Cellular and Molecular Life Sciences

, Volume 73, Issue 10, pp 2089–2104 | Cite as

Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals

  • Ludwig Heesen
  • Michael Peitz
  • Laura Torres-Benito
  • Irmgard Hölker
  • Kristina Hupperich
  • Kristina Dobrindt
  • Johannes Jungverdorben
  • Swetlana Ritzenhofen
  • Beatrice Weykopf
  • Daniela Eckert
  • Seyyed Mohsen Hosseini-Barkooie
  • Markus Storbeck
  • Noemi Fusaki
  • Renata Lonigro
  • Raoul Heller
  • Min Jeong Kye
  • Oliver Brüstle
  • Brunhilde Wirth
Original Article


Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier.


F-actin dynamics Growth cones Gene modifier Gene expression Sendai virus 



Plastin 3


Spinal muscular atrophy


Survival motor neuron 1


Survival motor neuron 2


Induced pluripotent stem cell


Central nervous system






Neural stem cell




Rho-associated coiled-coil containing protein kinase


Sendai virus



We thank Dr John Dimos (iPierian) for generating the HGK13 and HGK16 iPSC clones. We thank M. Segschneider, C. Thiele, R. Konang and V. Poppe from the Institute of Reconstructive Neurobiology (RNB, Bonn, Germany) for excellent technical support and A. Leinhaas for carrying out the teratoma assays. Dr A. Schauss and I. Hensen of the Imaging Facility of the Cluster of Excellence in Cellular Stress Responses in Aging-associated Diseases (CECAD, Cologne, Germany) supported the confocal microscopy. This work has been supported by the European Community’s Seventh Framework Program FP7/2007-2013 under grant agreement no 2012-305121 (NeurOmics), the 7FP project SCR&Tox (HEALTH-F5-2010-26675), BIO.NRW (Project StemCellFactory, #005-1403-0106), the Center for Molecular Medicine Cologne (grant no C11), the Hertie Foundation and the Deutsche Forschungsgemeinschaft (Wi 945/13-1; Wi 945/16-1) to B.W.

Compliance with ethical standards

Conflict of interest

No competing financial interests exist.

Supplementary material

18_2015_2084_MOESM1_ESM.tif (60.7 mb)
Supplementary material 1: Figure S1: Validation of human iPSCs from SMA III and asymptomatic siblings. (A) Typical flat colony morphology with dense centre in phase contrast was shown for one representative asymptomatic (HGK21.1) and one representative SMA III (HGK22.17) iPSC line. Pluripotency was proven by AP activity (blue) and via immunostaining with pluripotency surface markers SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 (all red). Nuclei were counterstained with DAPI (blue). Scale bars 200 µm, valid for all images. (B) Exemplary karyograms of human iPSC lines HGK21.1 (asymptomatic) and HGK22.17 (SMA III) with respective original fibroblast lines schematically displayed every chromosome with the corresponding B allele frequency (upper panel) and Log R ration (lower panel). (C) Semi-quantitative analysis of SeV nucleocapsid (NP) mRNA expression in representative iPSC lines in comparison to 18S rRNA expression as loading control. SeV vector activity vanished at later passages (> p7). Only the positive control (pos ctrl) depicted a strong NP expression (TIFF 62136 kb)
18_2015_2084_MOESM2_ESM.tif (41.1 mb)
Supplementary material 2: Figure S2: Validation of human iPSCs from SMA III and asymptomatic siblings. (A) Spontaneous undirected differentiation of human iPSCs delivered progeny of all three germ layers in representative cell lines showing endodermal cells (α-fetoprotein AFP, green), mesodermal cells (smooth muscle actin SMA, red) and ectodermal cells (β III-tubulin, red) upon immunocytochemical labelling. (B) Teratoma formation assay confirmed pluripotency in iPSCs by generating derivatives of all three germ layers in vivo. Immunohistologic haematoxylin/eosin (HE) staining in representative samples displayed endoderm (glandular epithelium), mesoderm (cartilage) and ectoderm (neural rosettes). Nuclei were counterstained with DAPI (blue) (A). Scale bars 200 µm (A and B), valid for all images (TIFF 42104 kb)
18_2015_2084_MOESM3_ESM.tif (30 mb)
Supplementary material 3: Validation of smNPCs. iPSC-derived smNPC lines clearly disclosed typical NSC markers PAX6 (green), SOX1 (red), SOX2 (green) and nestin (red) in four representative cell lines upon immunolabelling. Expression of anterior marker FORSE-1 (green) was visualised as well. smNPCs expressed characteristic rosette-markers PLZF (green) and ZO-1 (red), however, lacked the typical petal-like arrangement underscoring their early pre-rosette NSC fate. Nuclei were counterstained with DAPI (blue). Scale bar 100 µm, valid for all images (TIFF 30730 kb)
18_2015_2084_MOESM4_ESM.tif (14.9 mb)
Supplementary material 4 Immunocytochemistry of four smNPC-derived cultures subjected to MN differentiation. Cultures representing the respective phenotype classes (control 30 m-r12, SMA I patient HGK1, SMA III patient HGK27.13 and asymptomatic sister HGK28.9) proved correct pMN-regionalisation on day 8 of the differentiation protocol. Pan-NSC markers SOX2 (green) and nestin (red) were clearly visible while early CNS marker PAX6 (green) was moderately expressed. Cells broadly exhibited pMN-marker OLIG2 (green) and NKX6.1 (red) whose evident co-localisation confirmed true MN progenitor identity. Moreover, expression of HOXB4 (red) verified sufficient posteriorization. Presence of only few NKX2.2+ cells (green) marked correct domain boundaries meaning that cells were not too ventralised into domain p3. Insets showed corresponding pictures with stained nuclei. Nuclei were counterstained with DAPI (blue). Scale bar 100 µm, valid for all images (TIFF 15294 kb)
18_2015_2084_MOESM5_ESM.tif (2.2 mb)
Supplementary material 5: Quantification of MN marker expression in smNPC-derived MN cultures on day 8 and day 27. For each individual cell line, MN numbers are given for (A) HB9+ MNs on day 8, (B) ISL1+ MNs on day 8, (C) HB9+ MNs on day 27 and (D) ISL1+ MNs on day 27. Error bars represented ± SEM of two (controls 35 m-r1, 61f-s2, 51f-s6, 62 m-s4) to three (all other lines) independent MN differentiation experiments (A and B) and n = MN differentiation experiments as outlined in tables below (C and D). Significance was calculated relative to 30 m-r12 (Kruskal–Wallis test, p < 0.05 = *) (TIFF 2297 kb)
18_2015_2084_MOESM6_ESM.tif (4.3 mb)
Supplementary material 6: Determination of gem numbers in different cell populations. Quantification of individual cell lines was displayed in left hand panels in (A) fibroblasts, (B) iPSCs, (C) smNPCs, (D) early MN culture (day 8) and (E) late MN culture (day 27). Right hand panels show grouped phenotypes. Error bars represented ± SEM of duplicates in fibroblasts (n = 2) and triplicates in all other cell populations (n = 3). MN cultures displayed data of two to three independent MN differentiation experiments (n = 3). Significance was calculated relative to 30 m-r12 (individual cell lines) and grouped five healthy controls (phenotype groups), respectively (Kruskal-Wallis test, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****) (TIFF 4443 kb)
18_2015_2084_MOESM7_ESM.tif (3.5 mb)
Supplementary material 7 Quantitative analyses of SMN mRNA expression levels. (A) SMN mRNA expression occurred in a similar pattern in fibroblasts, iPSCs and smNPCs with controls at the top, SMA III and asymptomatic in the middle and SMA I at the lowest range, matching the SMN1/SMN2 copy numbers. In MN culture, SMA III and asymptomatic exhibited an elevated SMN level in comparison to control. Quantification of SMN mRNA expression in individual lines of (B) fibroblasts, (C) iPSCs, (D) smNPCs and (E) MN cultures (day 27) is shown. Expression was normalised to total RNA levels and significance calculated relative to 30 m-r12 (individual cell lines) and grouped five healthy controls (phenotype groups), respectively (Kruskal–Wallis test, p < 0.05 = *, p < 0.01 = **). Error bars represented ± SEM of duplicates (n = 2). MN culture displayed data of one to three MN differentiation experiments (TIFF 3619 kb)
18_2015_2084_MOESM8_ESM.tif (3.6 mb)
Supplementary material 8: Quantitative analyses of PLS3 mRNA expression (A) In fibroblasts, PLS3 mRNA expression levels hardly differed among the classes. Expression rates in iPSCs showed a wider spreading with asymptomatic at the top, SMA I and controls following closely and SMA III at lowest level. While asymptomatic prevailed as strongest expressing group underlining putative overexpression, PLS3 expression continuously declined in controls resulting in barely any detectable PLS3 mRNA in MN cultures. Expression in SMA I also dropped to levels equal to SMA III in MN cultures. Importantly, PLS3 levels in asymptomatic were significantly elevated in comparison to controls. Quantification of PLS3 mRNA expression in individual lines of (B) fibroblasts, (C) iPSCs, (D) smNPCs and (E) MN cultures (day 27) is shown. Expression was normalised to total RNA levels and significance computed relative to 30 m-r12 (individual cell lines) and grouped five healthy controls (phenotype groups), respectively (Kruskal–Wallis test, p < 0.05 = *, p < 0.01 = **). Error bars represented ± SEM of duplicates (n = 2). MN culture displayed data of one to three MN differentiation experiments (TIFF 3735 kb)
18_2015_2084_MOESM9_ESM.tif (4 mb)
Supplementary material 9 Quantitative analyses of SMN protein expression. Western blot analysis from healthy controls, SMA I- and SMA III-affected and asymptomatic discordant siblings in (A) fibroblasts, (B) iPSCs, (C) smNPCs, (D) MN cultures (day 27). Expression was normalised to β-actin protein levels and significance calculated relative to 30 m-r12 (individual cell lines, left hand panels) and grouped five healthy controls (phenotype groups, right hand panels), respectively (Kruskal-Wallis test). In fibroblasts, iPSCs and smNPCs error bars represented ± SEM of duplicates (n = 2). In MN culture (day 27) error bars represented ± SEM of two to three independent MN differentiation experiments (TIFF 4061 kb)
18_2015_2084_MOESM10_ESM.tif (3.8 mb)
Supplementary material 10 Quantitative analyses of PLS3 protein expression. Western blot analysis from healthy controls, SMA I- and SMA III-affected and asymptomatic discordant siblings in (A) fibroblasts, (B) iPSCs, (C) smNPCs and (D) MN culture (day 27). Note that all asymptomatic cell lines except for HGK16 strikingly elevated PLS3 expression in comparison to SMA III and controls. Expression was normalised to β-actin protein levels and significance calculated relative to 30 m-r12 (individual cell lines, left hand panels) and grouped five healthy controls (phenotype groups, right hand panels), respectively (Kruskal–Wallis test, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****). In fibroblasts, iPSCs and smNPCs error bars represented ± SEM of duplicates (n = 2). In MN culture (day 27) error bars represented ± SEM of two to three independent MN differentiation experiments (TIFF 3854 kb)
18_2015_2084_MOESM11_ESM.tif (2.9 mb)
Supplementary material 11 Protein expression levels in control cell lines. (A) Representative Western blots from male and female controls visualising SMN and PLS3 protein expression in MN culture on day 27. (B) Graphs visualise PLS3 expression on RNA and protein level in MN cultures on day 27 according to gender of controls. Note that no different PLS3 protein expression was detected between male and female controls. (C) PLS3 in vitro siRNA-mediated knock-down shows the upper band as the correct specific PLS3 signal (marked with an arrow). The lower band is unspecific (TIFF 2958 kb)
18_2015_2084_MOESM12_ESM.tif (32.7 mb)
Supplementary material 12: Analysis of growth cones in mixed MN cultures on day 27. (A) Growth cones of male control 30 m-r12, female control 51f-s6, SMA III HGK27.13 and asymptomatic HGK21.8 visualising PLS3 (green) and actin (red) in growth cones and MAPT (magenta) in axons. Growth cones were magnified (dashed lines) in PLS3/actin merge in insets. Scale bar 10 µm, valid for all images. (B) Average area size was measured by encircling every growth cone individually. In comparison to controls, average area size was increased in SMA III and asymptomatic yet only in asymptomatic the difference was significant. (C) Co-localisation of PLS3 and actin was calculated by Pearson’s coefficient. However, Pearson’s coefficient accounted for a comparable value (control: 0.52; SMA III: 0.52; asymptomatic: 0.47) among all three phenotype groups indicating a medium co-localisation between PLS3 and actin. Growth cones were analysed from four independent MN differentiation experiments (n = 4). In total, n = 28 (controls 30 m-r12, 35 m-r1, 51f-s6), n = 22 (SMA III HGK27.10, HGK27.13) and n = 20 (asymptomatic HGK21.1, HGK21.8, HGK28.9) growth cones were analysed. Significance was calculated relative to grouped three healthy controls (Kruskal-Wallis test, p < 0.05 = *) (TIFF 33,462 kb)
18_2015_2084_MOESM13_ESM.tif (2.9 mb)
Supplementary material 13: Overview table listed specific features of iPSC lines such as originating parental fibroblast line, SMA phenotype, age, sex, SMN1/SMN2 copy number, elevated (↑) PLS3 expression levels in blood, reprogramming technique and validation criteria. Note that numbers in HGK denote individual line and additional clone number of the same line (TIFF 2953 kb)
18_2015_2084_MOESM14_ESM.tif (1.8 mb)
Supplementary material 14: Primer nucleotide sequences (TIFF 1860 kb)
18_2015_2084_MOESM15_ESM.tif (2.9 mb)
Supplementary material 15: List of primary antibodies and respective secondary antibodies (gt = goat, ms = mouse, rb = rabbit) (TIFF 2967 kb)


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Copyright information

© Springer Basel 2015

Authors and Affiliations

  • Ludwig Heesen
    • 1
    • 2
  • Michael Peitz
    • 2
    • 6
  • Laura Torres-Benito
    • 1
  • Irmgard Hölker
    • 1
  • Kristina Hupperich
    • 2
  • Kristina Dobrindt
    • 2
  • Johannes Jungverdorben
    • 2
    • 6
  • Swetlana Ritzenhofen
    • 2
  • Beatrice Weykopf
    • 2
    • 6
  • Daniela Eckert
    • 2
  • Seyyed Mohsen Hosseini-Barkooie
    • 1
  • Markus Storbeck
    • 1
  • Noemi Fusaki
    • 3
  • Renata Lonigro
    • 4
    • 5
  • Raoul Heller
    • 1
  • Min Jeong Kye
    • 1
  • Oliver Brüstle
    • 2
    • 6
  • Brunhilde Wirth
    • 1
  1. 1.Institute of Human Genetics, Institute of Genetics and Center for Molecular Medicine CologneUniversity of CologneCologneGermany
  2. 2.Institute of Reconstructive Neurobiology, LIFE & BRAIN CenterUniversity of BonnBonnGermany
  3. 3.Keio University School of Medicine and JST PRESTOTokyoJapan
  4. 4.Department of Biological and Medical SciencesUniversity of UdineUdineItaly
  5. 5.Institute of Clinical PathologyA. O. UUdineItaly
  6. 6.DZNE, German Center for Neurodegenerative DiseasesBonnGermany

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