3.1 Introduction
3.1.1 Differentiation potential of stem cells
One of definitions of stem cells is multidifferentiation potential. Also the degree of their stemness is determined by their differentiation potential. According to results of section 2, sphere forming cells expressed pluripotent cell markers. Therefore in this section, we aimed to confirm their differentiation potential in vivo and in vitro.
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3.2 Experimental<→Experiment>
3.2.1 In vitro Differentiation Assays.
In vitro differentiation assays were examined following the published differentiation culture conditions for murin ES cells.
Mesoderm lineage differentiation assay. Dissociated muscle cells were stained with anti-αmooth muscle actin antibody, anti-Myosin antibody and anti-Desmin antibody. Chondrocyte were stained with Safranin-0 and Fast Green. Osteocytes were stained with ALIZARIN RED S. After 21 days, adipocytes were stained with Oil Re 0.
Ectoderm lineage (Neural lineage) differentiation assay. Cells were plated on ortinin-coated chamber slides and incubated with anti-βIII Tubuin mouse monoclonal, anti-O4 mouse monoclonal antibody and anti-GFAP mouse monoclonal antibody.
Endoderm lineage (Hepatic) differentiation assay. Differentiated cells were detected by immunohistochemistory using anti-αfetoprotein mouse monoclonal antibody, anti-Albumin goat polyclonal antibody and anti-Cytokeratin 18 mouse monoclonal antibody. Results from immunohistochemistry were confirmed by RT-PCR.
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3.2.2 In Vivo Differentiation.
Spheres were seeded onto biodegradable scaffolds and implanted into subcutaneous of NOD/SCID mice (Charles River laboratories). After 6 weeks, the implants were harvested and fixed with 10% formaldehyde, then examined by immunocytochemistry.
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3.3Results
3.3.1 Differentiation potential of cells in vitro
When representative bone marrow 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 alpha-fetoprotein (AFP, endoderm) (Fig. 10).The addition of a neural differentiation medium to the in vitro environment of cells from bone marrow spheres, resulted in expression of pIII tubulin (a marker for neuron) (Fig. 11).
Alternatively, the addition of 20% fetal calf serum to the media resulted in the expression of markers representative of mesoderm; that is, a-smooth muscle actin (Fig. 11) as well as the mesenchymal cells, chondrocytes, osteocytes and adipocytes (Fig. 12). Thus, cells from spheres differentiated into all cell types of neural (neurons, oligodendrocytes and ghas) and mesenchymal stem cell lineage (chondrocytes, osteocytes and adipocytes). When exposed to a hepatocyte differentiation media the expression of a-fetoprotein (Fig. 11), was seen, suggestive of differentiation into endodermal tissue.
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3.3.2 Differentiation potential in vivo.
Bone marrow spheres and ES cells were transplanted subcutaneously into immune deficient mice to examine their tumor-initiating capacity. As a result, after 6 weeks ES cells formed a tumor. Spheres did not form tumor as big as ES cells did. We concluded that the proliferative potential of sphere cells was much weaker than that of ES cells (Fig. 13).
Next, we investigated if transplanted cells differentiated in vivo after transplantation. Transplanted cells were harvested after 6 weeks, and processed for immunohistochemical analyses. According to results of immunohistochemical analyses, spheres differentiated into tissues derived from three germ layers in vivo (Fig. 14).
3.3.2 生体内分化能<訳注:目次では3.3.2 Differentiation potential of cells in vivoとなっているので、小保方さんがいろいろと編集中であることがわかる。これを見て草稿だと気づけない頭というのもどうなのか。>
骨髄のスフィア及びES細胞が、それらの腫瘍形成能力を調べるために、免疫欠損マウスに皮下移植された。その結果、6週間後にES細胞は腫瘍を形成した。我々はスフィア細胞の増殖能がES細胞よりもはるかに弱かったと結論付けた（図13）。
次に我々は移植された細胞が移植後に生体内で分化するかどうかを調査した。移植された細胞は6週間後に回収され、免疫組織化学的分析に供された。免疫組織化学的分析の結果によると、スフィアは生体内で三胚葉に由来した組織に分化した（図14）。
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3.4 Summary of section 3
♦ Spheres differentiated into cells derived from all three germ layers in vivo and in vitro
3.5 Discussion
Spheres differentiated into cells derived from three germ layers in vitro. It is yet answered that it was either differentiation or trans-differentiation. Also it is hard to refer the difference from in vitro differentiation potential of mesenchymal stem cells.
However, at least sphere forming cells enabled to generate various mature cells. In addition, in vivo differentiation assay proved that sphere forming cells were indeed stem cells, but distinct from ES cells in proriferative potential. The relationship between proliferative potential and differentiation potential has yet been understood. Spheres in this study showed differentiation potential which fulfill the critereia for both mesenchymal stem cells and neural stem cells. We believe that the spheres studied contain precursor cells to both mesenchymal and neural stem cells lineages.
It is important to note that cells described above, were propagated as non-adherent spheres, and are not known to exist in vivo. The in vitro behavior of cells in the spheres is likely to be very different from cells that reside in vivo. How these stem cells harbor in adult body and how they exert their potential
The spheres generated seemed to be composed of heterogenous 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. We believe that these differences also may be a function of the environment in which the cells were maintained.
3.5 討論
スフィアは試験管内で三胚葉に由来する細胞に分化した。それは分化または分化転換＜訳注:すでに分化した細胞が別の細胞種に転換する現象＞のいずれかであると分かっている。また間葉系幹細胞の試験管内分化能との違いに言及することは困難である。
しかし、少なくともスフィア形成細胞はさまざまな成体細胞の生成を可能にした。加えて、生体内分化実験はスフィア形成細胞が実際に幹細胞であることを証明したが、増殖能力においてES細胞と峻別された。増殖能と分化能との関係はもう理解されてきている。この研究におけるスフィアは間葉系幹細胞と神経幹細胞の両方の基準を満たす分化能を示した。私たちは研究しているスフィアに間葉系と神経系の両方の幹細胞系統への前駆細胞が含まれていると信じている。
上述の細胞が非接着性スフィアとして知られ、かつ生体内に存在することが知られていないことに留意することが重要である。スフィア内細胞の試験管内での挙動は生体内に存在する細胞とは非常に異なる可能性が高い。如何にこれらの幹細胞は成体の中にとどまり、そして如何に彼らの潜在能力を伸ばすのか。<訳注:構文が不完全なところも編集中の文章であることを示唆している。>
生成されたスフィアは、細胞の不均一な集団で構成されているようだった。同じ組織から単離された細胞から生成されていながら、同時に、いくつかのスフィアにはいくつかのマーカー発現が伴い、別のスフィアには他のマーカー発現があった。(訳注:ティシュー論文に同様の趣旨の記述がある。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. 小保方さんはティシュー論文を下地に博論を書こうとしていることがよくわかる。)我々は、これらの違いはその中で細胞が維持されている環境の関係かなと考えている。
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Figure 10 in differentiation of bone marrow spheres
After 6 weeks of culture, cells change their figurations into those of cells representative of three germ layers.
Figure 11 In vitro differentiation assay of cells from 3 germ layers.
Marrowspheres were dissociated and plated in each appropriate medium. Cells from spheres,differentiated into cells representative of the three germ layers. Neural cells (left), muscle cells (middle) cells<Translation machine note: a typographical duplication error>, hepatocytes (right). Neurons stained with plll tubuline (left),. Muscle cells stained with a-smooth muscle actin (middle). Hepatocytes were stained with a-fetoprotein (right).
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Figure 12 Mesenchymal lineage differentiation.
Dissociated spheres were plated into serum-containing medium and cultured for 14-21 days. Plated cells differentiated into mesenchymal lineage cells even plated cells were from spheres derived from endoderm or ectoderm tissues.
Marrow spheres differentiated into condrocytes (A), adipocytes (B) and osteocytes (C). Pnemospheres differentiated into condrocytes (D), adipocytes (E) and osteocytes (F). Spinalspheres differentiated into condrocytes (G) and adipocytes (H).
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Figure 13 Teratoma forming assay
10<to the power of 7> bone marrow cells and ES cells were injected subcutaneously into immunedificienl mice.
After 6 weeks of implantation, cell masses were harvested.
Figure 14 Teratoma like mass from bone marrow spheres contained nerve expressing betalll-tubuline (left)(ectoderm), muscle expressing desmin (middle)(mesoderm) and duct like structure expressing AFP (right)(endoderm).
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