinexpressing neurons are shown in green (i), GFAP-expressing glias are shown in red (ii), and O4-expressing oligodendrocytes are shown in red (iii). a-smooth muscle actin-expressing smooth muscle cells are shown in red (iv), myosin-expressing cells are shown in green (v), and desmin-expressing cells are shown in red (vi). AFP-expressing cells are shown in red (vii), albumin expressing cells are shown in red(viii),and CK18 are shown in green(ix).
Differentiation assay from myospheres(C) into neural cells(i,ii,and iii),muscle cells(iv,v,and vi)cells<Translation machine note: a typographical duplication error>,and hepatocytes(vii,viii,and ix).bIII tubulin-expressing neurons are shown in green (i), GFAP-expressing glias are shown in red (ii), and O4-expressing oligodendrocytes are shown in red (iii). a-smooth muscle actin-expressing smooth muscle cells are shown in red(iv),myosin-expressing cells are shown in green(v),and desmin expressing cells are shown in red (vi). AFP-expressing cells are shown in red (vii), albumin-expressing cells are shown in red (viii), and CK18 are shown in green (ix). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bars: 50mm<Translation machine note:For 'mm',read 'µm'. It's probably a typographical error.>.
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Differentiation potential of cells in vivo
When implanted subcutaneously into NOD/SCID mice, spheres generated from cells procured from each of the three germ layers demonstrated the potential to form tissue-like teratoma<Translation machine note:For 'tissue-like teratoma',read 'teratoma-like tissue'. It's probably a typographical error.>. containing cells representative of all three germ layers. The tissues generated were encapsulated and easily resected. Each explant was 〜25㎣(Fig. 6A). Individual explants contained cells representative of all three germ layers. Tissue generated from spinal spheres contained nerve (ectoderm; Fig. 6Bi, Bii), muscle (mesoderm; Fig. 6Biii, Biv), and duct-like tissue (endoderm; Fig. 6Bv, Bvi). Tissue generated from myospheres contained epithelium (ectoderm; Fig. 6Ci, Cii), muscle (mesoderm; Fig. 6Ciii, Civ), and ductlike tissue (endoderm; Fig. 6Cv, Cvi). Tissue generated from pneumospheres contained epithelium (ectoderm; Fig. 6Di, Dii), cartilage (mesoderm; Fig. 6Diii, Div), and gland (ectoderm; Fig. 6Dv, Dvi). Specific tissues were identified using immunohistochemical techniques. Nerves were identified using beta III-tubulin, epithelium identified using pancytokeratin, and muscle identified using desmin and myosin. Duct-like structures and gland were identified using FOXA2.
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Discussion
In one of the first reports, adult stem cells isolated from the central nervous system (an ectodermal-derived tissue) were shown to form spheres containing cells that express Nestin, a neural stem cell marker.[23] These cells are now well known to be capable of multi-lineage differentiation(multipotency) into neurons, astrocytes, and oligodendrocytes.[24] They reportedly have the potential to differentiate into all cells of the embryo body[25] and into cells representative of all three germ layers in vitro.[26] When injected into early stage embryos in vivo, they contribute to the generation of chimeric mice and contribute to tissue formation of the three germ layers.[25] These studies suggest that the cells procured from tissue derived from ectoderm, and propagated as neurospheres retain a fairly immature state. We believe that our report is the first description of spheres being generated from tissue representative of ectoderm, endoderm, and mesoderm having similar potentials.
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There are significant differences between the cells described in this report and ESCs. While the cells studied expressed many of the common cell markers associated with ESCs, they did not express all of them. Cells contained within the spheres did not express Rex1 (also known as Zfp42), a phenomenon also observed in the development of primitive ectoderm.[27],[28] During embryonic development, cavitation results from apoptotic cell death of the inner part of the epiblast cells. Cells surviving in the outer layer of the epiblast then form a columnar epithelium, defined as the primitive ectoderm (Oct4+, Rex1-, and Fgf5+), which gives rise to the embryo proper.[29] Formation of embryonic bodies (EB) using ESCs can mimic peri-implantation development in vitro, which has been utilized for both transient expression of primitive ectoderm-like gene expression (Oct4+, Rex1-, and Fgf5+) and induction of somatic cells.[30] Consequently, since ESCs pass though the primitive ectoderm state during the development of somatic tissue, primitive ectoderm may be the source for all adult stem cells found in somatic tissues. As many of the cells described in this report express markers associated with primitive ectoderm, in that they are Oct4+, Rex1-, and Fgf5+, we believe that the spheres described in this report contain common stem cells for all somatic cells. It has been controversial as to whether Oct4-expressing cells reside in adult tissue. While a few reports, which have not been well documented, support the existence of Oct4-expressing cells in BM, others have suggested that ES-like cells do reside in the adult body.[5]
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Since the spheres in this study contained cells with a differentiation potential that appear to fulfill the criteria for both mesenchymal stem cells and neural stem cells, we believe that the spheres contain precursor cells to both mesenchymal and neural stem cells lineages. The cells were propagated as nonadherent spheres, which are not known to exist in vivo. The in vitro behavior of cells contained in the spheres is likely to be very different from cells that reside in vivo, and the spheres generated seemed to be composed of heterogeneous populations of cells. The authors believe that it is important to the self-renewing, clonal analysis, and chimera studies to determine whether or not these are indeed true stem cells. Some markers were expressed in some spheres, whereas other markers expressed in different spheres generated from cells isolated from the same tissue, at the same time. We believe that these differences also may be a function of the environment in which the cells were maintained. Propagation of cells as nonadherent spheres in a basic serum-free, FGF-dependant media maintained cells expressing gene markers associated with immature cells for a short period, in contrast to the use of a pluripotent maintaining culture media containing leukemia inhibitory factor (LIF), which resulted in a higher proportion of cells expressing immature markers for a longer period.
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Although the spheres in this study described were capable of generating teratoma-like tissue, the transplanted cells did not form large teratomas as did ES cells, nor they did express Eras in vitro as ESCs,[31] which suggests that the teratoma-like tissue they generate may be very different from true teratomas generated from ESCs. In addition, the cells studied did not express the trophectoderm marker Cdx2[32 ]or also associated with ESCs. These differences of gene expression pattern may explain the differences of the biological function between ESCs and adult stem cells in this study.
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To fully document the true potential of these cells or that<Translation machine note:For 'that',read 'what'. It's probably a typographical error.> they exist in vivo is important issue for future studies. There exists a heterogeneous population of cells in adult tissues possessing properties more similar to those of ESCs than previously believed. There are, however, significant differences between this population of adult stem cells and ESCs. The population of cells described expresses many protein markers in common with ESCs and, when procured from tissues derived from each of the three germ layers, has the potential to mature into cells representative of the three germ layers, both in vitro and in vivo. The findings are consistent with recent reports that ESCs are heterogeneous rather than homogeneous when maintained in pluripotency culture conditions. Different cells within the spheres are likely to have different potentials. These observations suggest that status of both undifferentiated ES cells and the cells described in this report are in a constant state of fluctuation.[33] The environment in which the cells live may be the most crucial factor in how they develop, or which populations of cells in these heterogeneous spheres are selected to develop.
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Acknowledgments
The authors thank Selena Liao for comments on the article, Jason D. Ross, and Ana C. Paz, Ashley Penvose, and Peter Schow for extensive technical assistance and animal care. This work was supported by the Department of Anesthesiology, Perioperative and Pain Medicine at Brigham and Women’s Hospital in Boston.
Disclosure Statement
The corresponding author, Charles A. Vacanti, has applied for patents for some of the technology described, through the University of Massachusetts. Some of the pending patents have been licensed to a company, Celthera.
謝辞
著者は論文への助言に関してSelena Liaoに、幅広い技術援助と動物のケアに関してJason D. Ross、Ana C. Paz、Ashley Penvose、Peter Schowに謝意を表明する。この仕事は、ボストンのブリガム・アンド・ウィミンズ病院の麻酔、手術期管理および痛み医療学部によって支援された。
開示報告
責任著者、Charles A. Vacantiは、マサチューセッツ大学を通じて、記載されている技術のいくつかの特許を申請している。 保留中の特許の一部は、Celthera社にライセンス供与されている。
(英文)
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Address correspondence to: Charles A. Vacanti, M.D. Laboratory for Tissue Engineering and Regenerative Medicine Department of Anesthesiology Harvard Medical School Brigham and Women’s Hospital 75 Francis St. Boston, MA 02115
E-mail: cvacanti@partners.org
Received: June 30, 2010 Accepted: September 30, 2010
Online Publication Date: December 28, 2010
連絡先:Charles A. Vacanti、M.D.組織工学および再生医学研究所麻酔科ハーバードメディカルスクールBrigham and Women's Hospital 75 Francis St. Boston、MA 02115
Eメール:cvacanti@partners.org
受領日:2010年6月30日受諾:2010年9月30日
オンライン出版日:2010年12月28日