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Keywords:

  • Induced pluripotent stem cells;
  • Reprogramming;
  • Pluripotency;
  • Experimental models

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by progressive ossification of soft tissues. FOP is caused by mutations in activin receptor-like kinase 2 (ALK2) that cause its constitutive activation and result in dysregulation of BMP signaling. Here, we show that generation of induced pluripotent stem cells (iPSCs) from FOP-derived skin fibroblasts is repressed because of incomplete reprogramming and inhibition of iPSC maintenance. This repression was mostly overcome by specific suppression of ALK2 expression and treatment with an ALK2 inhibitor, indicating that the inhibition of iPSC generation and maintenance observed in FOP-derived skin fibroblasts results from constitutive activation of ALK2. Using this system, we identified an ALK2 inhibitor as a potential candidate for future drug development. This study highlights the potential of the inhibited production and maintenance of iPSCs seen in diseases as a useful phenotype not only for studying the molecular mechanisms underlying iPS reprogramming but also for identifying drug candidates for future therapies. STEM CELLS2012; 30:2437–2449


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues and muscles [1–3]. Severe debilitation, reduced life expectancy due to joint fusion, and restrictive ventilatory impairment with thoracic involvement are major symptoms of this disease. Patients with FOP have gradual worsening of pulmonary function and eventually die by 40 due to respiratory failure if they do not receive the appropriate support. There is no effective therapy for preventing the ectopic ossification associated with FOP. Recent studies have revealed that this disorder is caused by mutations in Activin A receptor type I, the gene encoding the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ALK2) [4–9]. The most common mutation is R206H, which is thought to alter ALK2 and confer constitutive activity to the mutant receptor. Mesenchymal cells derived from primary teeth of FOP patients showed elevated basal expression of RUNX2 and alkaline phosphatase (AP), which are involved in bone formation [4]. These data suggest that the dysregulation of BMP signaling seen in FOP patients results in ectopic expression of osteogenesis-related genes and aberrant ossification. Several other mutations in ALK2, such as G356D, underlie phenotypic variations of FOP and these also alter ALK2 and confer constitutive activity to the mutant receptor [10]. The weaker kinase activity of ALK2 (G356D) compared to that of ALK2 (R206H) suggests that clinical variation is due to differences in the bioactivity of ALK2 mutants [11].

Induced pluripotent stem cells (iPSCs) derived from patients with incurable diseases represent a powerful tool not only for biomedical research but also for investigating the effects of drugs on patient-derived cells [12–16]. These cells are derived from differentiated somatic cells and functionally resemble embryonic stem cells (ESCs) [17]. This process, known as reprogramming, is triggered by the expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, which are the same core factors underlying pluripotency in ESCs [17, 18]. This reprogramming process changes a cell's gene-expression profile from that of a somatic cell back to that of a pluripotent state [17]. It is well-known that iPSCs derived from somatic cells harboring pathogenic gene mutations represent the cellular phenotype of the disease [19–21].

Investigations of the process for generating iPSCs are valuable for understanding the molecular mechanisms underlying cellular reprogramming. Recent knockout-mouse studies have identified several genetic mutations that modify the efficiency of iPSC generation. For example, iPSCs can be generated with higher efficiency from p53- and Ink4a/Arf-null fibroblasts than from normal fibroblasts [22, 23]. A mutation in p21, which is a molecule involved in downstream p53 signaling, partially mimics this phenotype, suggesting that activation of p53 and Ink4a/Arf signals can inhibit cellular reprogramming. These results raise the notion that some genetic mutations underlying human diseases also affect the reprogramming processes and eventually abolished iPSC generation. However, it is still unclear how pathogenic gene mutations affect the cellular reprogramming required for the generation and maintenance of human iPSCs.

Here, we studied disease-derived iPSCs to elucidate how pathogenic gene mutations affect cellular reprogramming. We showed that iPSC generation from FOP-derived skin fibroblasts is repressed. The few FOP-derived iPSCs that we managed to isolate could not be maintained because they differentiated spontaneously into mesodermal and endodermal lineages. We showed that repression of iPSC generation results from inefficient reprogramming of FOP-derived fibroblasts and inhibition of iPSC maintenance. This repression was mostly overcome by specific suppression of ALK2 expression and treatment with an ALK2 inhibitor. Using this system in combination with in silico chemical library screening, we identified an ALK2 inhibitor as a potential drug candidate for future therapeutic applications. The inefficient production of iPSCs is a useful disease phenotype not only for understanding the mechanism of reprogramming but also for identifying drug candidates for future therapies.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Generation and Detection of Sendai Virus Vector

The Sendai virus (SeV) carrying Oct3/4, Sox2, Klf4, and c-Myc were generated as described previously [24]. To detect SeV genome, nested RT-PCR was performed. The sequences of primers and amplification conditions are listed in supporting information Table S1.

Cell Culture and iPSC Generation with SeV Vector

Fibroblasts from FOP patients and healthy volunteers were generated from explants of skin biopsy following informed consent under protocols approved by the ethics committee assigning authors. Skin samples were minced and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. After the fibroblast appeared, it was expanded for iPSC induction.

iPSCs were generated from human skin-derived fibroblasts as described previously [24]. Cells (5 × 105) of human fibroblast cells per well of six-well plate were seeded 1 day before infection and then were infected with SeV vectors at a multiplicity of infection of 3. Seven days after infection, fibroblasts infected were harvested by trypsin and replated at 5.4 × 104 cells per 60 mm dish on the mitomysin C (MMC)-treated mouse embryonic fibroblast (MEF) feeder cells. Next day, the medium was replaced in human iPSC medium. Thirty days after infection, colonies were picked up and recultured again in human iPSC medium.

Maintenance of Human iPSCs

Human iPSCs were maintained on MMC-treated MEF feeder cells in human iPS medium containing DMEM/F12 (SIGMA) supplemented with 20% KNOCKOUT serum replacement (Invitrogen), 2 mM L-glutamine, 1 × 10−4 M nonessential amino acids (SIGMA), 1 × 10−4 M 2-mercaptoethanol (SIGMA), 0.5% penicillin and streptomycin (Nacalai Tesque, Kyoto, Japan, http://www.nacalai.co.jp), and 5 ng/ml basic fibroblast growth factor (Wako, Osaka, Japan, http://www.wako-chem.co.jp/). In some experiment, ALK2 inhibitors such as LDN-193189 (STEMGNT, Cambridge, MA, http://www.stemgent.com/) and Dorsomorphin (DM; SIGMA) were added into the human iPS medium. DiPS used as a control iPSC line was kindly gifted by DNAVEC Corporation (Tsukuba, Japan, http://www.dnavec.co.jp/en/Index.html).

Chemical Library Screening

We extracted known inhibitors for homologous kinases including BMP receptor family from the CHEMBL database to determine the queries (https://www.ebi.ac.uk/chembldb/). The CHEMBL database, which contains 1,118,865 compounds and 4,668,202 activity data, is provided by European Bioinformatics Institute. We constructed a search engine to retrieve ALK family inhibitors and BMP inhibitors from the CHEMBL database. Using the search engine, 236 known kinase inhibitors were obtained. Finally, 153 compounds were selected from commercially available databases system and purchased (Namiki Shoji Co., Ltd., http://www.namiki-s.co.jp/english/; Kishida chemical Co., Ltd., http://www.kishida.co.jp/english/index.html). Unavailable seven compounds known as ALK2 inhibitor (Thomson Reuters IntegritySM) were prepared by ourselves. In total, 160 bioactive chemical compounds were evaluated by cell-based inhibition assay of BMP signaling. Other materials and methods are described in supporting information Supplemental Materials and Methods.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Generation of iPSCs from FOP-Derived Skin Fibroblasts

We attempted to generate iPSCs from skin fibroblasts from four patients with FOP and three healthy volunteers by the SeV method (Fig. 1A) [24–26]. Three patients had the common R206H mutation of ALK2 and the remaining patient had the G356D mutation (supporting information Fig. S1A) [10]. The SeV method is suitable for establishing disease-specific iPSCs because it is highly efficient, does not involve integration, and the SeV is easy to remove. The frequency of iPSC colony formation from FOP-derived fibroblasts was significantly lower than that from the normal controls (Fig. 1B, 1C). Almost all colonies generated from FOP-derived fibroblasts exhibited atypical morphologies compared to controls (Fig. 1D, 1E). Selected colonies of FOP-derived fibroblasts did not expand after several passages, exhibited a flat morphology, and disappeared. It is noted that these morphological changes are very similar to those observed in the induction of iPSC differentiation.

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Figure 1. Generation of induced pluripotent stem cells (iPSCs) from patients with FOP. (A): Summary of patients with FOP and healthy volunteers. (B): Efficiency of generation of the iPSCs from FOP patients and healthy volunteers. iPSC colonies were identified on day 30 of induction by the appearance of alkaline phosphatase-positive (AP+) colonies with undifferentiated colony morphology (typical iPSC morphology). The efficiency of iPSC generation from FOP-derived fibroblasts is substantially lower than that from healthy volunteers (controls). The data are means ± SD of three independent experiments. A one-way ANOVA followed by Tukey's multiple comparison test was performed to evaluate differences between groups. *, p < .05, when compared with each values of fibroblast N1, N2, and N3. (C): AP-staining of iPSC colonies in 60 mm dishes. (D): Colony morphology of iPSCs. AP+ colony with undifferentiated colony morphology was counted as a typical colony. Typical colonies were mostly observed in control cultures (upper left). Some typical colonies were detected in cultures derived from FOP patients (lower left). Atypical colonies are defined as those where only the center of the colony was AP-positive (upper right) or those where only the periphery of the colony was AP-positive (lower right). Scale bars = 200 μm. (E): The number and percentage of typical and atypical FOP-derived and control iPSC colonies. Almost all FOP-derived colonies exhibited an atypical morphology. Abbreviations: FOP, fibrodysplasia ossificans progressive; ND, not detected.

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Treatment with ALK2 Inhibitors Can Restore the Ability to Generate and Maintain FOP-Derived iPSCs

In FOP, the mutant ALK2 results in dysregulated BMP signaling via its constitutive activation [3, 5, 9]. We next examined the effects of ALK2 inhibitors on the generation and maintenance of iPSCs. Treatment with the ALK2 kinase inhibitor, LDN-193189 (LDN) [27], restores the colony formation capacity of iPSCs in dose-dependent manner (Fig. 2A). DM [28], another inhibitor for ALK2, also improves the efficiency of colony formation but not to the same extent as that by LDN. LDN enhances the generation of iPSCs from fibroblast F2 cells harboring the R206H mutation as well as from fibroblast F4 cells harboring the G356D, but with lower efficiency (Fig. 2A).

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Figure 2. Treatment with activin receptor-like kinase 2 (ALK2) inhibitors can restore the generation and maintenance of iPSCs from fibrodysplasia ossificans progressive (FOP) fibroblasts. (A): Improvement of iPSC colony generation by treatment with the ALK2 inhibitors, LDN-193189 (LDN) and Dorsomorphin. FOP-derived fibroblasts were treated with the inhibitors from day 8 to day 30 of iPSC inductions. iPS colonies were identified by the appearance of AP+ colonies with undifferentiated colony morphology (Fig. 1D). Left and right panels: F2 and F4 patients, respectively. (B): Phase-contrast picture of FOP-derived iPSCs generated from individual patients and treated with LDN. Scale bar = 200 μm. (C): RT-PCR analysis of SeV and human embryonic stem cell markers. The iPSC lines F1-1, F2-1, and F4-1 are derived from patients, F1, F2, and F4, respectively. 201B7: control human iPSC line. SeV(+): SeV-infected fibroblasts. (D): Immunofluorescence and AP staining of FOP-derived iPSC lines for pluripotency markers. Scale bars = 200 μm. (E): Tissue morphology of the hematoxylin and eosin-stained representative teratoma derived from FOP-derived iPSC line F2-3. The descendants of three germ layers are observed in the teratoma. G: gut-like structure (endoderm); C: cartilage (mesoderm); M: muscle tissue (mesoderm); CE: cuboidal epithelium (ectoderm); MP: melanin pigment (ectoderm). Scale bars = 200 μm. Abbreviations: AP, alkaline phosphatase; iPS, induced pluripotent stem; SeV, Sendai virus.

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In addition to improving the efficiency of colony formation, treatment with ALK2 inhibitors allows iPSC colonies to be maintained continuously without morphological alteration. The iPS colonies formed in the presence of LDN and DM exhibited a typical morphology (Fig. 2B and supporting information Fig. S1B). Individual colonies were cultured and maintained in the presence of LDN. Absence of the SeV DNA fragment following amplification with nested primers indicated that SeV was completely removed from the iPSC lines (Fig. 2C) [24]. RT-PCR and immunostaining analyses of these FOP-derived iPSC lines revealed that they expressed a set of markers typical of pluripotent cells (Fig. 2C, 2D) [17, 18]. To confirm the pluripotency of FOP-derived iPSC lines, we transplanted them into the subcutaneous tissues of the immunodeficient mice. Eight to twelve weeks after injection, FOP-derived iPSC lines tested formed teratomas that contained derivatives of all three germ layers (Fig. 2E and supporting information Fig. S2). Immunoblot analysis of phosphorylated Smad1/5/8, which are downstream molecules of ALK2 signaling, indicated that ALK2 kinase activity was higher in FOP-derived iPSCs than in controls (supporting information Fig. S3). We also demonstrated Smads dephosphorylation following treatment with ALK2 inhibitors thus confirming the ability of these inhibitors to suppress ALK2 activity (supporting information Fig. S3).

FOP-derived iPSCs Can Spontaneously Differentiate into Both Mesoderm and Endoderm Lineages Under Conditions for Maintaining iPSCs

The removal of ALK2 inhibitors from FOP-derived iPSC cultures caused the colonies to be disrupted and differentiation to be initiated even under conditions for maintaining iPSCs (Fig. 3A and supporting information Fig. S4). To define the differentiated cell type, the expression of a set of differentiation markers in control and FOP-derived iPSC lines was examined (Fig. 3B, 3C). We observed elevated expression of both mesodermal (MESP1, MESOGENIN, and BRACHYURY) and endodermal (SOX17 and FOXA2) markers in iPSC lines cultured in the absence of the ALK2 inhibitor [29, 30]. CDX2, a marker of trophectoderm [31], was also upregulated in FOP-derived iPSC lines cultured without LDN. In contrast, the expression levels of neuroectodermal markers, such as SOX1, NESTIN, and NEUROD1 [32], did not change, even in the absence of the inhibitor (Fig. 3B). The iPSC lines cultured in the absence of LDN expressed pluripotency markers such as OCT3/4 [33] and NANOG [34, 35] at a lower level than those in the presence of LDN (Fig. 3B). Immunostaining analysis also confirmed the elevated expression of mesodermal and endodermal markers (Fig. 3C). The expression patterns of these markers indicated that FOP-derived iPSCs tend to spontaneously differentiate into both mesodermal and endodermal cells rather than into ectodermal cells, despite being culturing under iPSC maintaining conditions.

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Figure 3. Spontaneous differentiation of fibrodysplasia ossificans progressive FOP-derived iPSCs into mesodermal and endodermal cells. (A): Spontaneous differentiation of FOP-derived iPSC lines in the absence of LDN. Undifferentiated (U) indicates an undifferentiated colony, partially differentiated (P) indicates a partially differentiated colony, and differentiated (D) indicates a completely differentiated colony (upper pictures). Number and ratio of the three types of colonies from FOP-derived iPSC lines treated or not treated with inhibitor (center and lower panels). Scale bar = 200 μm. (B): Quantitative RT-PCR analysis of the expression of pluripotency- and differentiation-related genes. The data are normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and represent expression levels relative to the 201B7 human iPSC line cultured without LDN. (C): Immunofluorescence staining of differentiation markers in FOP-derived iPSC lines cultured without LDN. Four iPSC lines were stained and the representative data obtained from the iPSC line F2-5 are shown here. Hoechst staining indicates the nuclear. Scale bars = 200 μm. Abbreviations: iPS, induced pluripotent stem; LDN, LDN-193189.

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Characterization of FOP-Derived iPSCs

We used microarray analysis to further characterize the FOP-derived iPSC lines. The patterns of global gene expression of three FOP-derived iPSC lines treated with LDN were different to those of cells not treated with LDN (Fig. 4A). We confirmed that the expression levels of mesodermal and endodermal markers were higher in iPSCs not treated with LDN than in those treated with LDN, whereas expression levels of neuroectodermal markers such as NESTIN and SOX1 [32] were similar (Fig. 4A). In contrast, LDN treatment had no effect on the expression levels of differentiation markers in control iPSC line (Fig. 4A). We also found that the expression levels of markers found to be elevated in FOP-derived iPSC lines not treated with LDN are enhanced by BMP-4 stimulation in control iPSC lines (supporting information Fig. S5). We performed principal component analysis (PCA) [36] and hierarchical clustering of all genes to determine overall differences in transcription levels between FOP-derived iPSC lines and control iPSC lines (Fig. 4B, 4C). We added the datasets for 201B7, normal iPSC line, and three ESC lines from Gene Expression Omnibus datasets, GSE29115, GSE22167, and GSE37258 into our analyses. The gene-expression profile of FOP-derived iPSC lines not treated with LDN was distinct from that of control iPSC lines and those treated with LDN. In contrast, the gene-expression profile of FOP-derived iPSC lines treated with LDN was grouped closely to control iPSC lines not treated with LDN as did normal iPSC lines (N3-1 and DiPS) treated with LDN.

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Figure 4. Characterization of fibrodysplasia ossificans progressive (FOP)-derived iPSCs. (A): Global gene-expression patterns of normal and FOP-derived iPSCs. Scattered plot representation of the global gene-expression patterns for the iPSC lines N3-1, F1-1, F2-1, and F4-1 in the presence and absence of LDN-193189 (LDN) were compared. N3-1: Normal iPSC line; F1-1, F2-2, and F4-1: FOP-derived iPSC lines. (B): Principal component analysis. All datasets were classified into three principal components; PC1 (53.17%), PC2 (32.59%), and PC3 (14.23%) and were simplified into three-dimensional scores. Percentage shows the portion of variance in each component. The position of fibroblasts (yellow group) and normal- and FOP-derived iPSCs (red and blue groups) are clearly separated. The position of FOP-derived iPSC lines without LDN treatment (blue group) is further separated from that of control iPSC and embryonic stem cell (ESC) lines (red group). FOP-derived iPSC lines with LDN treatment are closely positioned to normal iPSC lines (N3-1 and DiPS) and human ESC lines referred for Gene Expression Omnibus datasets, GSE29115, GSE22167, and GSE37258. (C): Hierarchical clustering of all genes. The datasets of all genes investigated were clustered according to Euclidean distance metrics. Then, the datasets of fibroblasts and those of iPSC lines were classified into separate branches (yellow vs. red and blue). Furthermore, FOP-derived iPSC lines with LDN treatment were close to that of normal iPSC lines (N3-1 and DiPS) with or without LDN treatment. In contrast, FOP-derived iPSC lines without LDN treatment were classified into different branches from those of normal iPSC and ESC lines and FOP-derived iPSC lines treated with LDN (red vs. blue). (D): Venn diagram for the genes that were upregulated or downregulated by twofold or more between FOP-derived iPSC lines with or without LDN treatment.comparing upregulated or downregulated gene profiles between FOP-derived iPSC lines with and without LDN treatments, the expression of 526 genes was shown to be commonly upregulated and those of 131 genes was shown to be downregulated. (E): Gene ontology (GO) analysis of commonly upregulated 526 genes. The top thirty-three of GO terms (percentage count are more than 5%) are listed. GO terms related to development and differentiation were frequently detected (underlines). Among the downregulated 131 genes, no GO terms were detected with a cutoff p-value of .1. Values are percentage count in all genes. Abbreviations: iPS, induced pluripotent stem; LDN, LDN-193189.

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As shown in Figure 4C, each dataset for 201B7 and three ESC lines are separated from that for normal iPSC lines (N3-1 and DiPS) and FOP-iPSCs. In PCA analysis (Fig. 4B), the datasets for iPSC (red group) are broadly varied in the y-axis (PC3) direction compared with fibroblast datasets (yellow group). Therefore, the separation seen in the cluster analysis may reflect the above-mentioned differences, and such differences are thought to depend on the methods of iPSC induction (between retrovirus and SeV) and/or those between ESCs and iPSCs.

Comparing each gene-expression profile of FOP-derived iPSC lines treated with LDN to those not treated with LDN revealed upregulated and downregulated genes with a fold change of >2. Overall, with these datasets combined, we identified 526 commonly upregulated and 131 commonly downregulated genes (Fig. 4D). In addition, gene ontology analysis revealed that the molecular signature related to development and differentiation was frequently detected in commonly upregulated genes (Fig. 4E). In contrast, no specific signature was found in downregulated genes with a p-value cutoff of .1. Taken together, these results suggest that constitutive activation of ALK2 can promote the differentiation of iPSCs.

Efficient iPSC Generation Requires ALK2 Inhibitor Treatment Within an Optimal Time Frame

To further explore the effect of constitutive activation of ALK2 on iPSC generation, we investigated the time frame of inhibition of iPSC generation by the ALK2 mutant. We treated cultures with the ALK2 inhibitor for various periods of time and then counted the number of AP-positive (AP+) colonies on day 30 after exposure (Fig. 5A). The highest number of AP+ colonies was detected when FOP-derived fibroblasts were treated with the inhibitor from day 8 to day 30 (Fig. 5B, 5C). Treatment from day 1 to day 30 gave a lower level of efficiency. Furthermore, colony-forming efficiency was not restored by treating iPSC-induction cultures from day 1 to day 7 only. To further examine the period from day 8 to day 30, we counted the number of AP+ colonies from day 8 to day 14, from day 15 to day 21, and from day 22 to day 30 (Fig. 5A, 5C). Unexpectedly, we counted few AP+ colonies in every period. Taken together, these results suggested that the formation of iPSC colonies from FOP fibroblasts requires the inhibition of mutant ALK2 activity from day 8 to day 30 of iPSC induction and not during the early phase from day 1 to day 7.

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Figure 5. Efficient generation of induced pluripotent stem cells (iPSCs) from fibrodysplasia ossificans progressive (FOP)-derived fibroblasts during the time frame of activin receptor-like kinase 2 (ALK2) inhibition. (A): Experimental design for determination of the time frame of ALK2 inhibition. FOP-derived fibroblast cultures for the induction of iPSCs were treated with LDN for the indicated periods. (B): Alkaline phosphatase (AP) staining of iPSC colonies from patient F2 (left) and healthy volunteer N1 (right). Colonies in 60 mm dishes were treated with LDN at 0 nM, 30 nM, and 200 nM for the indicated periods (day 1–day 7 [d1–d7], day 1–day 30 [d1–d30], and day 8–day 30 [d8–d30]). (C): Efficiency of formation of iPSC colonies from FOP-derived fibroblasts treated with LDN for the indicated periods. AP+ colony with undifferentiated colony morphology was counted as a typical colony on day 30. Abbreviations: LDN, LDN-193189; SeV, Sendai virus.

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Constitutive Activation of ALK2 in FOP Fibroblasts Inhibits Cellular Reprogramming

To elucidate the mechanisms underlying the inhibition of iPSC generation from FOP fibroblasts, we investigated whether the ALK2 mutation in FOP affects cellular reprogramming. We noticed that Tra-1-60, a pluripotency marker expressed on the iPSC surface, appeared during the early phase (∼ day 7) of iPSC induction (Fig. 6A). Expression of Tra-1-60 was reduced on day 14 but then was markedly increased again up to day 21. The proportion of Tra-1-60+ cells was lower in cultures of FOP-derived fibroblasts undergoing iPSC induction than in cultures of control fibroblast (Fig. 6A). However, Tra-1-60 expression was restored on day 21 in cultures treated with LDN. Consistent with this result, BMP-4 treatment inhibited the expression of Tra-1-60 during iPSC generation (Fig. 6A). To confirm that suppression of Tra-1-60 expression is due to the ALK2 mutation, we generated a shRNA expression construct specific for the ALK2 mutant R206H (Fig. 6B). The reporter plasmids are constructed by inserting synthetic oligonucleotides of normal and mutant ALK2 allelic sequence into 3′UTR of each luciferase genes [37]. The activity of luciferase carrying mutant ALK2 allelic sequence was specifically suppressed by shRNA expression (Fig. 6B). This shRNA effectively suppressed expression of the ALK2 mutant in FOP-derived fibroblasts and restored Tra-1-60 expression to control levels (Fig. 6C, 6D). Consistently, the generation of iPSC was partially improved by shRNA expression (supporting information Fig. S6A). Taken together, these results demonstrate that the suppression of Tra-1-60+ cell generation is due to the mutant ALK2 in FOP.

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Figure 6. Inhibitory mechanism of induced pluripotent stem cell (iPSC) generation from fibrodysplasia ossificans progressive (FOP)-derived fibroblasts. (A): Tra-1-60 expression during iPSC generation. After Sendai virus infection, the fibroblasts derived from FOP patients and healthy controls were cultivated without LDN [LDN(−)], with LDN [LDN(+)], and without LDN in the presence of BMP-4 [LDN(−) + BMP-4]. The expression of Tra-1-60, a pluripotency surface marker, was analyzed by fluorescence-activated cell sorting (FACS) on day 7, day 14, and day 21 of the iPSC induction period. Red line: expression pattern of healthy control (N1). Blue line: expression pattern of FOP (F2). (B): Assessment of specific inhibition of mutant activin receptor-like kinase 2 (ALK2) by shR206H. The silencing effect of shR206H was examined by luciferase and β-galactosidase assays. The expression levels of the Photinus (Wild) and Renilla (Mutant) luciferases and β-galactosidase were measured, respectively. The levels of both mutant and wild-type luciferase reporter allele activities were normalized to β-galactosidase activity. Values are average of four independent determinations. The ratios of the mutant and normal reporter activities in the present of shR206H were normalized to the control ratio obtained in the presence of the pSIREN-RetroQ-ZsGreen control plasmid. Error bars represent standard deviations. (C): Expression of ALK2 in FOP-derived fibroblasts treated with shRNA. The quantitative RT-PCR (qRT-PCR) analysis revealed that expression of ALK2 mRNA is significantly reduced by shRNA treatment (upper panel). Suppression of ALK2 expression was also confirmed by immunoblot analysis (lower panel). The data are means ± SD of triplicated cultures. Unpaired Student's t test was performed to evaluate differences between two groups. *, p < .05; **, p < .01, when compared with the values of respective control. (D): Treatment with shRNA specific for the ALK2 mutant R206H can restore expression of Tra-1-60. Virus carrying shRNA and ZsGreen cDNA was infected into both normal and FOP-derived fibroblasts. Tra-1-60 expression of ZsGreen+ cells is measured by FACS. The representative data are shown in the upper panel. The results are summarized in the lower graph. It is significantly recovered to normal level by treatment with the shRNA. *, p < .05. (E): Expression of pluripotent signature genes. On day 7 of iPSC induction, Tra-1-60+ and Tra-1-60 cells were purified by FACS and the expression of endogenous genes related to pluripotency was then measured by qRT-PCR. The expression of OCT3/4, NANOG, and SALL4 was exclusively detected in Tra-1-60+ cells but was significantly lower in FOP-derived Tra-1-60+ cells than in normals. PCLP1 encodes Tra-1-60. The data are means ± SD of three independent experiments. A one-way ANOVA followed by Tukey's multiple comparison test was performed to evaluate differences between groups. *, p < .05, **, p < .01, when compared with the values of day 7 (Tra-1-60+) from fibroblast N1. #, p < .05, ##, p < .01, when compared with the values of day 7 (Tra-1-60+) from fibroblast N3. (F): Incomplete reprogramming of fibroblastic markers. After Tra-1-60+ and Tra-1-60 cells were purified by FACS on day 7 of iPSC induction, the expression of fibroblastic markers was measured by qRT-PCR. The expression of PDGFRα and VIMENTIN of FOP-derived Tra-1-60+ cells was significantly higher than that of control Tra-1-60+ cells, suggesting that these genes are insufficiently downregulated during iPSC generation from FOP fibroblasts. The statistical analysis is described in (E). (G, H): BMPs can inhibit the colony formation of iPSCs during their induction. Colony morphology of iPSCs with alkaline phosphatase staining (G) and the number and percentage of typical and atypical iPSC colonies with or without BMP treatment (H). Almost all colonies are typical in cultures without BMPs or with both BMPs and LDN. In contrast, 50% of the colonies was atypical in cultures treated with BMPs. The concentrations of BMP-4 and BMP-7 are 10 ng/ml and 25 ng/ml, respectively. Typical and atypical colonies are defined as shown in Figure 1D. Scale bars = 200 μm. Abbreviations: BMP, bone morphogenetic protein; LDN, LDN-193189; NS, not significant.

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To further evaluate the reprogramming status of the fibroblasts, we purified Tra-1-60+ and Tra-1-60 cells from day 7 cultures by fluorescence-activated cell sorting and investigated the expression of other pluripotent and fibroblastic markers. Fibroblastic markers, such as PDGFRα and VIMENTIN, which are strongly expressed in fibroblasts, should be downregulated during iPSC generation because they are not expressed in iPSCs. The endogenous genes OCT3/4 and NANOG are expressed in day 7 Tra-1-60+ cells but not in day 7 Tra-1-60 cells in both control and FOP-derived cultures (Fig. 6E). The expression levels of these pluripotent markers are significantly lower in FOP-derived Tra-1-60+ cells than in controls. There was no expression of SOX2, REX1, KLF5, or DNMT3b in neither Tra-1-60+ nor Tra-1-60 cells (supporting information Fig. S6B). The expression of fibroblastic markers was downregulated in Tra-1-60+ cells. However, the expression levels of PDGFRα and β and VIMENTIN were significantly higher in FOP-derived Tra-1-60+ cells than in controls (Fig. 6F). These results suggest that the reprogramming status of FOP fibroblasts is incomplete, despite the appearance of pluripotency marker expression in these cells.

The inhibition of ALK2 by LDN during the later phase of iPSC induction can recover the colony-forming capacity of iPSCs. This finding suggests that newly generated iPSCs cannot be maintained due to the constitutive activation of mutant ALK2 in FOP-derived fibroblasts and that as a result iPS colonies cannot form. To prove this hypothesis, we investigated the effect of BMP signaling on the colony-forming capacity of iPSCs. We showed that BMP-4 and BMP-7 treatments reduced the area of AP+ cells in each iPSC colony (Fig. 6G). Morphological analysis revealed that this enforced BMP signaling disrupts the formation of iPSC colonies (Fig. 6G, 6H). Given the fact that BMP signals can induce the differentiation of iPSCs, these results suggest the existence of at least the two distinct mechanisms underlying the inhibition of iPSC generation; the first is incomplete reprogramming of pluripotent and fibroblastic genes, and the second is the forced differentiation of the cells during and after reprogramming.

Previous studies demonstrated that TGF-β/Activin signals affect iPSC generation [38]. Smad2 and 3, the downstream molecules of TGF-β/Activin receptors, are phosphorylated by the activation of these signals. However, we could not detect any change in Smad2 and 3 phosphorylations between normal and FOP-derived fibroblasts, suggesting that ALK2 mutation observed in FOP does not affect TGF-β/Activin signals during iPSC generation (supporting information Fig. S6C–S6F).

Inhibited Production and Maintenance of FOP-derived iPSC is a Useful Phenotype for Identifying Drug Candidates for Future Therapies

Our observation that treatment of FOP-derived fibroblasts with an ALK2 inhibitor allows iPSCs to be generated and maintained with pluripotency prompted us to use this system to identify candidate ALK2 inhibitors. We performed in silico screening by the multidirectional similarity search system and collected 160 bioactive compounds (see Materials and Methods). Our screen of the chemical library resulted in the identification of new candidate ALK2 inhibitors. One of them, RK-0071142 suppressed Smad1/5/8 phosphorylation (Fig. 7A, 7B). We used a cell-based assay of BMP signaling to show that the half-maximal inhibitory concentration (IC50) of RK-0071142 was 1.44 μM for BMP-6 and 4.08 μM for BMP-4 (Fig. 7C and supporting information Fig. S7). Treatment of FOP-derived fibroblasts with RK-0071142 restored their capacity to generate iPSCs but with efficiency lower than that of LDN (Fig. 7D). The spontaneous differentiation of FOP-derived iPSCs was suppressed by RK-0071142 at a similar level to that of LDN (Fig. 7E). These results, together with LDN analyses, indicate that the generation and maintenance of FOP-derived iPSCs is a useful system for evaluating the bioactivity of new candidates as ALK2 inhibitors.

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Figure 7. Screening for new candidates as ALK2 inhibitors. (A): Structures of RK-0071142, LDN-193189, and Dorsomorphin. (B): Immunoblot analysis of p-Smad1/5/8 and total Smad1 in fibrodysplasia ossificans progressive (FOP)-derived induced pluripotent stem (iPSC) lines with or without RK-0071142 treatment. RK-0071142 (1 μM) treatment suppressed the phosphorylation of Smad1/5/8. The relative phosphorylation level was normalized to the expression of Smad1 in each iPSC line. F1-1, F2-1, F2-2, F2-3, F2-4, F2-5, F4-1, and F4-2 are FOP-derived iPSC lines. Control iPSC line: Normal iPSC line. (C): Inhibitory activity of RK-0071142 and LDN-193189 for bone morphogenetic proteins (BMPs). The kinase activity of ALK2 was measured and determined by the promoter activity specific for ALK2 signals (supporting information Fig. S7). IC50 of bone morphogenetic protein-6 (BMP-6) (upper panel) and bone morphogenetic protein-4 (BMP-4) (lower panel) was calculated using a kinetic graph of ALK2 activity. (D): Alkaline phosphatase (AP)-staining of iPSC colonies from patient F1 and F2 in 60 mm dishes treated with or without RK-0071142 (top). Efficiency of formation of iPSC colonies in cultures treated with or without RK-0071142 (bottom). AP+ colony with undifferentiated colony morphology was counted on day 30 of iPSC induction. The cultures were treated with RK-0071142 from day 8 to day 30 of the induction period. *, p < .05. (E): The number and ratio of the three types of colonies (described in Fig. 3A) in FOP-derived iPSC lines treated with RK-0071142 at various concentrations. Spontaneous differentiation of FOP-derived iPSC lines was suppressed by RK-0071142 treatment. The suppression intensity of RK-0071142 was similar to that of LDN-193189. Abbreviations: ALK2, activin receptor-like kinase 2; BMP, bone morphogenetic protein; LDN, LDN-193189.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

In this study, we demonstrated that constitutive activation of ALK2 inhibits the generation and maintenance of human iPSCs. We also show that two distinct mechanisms underlie the inhibition of iPSC generation, the incomplete reprogramming of pluripotent and fibroblastic genes, and the forced differentiation of the cells during and after reprogramming.

Mutations in ALK2 underlie phenotypic variations of FOP and confer constitutive activity to the mutant receptor [3–9]. ALK2 acts as a type I receptor of BMPs, which are members of the TGF-β family [39]. TGF-βs and their family members are implicated in the generation and maintenance of iPSCs. Activation of TGF-β signaling blocks the cellular reprogramming required for the differentiation of a somatic cell into a pluripotent one and thus results in a reduction in the efficiency of iPSC generation [38]. Consistently, the blocking of TGF-β signaling by inhibiting ALK5 kinase, which is a receptor of TGF-β, augments the formation of iPSC colonies [40]. In contrast, a recent study using MEFs demonstrated that BMP signaling during the early-stage of iPSC induction (∼ day 8) can enhance the generation of iPSCs. This signal induces a set of miRNAs associated with the mesenchymal-to-epithelial transition (MET), which can accelerate the generation of iPSCs [41]. BMP signaling occurs via a tetrameric complex of two out of three type II receptors, namely, BMP receptor II (BMPRII), activin receptor type II (ActRII), and ActRIIB; and two out of three BMP type I receptors, namely, ALK2, 3, and 6 [42]. Suppression of BMPRII and ALK3 inhibits the generation of iPSCs, suggesting that enhancement of iPSC generation by BMP signaling is mediated by the receptor consisting of BMPRII and ALK3 [41]. Although these findings indicate that BMP signaling together with the receptors BMPRII and ALK3 are implicated in the generation of iPSCs, the role of other receptors, such as ALK2 and ALK6, in cellular reprogramming remains unknown. We show here that during the early phase of iPSC induction, from day 1 to day 7, treatment with an ALK2 inhibitor can suppress the generation of iPSCs from normal fibroblasts. Thus, BMP signaling mediated by ALK2 is necessary for reprogramming during the early phase of iPSC generation. In contrast to the previous findings for BMPRII and ALK3, we show here that constitutive activation of ALK2 affects both the upregulation of pluripotent markers and the downregulation of fibroblastic markers during the early phase of iPSC generation resulting in incomplete reprogramming. Signaling by BMP type I receptors appears to regulate iPSC generation through different mechanism; ALK3 signaling stimulated by BMPs can enhance the iPSC generation by promoting MET, whereas constitutive activation of ALK2 can suppress iPSC generation by reprogramming the pluripotent and fibroblastic markers insufficiently. The downstream molecules mediated by ALK2 are known to be similar but not identical to those mediated by ALK3. The BMP type I receptors, ALK2, 3, and 6, act as downstream components of type II receptors and phosphorylate Smad proteins [42, 43]. Whereas ALK3 and ALK6 phosphorylates Smad1, 5, and 8, ALK2 only phosphorylates Smad1 and 5 under physiological conditions [44]. In addition, ALK3 signaling has a functionally different effect from that of ALK2 and 6 on cellular apoptosis of hippocampal progenitors [45]. These studies can support our results that the effect of ALK2 on iPSC generation is different from that of ALK3.

We demonstrated that constitutive activation of ALK2 repressed the formation of iPSC colonies due to the forced differentiation of the cells during and after reprogramming. Consistently, LDN treatment at later stages of iPSC generation (from day 8 to day 30) restored the colony-forming capacity of FOP-derived iPSCs. Therefore, the molecular basis of the inhibitory effect on colony formation is the same as that causing repression of iPSC generation from FOP fibroblasts. BMP signaling is known to be important for maintaining the pluripotency of mouse ESCs. Mouse ESCs can be continuously cultivated with BMP-4 in the presence of leukemia inhibiting factor under serum-free conditions [46]. In contrast, in human ESCs and mouse epiblast stem cell studies, BMP-4 has been shown to induce trophoblastic lineage [47] as well as germ cell lineage differentiations [48]. In human ESCs, BMP-4 together with FGF2 can switch the cell lineage outcome to mesendoderm [49]. We demonstrated here that constitutive activation of ALK2 in the presence of FGF2 is forced to induce differentiation into both mesoderm and endoderm. This result suggests that BMP signaling mediated by ALK2 can synergy with FGF2 to direct iPSCs into both mesodermal and endodermal cells.

Several recent studies have reported that iPSCs established from patients can not only recapitulate some aspects of diseases but also can be used to better design and anticipate results from translational medicine [12–16]. Our study demonstrates that the aberrant molecular events occurring in diseases can impair the generation of iPSCs and that by restoring these events the efficiency of iPSC generation can also be restored. Thus, investigations into why iPSC production in diseases is inhibited can help unravel the underlying molecular and pathogenic events of these diseases.

This study also highlights the inefficient production of iPSCs as a useful disease phenotype not only for studying the molecular mechanisms underlying iPS reprogramming but also for identifying drug candidates for future therapies. In addition to the other ways to select drug candidates such as the measurement of kinase activity, the reprogramming process serves well as a validation tool for specific application. Although the system used here did not appear to be relevant to the symptoms of FOP, we showed that LDN, a known inhibitor of ALK2, could improve the efficiency of both the generation and the maintenance of iPSCs derived from FOP patients. In combination with in silico chemical library screening, we screened a chemical library to identify candidates with the ability to restore iPSC induction in FOP-derived fibroblasts. Using this approach, we identified a new ALK2 inhibitor candidate, RK-0071142, with potential for future therapeutic applications in the treatment of FOP. Since constitutive activation of ALK2 plays a pivotal role in the pathogenesis of FOP, screening for and evaluating new compounds as ALK2 inhibitors is expected to be the first step toward developing new drugs for the treatment of FOP. The patient-derived cellular model presented here has the potential to not only lead to the discovery of new compounds to treat FOP but also to be applied as a strategy to develop future therapies for other human diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

We would like to thank Masaki Takahashi for technical support, and Dr. Yuki Yanagihara and Dr. Kohei Miyazono for providing materials and helpful discussion. We also thank RIKEN Program for Drug Discovery and Medical Technology Platforms for providing the chemical compounds. This study was supported in part by grants from the Ministry of Health, Labor, and Welfare of Japan and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0288_sm_SupplTable1.pdf95KSupplementary Table 1
sc-12-0288_sm_SupplTable2.pdf33KSupplementary Table 2
sc-12-0288_sm_SupplTable3.pdf18KSupplementary Table 3
sc-12-0288_sm_SupplFigure1.tif2169KFigure S1. Confirmation of mutations in ALK2 gene and colony morphology of FOPderived iPS cells. (A): Genomic sequencing in ALK2 gene from FOP-derived iPSC lines. The mutations of 617G>A (R206H) and 1067G>A (G356D) were indicated by arrows. (B): Phase contrast picture of FOP-derived iPSCs with the treatment of Dorsomorphin (DM). Left panel: iPSCs derived from the patient, F2; right panel: iPSCs derived from the patient, F4. Scale bars, 200 μm.
sc-12-0288_sm_SupplFigure2.tif173KFigure S2. Gene expression of teratoma derived from FOP-iPSC line Normal and FOP-derived iPSC lines were transplanted into the subcutaneous tissues of the immunodeficient mice. Eight to twelve weeks after injection, the iPSC lines tested formed teratomas. After RNA was purified from the teratomas, the markers such as MESP1 and MESOGENIN (Mesoderm), FOXA2 and SOX17 (endoderm) and SOX1 and NEUROD1 (ectoderm) were examined by qRT-PCR. Both teratomas expressed the markers of three germ layers.
sc-12-0288_sm_SupplFigure3.tif356KFigure S3. Effects of LDN-193189 and Dorsomorphin on Smad 1/5/8 phosphorylation in FOP-derived iPSC lines. Immunoblot analyses of phosphorylated Smad1/5/8 of FOPderived iPSC lines (F1-1, F2-1, -2, -3, -4, -5, F4-1 and -2) and Normal iPSC lines (N3-1) treated with or without LDN-193189 (LDN) and Dorsomorphin (DM). The normal iPSC line, N3-1, was treated with BMP-4. The relative levels of Smad1/5/8 phosphorylation in each iPSC lines were suppressed by the treatment with LDN and DM. The density of all bands is measured by Image J software, and relative phosphorylation level of Smad 1/5/8 is normalized to Smad1.
sc-12-0288_sm_SupplFigure4.tif128KFigure S4. Effects of Dorsomorphin on colony formation and maintenance of iPSCs. The number and ratio of three types of colonies from FOP-derived iPSC lines. The iPSC F2-1 and iPSC F4-2 lines were cultured with or without DM. Undifferentiated (u), partially differentiated (p) and completely differentiated (d) colonies are defined as described in Fig. 3A. Scale bars, 200 μm.
sc-12-0288_sm_SupplFigure5.tif194KFigure S5. The effect of BMP-4 on the expressions of the pluripotency- and differentiation-related genes in normal iPSC lines. DiPS and N3-1 iPSC lines were cultured with or without 10 ng/ml of BMP-4 in the absence of bFGF for 5 days. The data are normalized to GAPDH and represent as relative expression levels to respective control of DiPS.
sc-12-0288_sm_SupplFigure6.tif732KFigure S6. Inhibitory mechanism of iPSC generation from FOP-derived fibroblasts. (A): The effect of shR206H on the iPSC colony formation. shR206H-infected fibroblast N1 and F2 were induced to iPSC by SeV vectors in the presence or absence of LDN from day 8 to day 30. At day 30 of induction, AP+ colony with undifferentiated colony morphology was counted as a typical colony. The atypical colony is described in Fig. 1D and 1E. (B): On day 7 of iPSC induction, Tra-1-60+ and Tra-1-60- cells were purified by FACS and then pluripotent marker genes (SOX2, GDF3, REX1, DNMT3b and KLF5) were measured by qRT-PCR. The data are means ± SD of three independent experiments. NS, not significant; ND, not detected. (C and D): Immunoblot analyses of the phosphorylation of Smad2 and 3. TGF-β3 stimulation can enhance the phosphorylation of Smad2 and 3 in HeLa cells. This result indicates that the system works well. The relative phosphorylation levels of Smad2 and 3 are summarized in (D). The density of all bands is measured by Image J software, and relative phosphorylation levels of Smad2 and 3 are normalized to total Smad2 and 3, respectively. (E and F): TGF-β/Activin signals during iPS cell generation from FOP-derived fibroblasts. Immunoblot analyses of phosphorylated Smad2 and 3 of normal and FOP-derived fibroblasts on day 7 of iPS cell induction. On day 7 of iPS cell induction, Tra-1-60+ and Tra-1-60- cells were purified by FACS and the phosphorylation of Smad2 and 3 was then measured by immunoblot analyses. The graph (F) shows relative phosphorylation levels of Smad2 and 3. Smad2 and 3 phosphorylations were not enhanced during iPS cell inductions. The density of all bands is measured by Image J software, and relative phosphorylation levels of Smad2 and 3 are normalized to total Smad2 and 3, respectively. PC: Positive control, TGFβ3-treated HeLa cell line
sc-12-0288_sm_SupplFigure7.tif158KFigure S7. Schematic representation of measurement ALK2 kinase activity for chemical screening. MDA-D-BREFluc/Rluc cell line which were cultured with ALK2 inhibitor candidates in the presence of BMP-4 or BMP-6. After 24 hr cultivation, luciferase activity (both Fluc and Rluc) were measured.
sc-12-0288_sm_SupplData.pdf157KSupplementary Data

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