Approximately 90% of LS cases, and 45% of NS, are caused by missense mutations in the PTPN11
gene that encodes the protein tyrosine phosphatase SHP2. PTPN11
is ubiquitously expressed, essential for normal development, and somatic mutations in this gene contribute to leukemogenesis in children3,4
. For LS, two mutations, T468M and Y279C, are most recurrent5
. Hipertrophic cardiomyopathy is the most common life–threatening cardiac anomaly in LS2
. Animal models of LS have been generated in Drosophila
, but the molecular pathogenesis of LS remains obscure.
Ectopic expression of four transcription factors (OCT4
) in adult human dermal fibroblasts can generate pluripotent iPSC8–10
. Together with defined in vitro
differentiation protocols, this suggests the possibility of developing reliable disease models11–14
. We have established iPSC lines from two LS patients, a 25-year-old female (L1), and a 34-year-old male (L2). A heterozygous T468M substitution mutation in PTPN11
is present in both.
Fibroblasts were transduced with OCT4-
- and c-MYC
-encoding VSV-pseudotyped Moloney-based retroviral vectors. Compact ESC-like colonies emerged after two weeks and TRA-1-81-positive colonies were clonally expanded to create stable LS-iPSC lines (Supplementary Fig. 1
. Three iPSC lines per patient were used for preliminary characterization: L1-iPS1, L1-iPS6, L1-iPS13, L2-iPS6, L2-iPS16 and L2-iPS18.
To verify that the iPSC originated from patient-derived fibroblasts, we performed DNA fingerprinting analysis (Supplementary Fig. 2a
). All iPSC had normal karyotypes of 46,XX (L1) and 46,XY (L2) (Supplementary Fig. 2b
and data not shown). In addition, they carried the expected T468M mutation (Supplementary Fig. 3a
). Restriction fragment length polymorphism analysis of an RT-PCR amplimer containing the mutation with BsmFI showed biallelic expression of PTPN11
(Supplementary Fig. 3b
). PCR and Southern blots indicated the presence of all four transgene proviruses in the LS-iPSC (Supplementary Fig. 4
) and quantitative RT-PCR (qRT-PCR) results confirmed efficient transgene silencing (Supplementary Fig. 5
To further characterize the LS-iPSC clones, expression of several HESC markers in two LS-iPSC lines from each patient (L1-iPS1, L1-iPS13, L2-iPS6 and L2-iPS16) was analyzed and compared to the HES2 HESC and a wt-iPSC line, BJ-iPSB5, derived in our lab from a normal human fibroblast line (BJ). The BJ-iPSB5 cell line was also karyotypically normal (46,XY), contained all four transgene proviruses, which were silenced (Supplementary Fig. 2b, 4b and 5b
). All LS and control iPSC lines exhibited high alkaline phosphatase activity, and expressed pluripotency markers, including surface antigens TRA-1-81, TRA-1-60, and SSEA-4, as well as the nuclear transcription factors OCT4 and NANOG (Supplementary Fig. 6
). Activation of a series of endogenous stemness genes (OCT4
) in iPSC was confirmed by qRT-PCR ( and Supplementary Fig. 7a
). Extensive demethylation of CpG dinucleotides in the OCT4
promoters compared to their parental fibroblasts was confirmed by bisufite sequencing ().
Gene expression profile in LS-iPSC is similar to HESC
We next examined genome-wide mRNA expression profiles of two LS-iPSC lines from each patient, the BJ-iPSB5 cell line, parental fibroblasts and HES2 cells. The resulting heat map and scatter-plot analyses indicated that iPSC lines shared a higher degree of similarity with HES2 cells than with their parental fibroblast cell lines ( and Supplementary Fig. 7b
Pluripotent HESC can differentiate into cell types representative of all three germ layers. We tested the differentiation abilities of our iPSC using an in vitro
floating embryoid body (EB) system, followed by replating on gelatin-coated dishes10,15
. Immunocytochemistry analyses detected expression of α-smooth muscle actin (α-SMA, mesoderm), desmin (mesoderm), α-fetoprotein (AFP, endoderm), vimentin (mesoderm), glial fibrillary acidic protein (GFAP, ectoderm) and βIII-tubulin (ectoderm) markers ( and Supplementary Fig. 8
). In order to determine pluripotency in vivo
, we injected LS-iPS, BJ-iPSB5 and HES2 cells into immune-compromised NOD-SCID mice. Histological analyses of the resulting teratomas showed cell types representative of the three germ layers, including pigmented cells (ectoderm), lung, respiratory and gut-like epithelia (endoderm), and mesenchyme, adipose tissue and cartilage (mesoderm) ( and data not shown).
LS-iPSC differentiate in vitro and in vivo into all three germ layers
As mentioned previously, hypertrophic cardiomyopathy is one of the major features of LS, affecting 80% of the patients. In addition, affected individuals occasionally manifest hematologic complications such as myelodysplasia and leukemia16,17
. Therefore, we asked if LS-iPSC were able to differentiate into hematopoietic and cardiac lineages. LS-iPSC from both patients differentiated into a variety of hematopoietic cell types including early hematopoietic progenitors (CD41+
, early erythroblasts (CD71+
, and macrophages (CD11b+
(Supplementary Fig. 9
and data not shown). The cardiac hypertrophic response includes induction of immediate-early genes (such as c-jun, c-fos and c-myc), an increase in cell size, and organization of contractile proteins into sarcomeric units21,22
. To have an appropriate control cell line to analyze some of these parameters, besides HESC, we generated a wt-iPSC line (S3-iPS4) from fibroblasts obtained from an unaffected brother of L1 without the T468M mutation (Supplementary Fig. 10
and Supplementary Fig. 11
). Using a well-established cardiac differentiation protocol23
, we observed contracting EBs emerging around day 11 of differentiation (Supplementary Movies 1–7
). In order to monitor cardiac development, we analyzed cardiac troponin T (cTNT) expression on day 18 of differentiation by flow cytometry (data not shown). Replated cells from beating EBs were processed as described in Material and Methods. Briefly, cells were fixed, immunostained for cTNT (), and 50 cardiomyocytes were randomly chosen from each sample for surface area measurement using a computerized morphometric system (ImageJ software, NIH). Cardiomyocytes derived from LS-iPSC lines: L1-iPS13, L1-iPS6 and L2-iPS10 (Supplementary Fig. 10
and Supplementary Fig. 11
), had a significantly increased median surface area compared to wt-iPSC cardiomyocytes; 1.8 times, 2.5 times and 4.8 times larger, respectively, whereas the area median of the cardiomyocytes obtained from HESC was similar to wt-iPSC cardiomyocytes (). We also observed increased sarcomere assembly in L1-iPS6 and L2-iPS10 cells when compared to wt S3-iPS4 cells (). Recently, the calcineurin-NFAT pathway has been shown to be an important regulator of cardiac hypertrophy. Active calcineurin dephosphorylates NFAT transcription factors, resulting in their nuclear translocation22,24
. We analyzed the localization of NFATc4 using immunocytochemistry in 50 cTNT-positive cardiomyoctes derived from the L2-iPS10 cell line, which produced the largest cardiomyocytes, and wt S3-iPS4. We observed a significantly higher proportion of LS cardiomyocytes with nuclear NFATc4, (~80% versus ~30%, respectively) ().
Cardiomyocytes derived from LS-iPSC show hypertrophic features
In order to identify potential molecular targets that could be affected by the T468M PTPN11 mutation, protein extracts from LS-iPS, wt BJ-iPSB5 and HES2 cells were analyzed using a phosphoproteomic microarray chip containing approximately 600 pan and phospho-specific antibodies (Kinexus Bioinformatics Corporation). We established eight groups for comparison, each of the LS-iPSC lines versus one control cell line, either HES2 or wt-iPSC. Proteins with a 1.5 fold change were filtered, and those that were conserved in most of the groups were represented in a heat map (). Some of the proteins were more abundantly present in LS-iPS when compared to either HES2 (Tyro10, Tyk2 and Haspin) or wt-iPSC (p-MARCKs, p-Synapsin1, p-NMDAR2B, p-MSK1, p-RSK1/3 and p-p53). The phosphorylation of other proteins was increased (p-Caveolin2, p-MEK1, p-EGFR and p-FAK) or decreased (p-Vinculin, p-S6 and p-Lck) in LS-iPSC when compared to control cell lines. In order to eliminate false positives, we verified the phosphoproteomic results by Western blot (WB) for three of the most altered proteins (p-S6, p-EGFR and p-MEK1) in four LS-iPSC lines, in comparison to wt-iPSC. While we did not confirm a major change in the phosphorylation status of S6 protein (data not shown), WB confirmed that the phosphorylation of EGFR and MEK1 proteins was considerably increased in the LS-iPSC samples ().
Phosphoproteomic and MAPK activation analyses
RAS-MAPK represents the major signaling pathway deregulated by SHP2 mutants. NS mutants increase basal and stimulated phosphatase activity, whereas LS mutants are catalytically impaired and have dominant-negative effects, inhibiting growth factor-evoked ERK1/2 activation25
. We analyzed the ability of LS-iPSC to respond to external growth factors. We used bFGF (basic Fibroblast Growth Factor), the main growth factor in the maintenance of HESC, to induce the stimulation of the MAPK signaling pathway. bFGF treatment increased the phosphorylation of ERK1/2 (p-ERK) levels over time in HES2 and wt S3-iPS4 cells (). Although the LS-iPSC expressed the four FGF receptor (FGFR) family members (Supplementary Fig. 12a
), bFGF stimulation did not cause any substantial change in p-ERK levels ( and Supplementary Fig. 12b-c
). However, the LS-iPSC lines had higher basal p-ERK levels compared to HES2 and S3-iPS4 cells (Supplementary Fig. 12b-c
), in accordance with the increased pMEK1, ERK upstream kinase, levels found in LS-iPSC samples in phosphoproteomic array results.
In summary, we have generated and characterized LS patient-specific iPSC, providing a new system for the study of disease pathogenesis. Some of the standard procedures to analyze cardiomyocyte hypertrophy (e.g., protein synthesis rate, re-activation of the fetal gene program) could not be reliably assessed due to the variably mixed population of cells obtained using this cardiac differentiation procedure (e.g. endothelial, cardiomyocytes), and the lack of a reliable cell surface marker for cardyomyocyte purification. However, we observed increases in cell size, sarcomeric organization and nuclear NFATc4 localization in LS-iPSC-derived cardiomyocytes, when compared to HESC and wt-iPSC-derived cardiomyocytes. These results would be consistent with cardiac hypertrophy, a condition commonly found in LS patients2
, and suggest that this abnormality occurs through a cell autonomous mechanism due to the PTPN11
mutation. Since many human cell types, such as cardiomyocytes, cannot be propagated readily in cell culture, iPS-derived cells exhibiting disease-relevant phenotypes provide the requisite resource for precisely elucidating pathogenesis and pursuing novel therapeutic strategies.
In our studies, we also attempted to provide insights into the molecular events that could be affected by the PTPN11
mutation in the pluripotent iPSC using antibody microarrays. We found that the phosphorylation of certain proteins was increased in LS-iPSC when compared to wild-type HESC and iPSC. Further analysis will be required to elucidate if these proteins/signaling pathways are involved in the development of the disease phenotype. Interestingly, one of the more upregulated phosphoproteins was MEK1, the upstream kinase of ERK1/2, whose gene is sometimes mutated in the related disorder, cardiofaciocutaneous syndrome. PTPN11
mutations underlie 45% and 90% of NS and LS, respectively. It is not well understood how mutations that provoke opposite effects on SHP2 phosphatase activity cause syndromes with similar features26
. In concordance with observations in the Drosophila
, basal p-ERK levels were increased in LS-iPSC. Of note, receptor tyrosine kinase stimulation with bFGF in LS-iPSC failed to elicit further activation of ERK, as previously observed in a different cellular model25
. Interestingly, this result demonstrates for the first time that RAS-MAPK signal transduction is perturbed in LS as early as the pluripotent stem cell stage.
Taken together, this is the first described human model of an inherited RAS pathway disorder.