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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) <t>PAX6</t> combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.
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Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) PAX6 combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.

Journal: Frontiers in Immunology

Article Title: Generation and characterization of human induced pluripotent stem cells from neuropathologically confirmed multiple system atrophy patient-derived fibroblasts

doi: 10.3389/fimmu.2026.1641981

Figure Lengend Snippet: Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) PAX6 combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.

Article Snippet: Rabbit monoclonal anti-paired box 6 (PAX6) , Cell Signaling , Cat#60433; RRID: AB_2797599.

Techniques: Derivative Assay, Functional Assay, Cell Culture, Generated, Immunofluorescence, Imaging, Expressing