2nd iPSC Consortia Executive Summary

2^nd IPSC Consortia Workshop

Hyatt Regency, Bethesda, Maryland
December 15, 2010
Executive Summary
Workshop   Agenda   Participant List

Participants: 

The invited participants included principal investigators and post doctoral fellows from the three NINDS sponsored RC2 iPSC consortia, representatives from industry, non-government organizations, Coriell repository staff, NINDS program staff and other NIH IC program representatives.

Introduction: 

In 2009, through the American Recovery and Reinvestment Act (ARRA), NINDS funded three consortia, to develop well-characterized, publically available, induced pluripotent stem cell (iPSC) lines for familial forms of Parkinson’s Disease (PD), Huntington’s Disease (HD) and Amyotrophic Lateral Sclerosis (ALS).  The first iPSC consortia workshop was held in February of 2010, and during the last 10 months, over 87 fibroblast lines (12–ALS, 18-HD, 29-PD, 1-GBA, 3-FTD, 24-population or unaffected family controls) and 25 iPSC lines have been developed and will be publically available through the NINDS repository at Coriell. Information on fibroblasts currently available for academic and industry research can be found at: http://ccr.coriell.org/Sections/Collections/NINDS/Fibro.aspx?SsId=10&coll=ND.   

The goals of the 2^nd iPSC consortia workshop were to: 1) share progress; 2) methodologies; and 3) phenotypic analyses being utilized; 4) develop a standard report card for the iPSC lines; and 5) identify and resolve any remaining roadblocks that would delay successful completion of the proposed research in the accelerated two year time frame. The workshop included an industry perspective and highlighted collaborations and contributions from non-government organizations (NGOs) in support of consortia effort.

Amyotrophic Lateral Sclerosis iPSC Consortium

The Amyotrophic Lateral Sclerosis (ALS) consortium is directed by Dr. Jeffrey Rothstein (Johns Hopkins University) and includes four principal investigators, working in tight collaboration, to generate and evaluate familial ALS (fALS) iPSC lines. The principal investigators include Drs. Kevin Eggan (Harvard University), Chris Henderson (Columbia University), Merit Cudkowicz (Massachusetts General Hospital) and Tom Maniatis (Harvard University).  To generate iPSC lines, this consortium subcontracted with iPierian, a San Francisco-based biopharmaceutical company, to generate iPSC lines from ALS patient fibroblasts using retroviral delivery of Klf4, Sox2, Oct4 and c-Myc (OKSM)⁽¹⁾. Putative iPSC lines were characterized for stability and pluripotency by: 1) morphological analysis; 2) antigen expression analysis; 3) gene expression analysis; 4) embryoid body analysis; 5) viral vector silencing; 6) epigenetic analysis of selected loci; 7) karyotype analysis; and 8) teratoma formation.  In an independent effort, Dr. Eggan’s laboratory has developed a lineage assay to determine the propensity of individual lines to differentiate along various cell lineages.  Fibroblasts and iPSC lines available through the ALS consortium efforts are listed in Table 1.  Clinical information for each fibroblast and iPSC line includes demographics, subject ALS history (date of onset), family ALS history (gene and mutation), ALS diagnosis (Escorial Criteria, site of onset, date of diagnosis, clinical findings, EMG findings), concomitant medications, medical history, and smoking history.  Additional fibroblast lines available for future iPSC development include biopsies from sporadic ALS patients with typical progression (<5 years), sporadic ALS patients with slow progression (> 5 years), hereditary spastic paraplegia patients, primary lateral sclerosis patients, and patients with non familial ALS/FTD.  The consortium is currently using the iPSC lines to determine molecular, biochemical and electrophysioloigcal phenotypes associated with the various mutations in both iPSC-derived motor neuron and astrocyte cultures.

Table 1: Fibroblast and iPSC lines developed by the ALS Consortium

Clone ID	Mutation	Method	Fibroblast	iPSC line
001 HS	SOD1 N139K	OKSM	√	√
002 LWM	SOD1 A4V	OKSM	√	√
003TMS	SOD1 I112T	OKSM	√	√
004 MJF	SOD1 D90A	OKSM	√	√
005 D-M	Population control	OKSM	√	√
006 GDP	Population control	OKSM	√	√
007 MBN	Presym/SOD1 A4V	OKSM	√	√
008 T-M	Presym/SOD1 A4V	OKSM	√	√
009 DCM	V148G	OKSM	√	√
010 LBB	Population control	OKSM	√	√
013 M-P	SOD1 A4V	OKSM	√	√
014 M-R	FIG 4	OKSM	√	√
015 RGO	SOD1 L144P	OKSM	√	√
016 DTM	SOD1 I1113T	OKSM	√	√
017 DMN	SOD1 D91A	OKSM	√	√
018 RRN	Population control	OKSM	√	√
021 JZL	SOD1 C38G	OKSM	√	√
023 JFV	FUS	OKSM	√	√
024 TLC	SOD1 E49K	OKSM	√	√
025 AGB	FUS	OKSM	√	√
026 JAP	SOD1 E100G	OKSM	√	√
027 CDG	ANG	OKSM	√	√
028 NCB	SOD1 D90A	OKSM	√	√
031 FVW	FUS	OKSM	√	√
033 MJM	Presym/SOD1G86R	OKSM	√	√

 

Huntington’s Disease iPSC Consortium

The Huntington’s Disease iPSC consortium is lead by Dr. Leslie Thompson from UC Irvine.  Members of the consortium include Drs. Steven Finkbeiner (J.D. Gladstone Institute), Jim Gusella (Massachusetts General Hospital), Clive Svendsen (Cedar-Sinai Medical Center), Chris Ross (Johns Hopkins University), Hongjun Song (Johns Hopkins University), Vanessa Wheeler (Massachusetts General Hospital) and Marcy MacDonald (Massachusetts General Hospital), Nick Allen (University of Cardiff), Elena Cattaneo (University of Milan), Marco Onorati (University of Milan), Paul Kemp (University of Cardiff), and Kwang-Soo Kim (McLean Hospital).  The HD iPSC consortia has created a series of iPSC lines from both control and HD patient fibroblasts through lentiviral transduction of Oct4, Sox2, Nanog , Lin28, c-Myc and Klf4 (OKSMLN).  The fibroblast and iPSC lines have HTT exon 1 CAG repeat lengths ranging from 20 to 180 triplet repeats. The HD mutation does not affect reprogramming efficiency and all iPSC lines express the full range of pluripotency markers and can be expanded indefinitely in culture. Using a novel protocol, primitive multipotent neural stem cells (EZ spheres) were generated from a subset of iPSC lines (180 CAG repeats, 66 CAG repeats and 33 CAG repeats) to enable easy expansion and distribution of reprogrammed lines to members of the consortium. Upon differentiation in the presence of specific morphogens, EZ spheres consistently gave rise to forebrain neurons, some of which expressed striatal-specific markers. iPSC and EZ sphere lines were assessed for stability and pluripotency by: 1) karyotype analysis; 2) expression of pluripotency markers; 3) silencing of reprogramming transgenes; 4) embryoid body analysis; 5) CAG repeat stability; and 6) teratoma formation. Using a battery of different methods the consortium has demonstrated CAG repeat dependent differences in (i) dentritic outgrowth, (ii) electrophysiology, (iii) vulnerability to neurotoxic insult and iv) gene expression.  These observations are currently being replicated among the consortium investigators using both the EZ spheres and iPSC lines.  Table 2 outlines the fibroblasts and iPSC/EZ spheres developed by HD consortium members.  Additional iPSC and EZ sphere lines are currently under development using both the lentiviral-driven OKSMLN method⁽²⁾, as well as an episomal non-integrating methodology for IPSC derivation⁽³⁾. Additional fibroblast lines developed by the consortium are available through the NINDS Repository at Coriell and are listed in Table 4.  Clinical data for the fibroblast and iPSC lines includes: demographics, medical history (motor, cognitive, psychiatric, oculomotor), family history, subject status, UHDRS total motor score, UHDRS total behavior score, and UHDRS total functional capacity score.   Methodologies including iPSC monolayer culturing techniques, improved differentiation protocols for medium spiny neurons, chip (PCR-based) technology for cell type assessment, and establishment of unbiased high content phenotypic assays are currently being developed by consortium members.

Table 2: Fibroblast and iPSC lines developed by the HD Consortium

Clone ID	CAG repeat length	Method	Fibroblast	iPSC
MAA783	21		v
MAA773	22		v
GM02183	33	OKSMLN iPSC and EZ sphere	v	v
MAA753	42		v
MAA787	44		v
LMT001	50		v
LMT005	60		v
GM03621	60		v
LMT003	66	OKSMLN iPSC and EZ sphere	v	v
DLS	77		v
OMR	109		v
GM09197	180	OKSMLN iPSC and EZ sphere	v	v

 

Parkinson’s Disease iPSC Consortium

The PD iPSC consortium is lead by Dr. Ole Isacson (McLean Hospital).  Members of the consortium include Drs. Jim Surmeier (Northwestern University), Ted Dawson (Johns Hopkins University), Jian Feng (University of Buffalo), Karen Marder (Columbia University), Serge Przedborski (Columbia University), Zbigniew Wszolek (Mayo Clinic Jacksonville), Owen Ross (Mayo Clinic Jacksonville), Virginia Lee (University of Pennsylvania), Dimtri Krainc (MGH, Boston), and John Trojanowski (University of Pennsylvania).  The PD iPSC consortium has generated 12 genetic PD iPSC lines from 4 genotypes (PINK1, LRRK2 G2019S, LRRK2 R1441C, and SNCA triplication) and 7 control iPSC lines (2 normal, 5 PD idiopathic). Fibroblasts were transformed with either retroviral vectors expressing Oct4, Klf4 and Sox2 or Oct4, Klf4, Sox2 and c-Myc in the presence or absence of valproic acid (VPA). The stability and pluripotency of the iPSC lines was verified by: 1) karyotype analysis; 2) immunocytochemistry for pluripotency markers; 3) transgene silencing; 4) in vitro germ layer analysis: 5) teratoma formation, and 6) FOXA2 immuno-positive (FOX2A⁺), dopamine (DA) neuronal differentiation.  Clinical data available for the fibroblasts and iPSC lines includes demographics, clinical diagnosis (presence or absence of bradykinesis, activation or resting tremor, postural instability, rigidity, and gait disturbances), family history of PD, responsiveness to anti-Parkinsonism therapy, genotype data, smoking history, UPDRS total motor score, Hoehn and Yahr score, mini-mental status score and family medical history.  Phenotypic analysis of the mutant iPSC lines demonstrates differential sensitivity to neurotoxic insult and cellular biology relevant to the mutation analyzed.  The PD consortium has refined differentiation assays to demonstrate that SAG can substitute for recombinant SHH-C2411 during differentiation of FOX2A⁺ human neuronal progenitor cells.  The consortium members are currently validating observed phenotypes across laboratories and continuing to develop new iPSC lines for analysis.

Table 3: Fibroblast and iPSC lines developed by the PD Consortium

Clone ID	Mutation	Method	Fibroblast	iPSC
PD1 -9.1	SNCA triplication	OKS	√	√
PD1-9.2	SNCA triplication	OKS	Same as above	√
PD1-A	SNCA triplication	OKSM/VPA	Same as above	√
PD2 -D	LRRK2 R1441C	OKSM/VPA	√	√
PD2-E	LRRK2 R1441C	OKSM/VPA	Same as above	√
PD2-F	LRRK2 R1441C	OKSM/VPA	Same as above	√
PD3-A	LRRK2 R1441C	OKSM/VPA	√	√
PD3-C	LRRK2 R1441C	OKSM/VPA	Same as above	√
PD3-D	LRRK2 R1441C	OKSM/VPA	Same as above	√
PD9-A	LRRK2 G2019S (homo)	OKSM/VPA	√	√
PD21 L2122	PINK1 c.1366C>T	OKSM	√	√
PD22 L2124	PINK1 c.1366C>T	OKSM	√	√
HS7 L2131	Unaffected Family control	OKSM	√	√
HS8 L2135	Unaffected Family control	OKSM	√	√

 

Workshop Summary: 

In 2006, the generation of induced pluripotent stem cells (iPSCs) from somatic cells through retroviral-driven expression of four embryonic transcription factors (c-Myc,Oct4, Sox2 and Klf4)(4) initiated a revolution in resource development for therapeutic discovery. Development of iPSC lines from fibroblasts of patients with early onset neurological disorders such as, Spinal muscular atrophy(5), Friedreich’s ataxia(6), Rett syndrome(7), Fragile X syndrome(8), and Angelman syndrome(9) demonstrated that the genetic defects carried by these cells could model certain aspects of the disease phenotype.  In 2009, through the American Recovery and Reinvestment Act (ARRA), NINDS funded three consortia, to develop well-characterized publically available, induced pluripotent stem cell (iPSC) lines for familial forms of Parkinson’s Disease, Huntington’s Disease, and Amyotrophic Lateral Sclerosis.  This consortium approach enabled rapid resource development and the initial identification of cellular phenotypes associated with late-onset neurodegenerative disease in iSPC-derived neuronal cultures.  To facilitate the use of these resources for translational research, each consortium was asked to identify research challenges where partnerships with industry could help facilitate rapid progress towards utilization of iPSC lines for therapeutic development.

Challenges and areas for development identified by iPSC consortia members included:

1. Development of methodologies and reagents that would enable cost-effective large-scale growth of iPSCs and differentiated cell types to enable distribution, standardization and quality assurance of reagents being used in phenotypic analyses across laboratories.
2. Development of methodology and standardization for storage of differentiated cells.
3. Improvement of differentiation protocols to increase efficiency of specific cell-type derivation and minimization of the length of time in culture.
4. Development of Chip-based assays for standardization and quality assurance of cell derivation approaches across laboratories and platforms.
5. Development of  reference compound libraries for testing specific cellular pathways
6. Identification of criteria for the development of cell based platforms that meet requirements of industry for target validation and secondary screening.
7. Validation of phenotypes through genetic or pharmacological rescue and across laboratories and platforms
8. Expansion of the number of available iPSC lines for ALS, HD and PD research.

Industry Perspective:

To facilitate discussions with industry leaders and consortium members, part of the meeting agenda was devoted to presentations and discussion focused on the industry perspective for the utility of iPSC lines in the drug discovery process and what challenges exist in utilizing this technology for the development of therapeutics for neurodegenerative diseases. Representatives from Lundbeck, GSK and Pfizer gave presentations.

Several of the key issues raised by the industry representatives included:

1. Current  hurdles to drug discovery in neuroscience:
   1. Poor understanding of disease biology
   2. Paucity of tractable CNS targets
   3. Poor predictable value of animal models
   4. Difficulty in delivering drugs across the BBB
   5. Lengthy and Large clinical trials
2. iPSC Challenges in Drug Screening
   1. Lack of available iPSC lines from patients for industrial “research” use
   2. Low proportion of neuronal cells in the ”differentiated” iPSC culture
   3. Lack of disease phenotype in neurons differentiated from iPSC lines
   4. Difficulty in adaption to HTS assay requirements which include: consistency in differentiation; percentage of neurons; and need for large quantity of cultures
   5. Ability to genetically manipulate iPSCs for target identification
3. Potential iPSC utility in drug discovery process
   1. Representation of human in vitro model
   2. Use of non-neuronal cells for safety studies
   3. Target identification through use of disease-relevant human cell population and identification of disease-relevant readout
   4. For early stage discovery projects advantages include: 1) de-convolution of multiple disease etiologies,; 2) development  of assays for evaluating structure-activity relationships of compounds where the target is the protein encoded by the genetic variant; 3) development of in vitro mechanistic or disease-relevant assay to establish effect of compounds in cells
   5. For late stage discovery projects advantages include: 1) potential biomarker identification; 2) stratification marker identification; 3) broadening of therapeutic area
4. Questions that could be addressed using iPSC technology
   1. What are the cellular phenotypes of neurons and astrocytes differentiated from iPSC lines from patients with neurodegenerative diseases?
   2. Is there a common signature in signaling pathways, differentiation capacity or survival?
   3. Are there differential effects of mutations on neurons derived from idiopathic versus familial forms of disease?
   4. In developing a panel of cells, what criteria should be applied and how many different lines are needed to account for variability?

Foundation Perspective:

Since commencement of the iPSC consortia efforts in 2009, non-government organizations (NGOs) have played a vital role in enabling the rapid progress demonstrated by all consortia to date.  CHDI, The Michael J. Fox Foundation, and the Amyotrophic Lateral Sclerosis Association have contributed funding to support collaborative efforts among researchers from the USA and Europe and consortia investigators.  These collaborative efforts have contributed to new assay development and culture methodologies, expansion of available iPSC lines, and the development of reporter lines for monitoring of cell-type specific differentiation.  Representatives from the Amyotrophic Lateral Sclerosis Association, Association for Frontotemporal Dementias, CHDI, Foundation for the National Institutes of Health, the Hereditary Disease Foundation, Huntington’s Disease Society of America, Michael J. Fox Foundation for Parkinson’s Research, Parkinson’s Action Network, Parkinson’s Disease Foundation, Project ALS, and the Robert Packard Center for ALS Research attended the meeting. Representatives from the following NGOs (Amyotrophic Lateral Sclerosis Association, The Hereditary Disease Foundation, CHDI Foundation Inc, The Michael J. Fox Foundation for Parkinson’s Research and the Parkinson’s Disease Foundation) gave presentations focused on the potential use of iPSC lines for therapeutic development and the existing challenges that need to be addressed for use of these lines in drug development.

The NGOs identified the following opportunities and challenges for iPSC utilization in the drug discovery process:

1. Potential uses for iPSC lines would include:
   1. Humanized in vitro system for target validation and screening
   2. Evaluation of off-target effects in peripheral cell types
   3. Identification of new disease targets
   4. Development of drug screening assays
2. What is still needed to move iPSC technology forward for drug development application:
   1. Development of large-scale, fast, efficient and reproducible differentiation protocols
   2. Identification of disease-relevant phenotypes
   3. Development of reporter lines for monitoring cell differentiation
   4. Development of isogenic lines for assay development and target identification

Next Steps for the iPSC Consortia include:

1. Standardization of criteria for iPSC banking, providing Coriell with relevant information needed for banking, and activation of a plan to begin submission of iPSC lines to Coriell by late January. 
2. Lift embargoes on all fibroblast lines banked with the NINDS Repository at Coriell
3. Coordinate analysis of lineage potential for all GO grant iPSC lines submitted to Coriell using  lineage assay developed by  Dr. Kevin Eggan’s laboratory.
4. Create NINDS website with information on protocols, iPSC characterization, and a link to the NINDS Repository at Coriell for access to lines.
5. Include language in the NINDS Repository Materials Transfer Agreement form that defines DNA and cell lines assessed through the repository as non-human research.
6. Coordinate meeting with industry and foundations in February/March to develop “industry white paper” highlighting potentials and challenges for use of iPSC lines in drug development process.

References

1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007, 131:861–72.
2. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007, 318, 1917–1920.
3. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA.  Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009,  8;324(5928):797-801.
4. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006, 126: 663–676.
5. Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN. Induced pluripotent stem cells from a spinal muscular atrophy patient.Nature. 2009 Jan 15;457(7227):277-80
6. Ku S, Soragni E, Campau E, Thomas EA, Altun G, Laurent LC, Loring JF, Napierala M, Gottesfeld JM.Friedreich's ataxia induced pluripotent stem cells model intergenerational GAA⋅TTC triplet repeat instability. Cell Stem Cell. 2010 Nov 5;7(5):631-7.
7. Marchetto MC, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell. 2010 Nov 12;143(4):527-39.
8. Urbach A, Bar-Nur O, Daley GQ, Benvenisty N. Differential modeling of fragile X syndrome by human embryonic stem cells and induced pluripotent stem cells. Cell Stem Cell. 2010 May 7;6(5):407-11.
9. Chamberlain SJ, Chen PF, Ng KY, Bourgois-Rocha F, Lemtiri-Chlieh F, Levine ES, Lalande M. Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes. Proc Natl Acad Sci U S A. 2010 Oct 12;107(41):17668-73.

 

Table 4: Fibroblasts available through the NINDS repository at Coriell

Diagnosis	Genotype	Biopsies Submitted	Fibroblasts Submitted	QC completed, available at Coriell
ALS	ANG	1	0	1
ALS	FUS G-->A 1566	1	0	1
ALS	FUS T-->C 198	1	0	1
ALS	SOD1 D90A	2	0	1
ALS	SOD1 D91A	1	0	1
ALS	SOD1 E100G	1	0	1
ALS	SOD1 I113T	1	0	1
ALS	SOD1 L144P	1	0	1
ALS	SOD1 L38V	1	0	1
ALS	SOD1 A4V	1	0	1
ALS	TDP-43 G298S	0	1	In progress
Population control	N/A	11	0	11
PD	parkin: WT; A82E (parkin SNP), G2019S	1	0	1
PD	parkin: WT; G2019S	4	0	4
PD	parkin: compound het; 255delA, del exon 3-4	1	0	1
PD	parkin: WT; N370S	3	0	3
PD	parkin: heterozygous; Q34R	1	0	1
PD	parkin: heterozygous; R275W	3	0	3
PD	parkin: WT; L444P	1	0	1
PD	parkin: compoun het; R42P, del exon 3	1	0	1
PD	Parkin  Arg42Pro (het)	1	0	1
PD	GBA N370S	1	0	1
PD	Parkin 81G>T  (het)   this is still being reconfirmed	1	0	1
PD	LRRK2 G2019S	2	0	2
PD	Idiopathic	10	0	10
PD	Unaffected control for LRRK2:R1141C	0	2	In progress
PD	Unaffected control for MAPT:N279K	0	1	In progress
PD	Unaffected control for LRRK2: G2019S	0	1	In progress
PD	LRRK2:G2019S HOMOZYGOTE	0	3	In progress
PD	LRRK2 R1441G	0	2	In progress
PD	LRRK2 G2019S	0	5	In progress
PD	c2239C>T (pR747W) PLA2G6	0	1	In progress
PD at risk	LRRK2 R1141C	0	2	In progress
PD at risk	LRRK2 G2019S	0	1	In progress
Perry Syndrome	DCTN1: T72P	0	1	In progress
FTDP-17	MAPT:P301L	0	2	In progress
FTD	MAPT:V337M	0	1	In progress
FTD (at risk)	MAPT:N279K	0	1	In progress
FTD (at risk)	PGRN:C26C>A	0	1	In progress
HD (affected)	CAG (TBD)	11	0	11
HD (at risk)	CAG (TBD)	7	0	7