Myogenic differentiation of VCP disease-induced pluripotent stem cells: A novel platform for drug discovery

Abstract

Valosin Containing Protein (VCP) disease is an autosomal dominant multisystem proteinopathy caused by mutations in the VCP gene, and is primarily associated with progressive muscle weakness, including atrophy of the pelvic and shoulder girdle muscles. Currently, no treatments are available and cardiac and respiratory failures can lead to mortality at an early age. VCP is an AAA ATPase multifunction complex protein and mutations in the VCP gene resulting in disrupted autophagic clearance. Due to the rarity of the disease, the myopathic nature of the disorder, ethical and practical considerations, VCP disease muscle biopsies are difficult to obtain. Thus, disease-specific human induced pluripotent stem cells (hiPSCs) now provide a valuable resource for the research owing to their renewable and pluripotent nature. In the present study, we report the differentiation and characterization of a VCP disease-specific hiPSCs into precursors expressing myogenic markers including desmin, myogenic factor 5 (MYF5), myosin and heavy chain 2 (MYH2). VCP disease phenotype is characterized by high expression of TAR DNA Binding Protein-43 (TDP-43), ubiquitin (Ub), Light Chain 3-I/II protein (LC3-I/II), and p62/SQSTM1 (p62) protein indicating disruption of the autophagy cascade. Treatment of hiPSC precursors with autophagy stimulators Rapamycin, Perifosine, or AT101 showed reduction in VCP pathology markers TDP-43, LC3-I/II and p62/SQSTM1. Conversely, autophagy inhibitors chloroquine had no beneficial effect, and Spautin-1 or MHY1485 had modest effects. Our results illustrate that hiPSC technology provide a useful platform for a rapid drug discovery and hence constitutes a bridge between clinical and bench research in VCP and related diseases.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Differentiation of patient and control VCP iPSC into myogenic lineages.

(A) Schematic of early hiPSCs and embryoid bodies commitment (Days 0–50) into early mesenchymal stem/stromal cells (MSC) and differentiation into myoblasts (Day 64). (B) Myogenic differentiation of human iPSC at Days 0, 21, 50, and 69 with (C) primary myoblast cells from a 57-year old patient diagnosed with IBMPFD. (D) Control derived myogenic precursors at Days 0, 21, 50, and 69 with (E) primary myoblast cells from age matched healthy control. Differential interference contrast (DIC) microscopy images of differentiated primary mature myoblasts from iPSCs. Scale: Bar = 1000 μm.

Fig 2. Validation of myogenic differentiation in patient and control-derived iPSCs.

Human iPSC from a 57-year old patient diagnosed with VCP disease-derived myogenic precursors were stained at Day 21 and Day 50 with (A-B) MYF-5, desmin, and Pax7; (C-D) MyoD and MYH2. Representative merged overlay images of stained iPSC with DAPI. Scale: Bar = 50 μM. (E) Western blot analysis of myoblast differentiation markers at Day 0, 7, 21, 35, and 50 with anti-Oct3/4, Nanog, desmin, MYF5, MyoD, MyoG and MYH2. GAPDH was used as a positive loading control. (F) FACS analysis of iPSC-derived MSCs with pluripotent marker (CD34) and myoblast marker (CD56). (G) CD34 isotype control. (H) CD65 isotype control.

Fig 3. Characterization of autophagy signaling cascade in control and patient VCP iPSC-derived myoblast lineages.

Differentiated (A) control and (B) iPSC-derived myoblast lineages were immunostained with TDP-43, LC3, and p62/SQSTM1. Representative merged overlay images of stained iPSC with DAPI. Scale: Bar = 50 μM. (C) Western blot analysis of iPSC-derived control and patient myoblasts with anti-TDP-43, LC3I/II, p62/SQSTM1, and ubiquitin. GAPDH was used as a positive loading control. (D) Densitometry analyses confirmed these Western blot results. Statistical significance is denoted by *p<0.05, **p<0.005 and ***p<0.001. (E) Western blot analysis of cytoplasmic (Cy) and nuclear (Nu) factions of iPSC-derived control and patient myoblasts with anti-TDP-43.

Fig 4. Drug screening with autophagy inducers Rapamycin, Perifosine and AT101 in patient VCP iPSC-derived myogenic lineages.

(A) Schematic of intervention with autophagy modifying agents. Green arrows show the active location of autophagy activators Rapamycin, Perifosine and AT101. Red arrows show the active location of autophagy inhibitors chloroquine, Spautin-1 and MHY1485. VCP is indicated to have an interactive role with Akt and as a chaperone protein with ubiquitin. (B) Untreated differentiated control and (C) untreated patient derived myogenic lineages. Patient derived myogenic lineage were treated with either (D) Rapamycin (10 μM), (E) Perifosine (80 μM) or (F) AT101 (10 μM) for 24 hours. Subsequently, cells were stained with TDP-43, LC3 or p62/SQSTM1 antibodies. Representative merged overlay images of stained iPSC with DAPI. Scale: Bar = 25 μm. White dotted lines represent areas of increased or decreased expressions. (G) Western blot analysis of iPSC-derived control (C) and patient (P) myoblasts with mTOR, TDP-43, LC3I/II and p62/SQSTM1. GAPDH was used as a positive loading control. (H) Densitometry analyses of the Western blot. Black dotted line indicates expression over baseline control sample. Statistical significance is denoted by *p<0.05, **p<0.005 and ***p<0.001.

Fig 5. Drug screening with autophagy inhibitors chloroquine, Spautin-1, and MHY1485 shows in patient VCP iPSC-derived myoblast lineages.

(A) Untreated differentiated control and (B) untreated patient derived myogenic lineages. Patient derived myogenic lineage were treated with either (C) chloroquine (10 μM), (D) Spautin-1 (10 μM) or (E) MYH1485 (2 μM) for 24 hours. Subsequently, cells were stained with TDP-43, LC3 or p62/SQSTM1 antibodies. Representative merged overlay images of stained iPSC with DAPI. Scale: Bar = 25 μm. White dotted lines represent areas of increased or decreased expressions. (F) Western blot analysis of iPSC-derived control and patient myoblasts probed against TDP-43, LC3-I/II, and p62/SQSTM1 antibodies. GAPDH was used as a positive loading control. (G) Densitometry analyses from Western blot. Black dotted line indicates expression over baseline control sample.