Transplantation efficacy demonstrated by the percentage of human nuclei in the transplanted muscles.

<p>The number of cells of human origin was divided by the number of total nuclei stained by DAPI. The result also appears in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051638#pone-0051638-g004" target="_blank">Figures 4C and 4D</a>. SD: standard deviation. ND: not detected. *, ** indicate the cell numbers transplanted at the site: * 5.0×10⁵ cells/site, ** 1.0×10⁵ cells/site. †: One mouse transplanted with 253G4-derived cells died accidentally before analysis. NS: statistically not significant.</p

Schematic presentation of the differentiation protocol.

<p>Two different differentiation protocols were compared (Left: embryoid body (EB) culture, Right: Dissociation culture) and were exactly the same until the first 21 (7+14) days of culture. On the left side, EBs continued to be incubated in serum-containing medium without specific manipulation until the end of culture. On the right side, EBs and their outgrowth cells were dissociated and seeded onto collagen type I-coated tissue culture plates in serum-containing medium. The medium was changed to serum-free ITS medium on day 49 (7+14+28). In some experiments, the cells were harvested and used as donor cells for the transplantation assay at this time point.</p

Engraftment of myogenic progenitors in damaged muscles of immunodeficient mice.

<p>(A) Human nuclei labeled with human-specific lamin A/C localized mainly inside muscle fibers surrounded by laminin. (B) Muscle reconstruction by transplanted human cells was demonstrated by the detection of human-specific laminin-alpha 2. (C) The proportion of myofibers containing human nuclei at 4, 12, and 24 weeks after transplantation. (D) The proportion of myofibers containing human nuclei in reinjured (3+1 weeks) and in non-reinjured mice (4 weeks) at 4 weeks after transplantation. In C and D, data are presented as the mean ± standard deviation. (E) Distribution of the transplanted cells at 24 weeks after transplantation. Typical central nuclei of human origin were observed (outlined arrowheads). Some human cells located within the lamina rara beneath the basal lamina, indicating engraftment of the transplanted cells into a satellite cell compartment (white arrowhead). (F) Triple-staining for human Lamin A/C, PAX7, and pan-Laminin clearly demonstrated the existence of PAX7-positive human nuclei indicating the transplanted cells engrafted as satellite cells (white arrowhead). Human lamin A/C-negative host satellite cells were also detected (outlined arrowhead). Laminin was stained by a polyclonal antibody that recognizes both human and murine laminin, and was subsequently visualized with fluorescein isothiocyanate (FITC) (Green); human lamin A/C and human-specific laminin, with Cy3 (red). Nuclei were counterstained with DAPI (blue). Scale bars  =  (A) 100 µm, (B) and (E) 50 µm.</p

Characterization and differentiation of the derived myogenic mesenchymal cells.

<p>(A) Morphology of the derived myogenic mesenchymal progenitors 2 days (day 7+14+2) and 4 weeks (day 7+14+28) after replating. Homogeneous spindle-shaped fibroblastic cells were observed. (B) Surface marker analysis of myogenic mesenchymal progenitors. Representative data from KhES1 differentiation are shown. Note that CD56 in addition to mesenchymal markers CD73, CD105, CD166, and CD29 was exclusively expressed. (C) Changes in the expression of myogenic markers were analyzed by immunofluorescence. The number of Cy3-positive nuclei was divided by the total number of nuclei stained by DAPI. The expression of myogenic progenitor markers decreased after exposure to serum-free medium, whereas the number of MYOG-positive cells substantially increased after serum deprivation. (D) Changes in the number of MYOG-positive nuclei were observed up to 3 weeks after serum deprivation. hES/iPS-derived myofibers tended to detach from tissue culture plates during long-term culture in serum-free medium. (E) Serum deprivation increased the number of skeletal myosin-positive fibers and MYOG-positive nuclei for more than 2 weeks. KhES1 was used in this figure. (F) Multinucleated myofibers denoted by MYOG myogenin-positive nuclei aligned in skeletal myosin-positive fibers. (G) Morphology of mature myofibers, which were stained with skeletal myosin, MYOG, and dystrophin, from both KhES1 and 253G4 cells. Skeletal myosin was visualized with fluorescein isothiocyanate (FITC) (Green), myogenin was visualized with Cy3 (red), and nuclei were counterstained with DAPI (blue). Scale bars  =  (C, E) 100 µm, (D) 50 µm.</p

Skeletal muscle development from human embryonic stem (<b>hES</b>) <b>and human induced pluripotent stem</b> (<b>hiPS</b>) <b>cells by the EB culture method.</b>

<p>(A) PAX3- and PAX7-positive nuclei emerged in the proximal area of the embryoid body (EB)-outgrowth cells derived from hES KhES1 cells. (B) Simultaneous derivation of neural and cardiac cells in the EB-outgrowth cells derived from hES KhES1 cells. Upper: TUJ1-positive neural cells observed on day 7+28. Lower: Neural cells (outlined arrowheads) and colonies of beating cardiomyocytes (white arrowhead) appeared on day 7+28. (C) Skeletal myosin-positive myofibers in the EB-outgrowth cells derived from human embryonic stem (hES) KhES1 cells detected on day 7+42. (D) Sequential analysis of undifferentiated and skeletal myogenesis-related gene expression by semi-quantitative RT-PCR. (E) Skeletal myosin-positive fibers from human induced pluripotent stem (hiPS) cells. Four hiPS cell-lines were used. hiPS 201B6 on day 7+105, hiPS 201B7 on day 7+105, hiPS 253G1 on day 7+77, and hiPS 253G4 on day 7+56. (F) Sequential analysis of undifferentiated and skeletal myogenesis-related gene expression by semi-quantitative RT-PCR. In (A-C) and (E), antibodies were visualized using Cy3 (red). Nuclei were counterstained with DAPI (blue). Scale bars  = 100 µm.</p

Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

<div><p>Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×10⁶±0.3×10⁶ floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.</p> </div

Phenotype analysis and gene expression pattern of monocytic lineage cells derived from pluripotent stem cells.

<p>(A) Flow cytometric analysis of monocytic lineage cells derived sequentially from pluripotent stem cells. An analysis of adherent cells on day 6 and supernatant cells on day 13 and 18 is shown. (B) May-Giemsa staining of CD14⁺ monocyte-like cells derived from KhES1 on day 16 (left) and primary human monocytes (right). (C) Esterase staining for CD14⁺ monocyte-like cells derived from KhES1 on day 16. (D) The percentage of CD14⁺ cells within the total floating cells derived from KhES1/iPS-201B7 was evaluated from day 13 to day 28. (E) May-Giemsa staining (left) and phase contrast image (right) of mature DCs derived from pluripotent stem cells. (F) Flow cytometric analysis of immature/mature DCs derived from pluripotent stem cells. (G) Phase contrast image and flow cytometric analysis of macrophages derived from pluripotent stem cells.(H) RT-PCR analysis of monocytic lineage cells derived from KhES1/iPS-201B7 clones for expression of monocytic lineage marker genes (<i>PU.1, c-MAF, TLR4, CCL17</i> and <i>CCL18</i>). Peripheral blood monocytes and peripheral blood monocyte-derived mature DCs were used as positive controls.(A–C, E–G) The data from KhES1-derived cells are shown as representative.</p

Functional assays for monocytes derived from pluripotent stem cells.

<p>(A) The levels of IL-6 and TNFα in supernatants of PS-Mo culture medium 4 hours after LPS stimulation. The levels of IL-1β were measured 4 hours after LPS stimulation with/without an additional 30-minute ATP stimulation. (B) Flow cytometric analysis of CX3CR1 on PS-Mo. (C) Chemotaxis assay of PS-Mo for CX3CL1 (fractalkine) using a trans-well migration assay. After the addition of CX3CL1 into either the bottom or top of the trans-well chamber, PS-Mo were applied and incubated for 5 hours at 37°C. (D) Antigen uptake was evaluated in monocytes, immature DCs and mature DCs derived from pluripotent stem cells by examining the fluorescence intensity of Alexa fluor 488-conjugated ovalbumin 45 minute after incubation at 37°C (black). Control samples (white) were kept on ice. (B–D) The data of KhES1-derived cells are shown as representative. PS-Mo: monocyte derived from pluripotent stem cells.</p

Functional assays for M1/M2 macrophages derived from pluripotent stem cells.

<p>(A) Flow cytometric analysis of M1/M2 macrophages derived from pluripotent stem cells. (B) The levels of IL-12p70 and IL-10 in supernatants of culture medium with M1/M2 macrophages derived from pluripotent stem cells 24 hours after LPS stimulation. The data of KhES1-derived cells are shown as representative.</p

Functional assays for dendritic cells derived from pluripotent stem cells.

<p>(A) Flow cytometric analysis of immature/mature DCs derived from pluripotent stem cells. (B) The levels of IL-10 and TNFα in supernatants of culture medium with PS-DCs 24 hours after LPS stimulation. (C) The proliferation of allogeneic naïve T cells (1×10⁵ cells per well) co-cultured with 40 Gy-irradiated stimulator cells for 3 days was evaluated. The proliferation of naïve T cells in the last 16 hours was measured by ³H-thymidine uptake. (A–C) The data of KhES1-derived cells are shown as representative.</p