Creation of disease model systems to understand and correct genetic diseases through gene or other therapy using iPS cells derived from somatic cells.

Embryonic stem cells (ESC) are self-renewing and pluripotent, which means they
can grow indefinitely in culture and can be differentiated into every cell type
of the human body. However, they are derived from blastocyst stage human
embryos, in the Netherlands surplus to requirements for in vitro fertilisation
as governed by the EmbryoWet 2002. No genetically defective human embryonic
stem cells have yet been derived under the EmbryoWet in the Netherlands. The
EmbryoWet states that should there be alternatives to destruction of human
embryos for deriving pluripotent cells then these sources should be used by
preference. A new development in basic stem cell research has recently
suggested that there may now be a candidate alternative. These are induced
pluripotent stem (iPS) cells (1, 2]. iPSC can be derived from adult somatic
cells by overexpression of specific transcription factors with the help of a
variety of integrating and non integrating vectors. POU domain class 5
transcription factor1 (OCT4), SRY- box containing gene 2 (SOX2), proto -
oncogene c-MYC and Kruppel - like factor 4 (KLF4) were the four factors
initially described needed but there are now multiple variant cocktails which
all seem to lead to iPSCs from both mouse and human that closely resemble mouse
and human ESCs respectively. In addition hiPSCs have been generated from a
variety of somatic cell sources. Whilst not yet suitable for transplantation
and cell therapy purposes because of teratoma risk and multiple other reasons,
hiPSCs are very interesting for deriving pluripotent stem cells carrying
specific genetic mutations or other disease phenotypes of presently unknown
origin. If derived from somatic tissue from patients this would i) obviate the
need for using IVF embryos "rejected" following preimplantation genetic
diagnosis ii) broaden the range of diseases from which it is possible to derive
pluripotent cells in the Netherlands (PGD is not allowed for all genetic
diseases at present) and iii) does not require CCMO approval since iPSCs do not
fall under the EmbryoWet. Most importantly however, it would enable the
generation of many different diseased cell types from patients. In the case of
cardiac diseases, for example, it would be possible to use a tissue biopsy from
a patient to derive iPS cells and differentiate them into cardiomyocytes in
order to investigate at the cellular level what effects the genetic mutation
has on heart cells, without the need for a heart biopsy. Likewise for other
cell types that are difficult to access through biopsies (eg brain, liver,
muscle etc) or for diseases that are very rare and for which the cells cannot
be cultured for an indefinite period human iPSC will be an excellent source of
tissue for disease research [3]. While mutant mouse models exist for some of
these diseases, they often exhibit different phenotypes than humans because
physiology differs so significantly in humans and mice. Just as examples: a
mouse heart beats at 500-600 times per minute whilst a human heart only beats
at 60 times per minute; some mutations causing muscular dystrophy in humans do
not have orthologous regions of the same gene in mice. In addition, disease
genes may only cause a disease phenotype in a human genetic background.
Therefore models which use human cells are in most cases superior to understand
the disease pathogenesis and devise therapeutic interventions. The use of human
stem cells promotes compliance with the 3Rs for animal experiments (reduce,
refine, replace).

Primary Objective: to derive iPSCs from patients with a variety of diseases
caused by genetic mutations or genetic predisposition to disease or of unknown
origin. The cells will be propagated and differentiated into a variety of
somatic cell types using in vitro differentiation protocols and compared with
control iPS cell derivatives.

Secondary objectives: (i) to identify the most suitable tissue source for each
reprogramming method used and (ii) to establish a genotype related phenotype,
which will allow study of the mechanisms underlying the disease pathogenesis,
and analysis of therapeutic interventions by genetic or drug mediated repair of
the defect (iii) to develop therapies for disease phenotypes captured by the
patient hiPSC (iv) to couple phenotype with related clinical manifestations of
disease, drug responses and whole genome sequencing. This may be in
collaboration with external researchers.

Acquisition of donor material Donor material will be collected from various
tissue sources and anonymised by key-coding. Skin will be obtained by 4 mm
punch biopsies; for isolation of blood cells maximally 20 ml of peripheral
blood will be collected. For children only non-invasive procedures (urine, milk
teeth) or minimally invasive procedures (peripheral blood with a maximum volume
of 10 ml, keratinocytes from hair) apply. Other tissues can only be obtained in
the context of necessary surgical procedures. Where possible non-invasive
collection of somatic tissues will apply for all patients and controls. Cord
blood will serve as an additional source for reprogramming if available;
derivative iPSC are suitable for studying disease in neonates. Fibroblasts,
keratinocytes, cardiac progenitor cells (CPCs), endothelial blood outgrowth
cells, erythroblasts or other blood cell progenitor, renal epithelial cells or
mesenchymal stromal cells will be isolated according to the tissue source,
expanded and frozen stocks will be prepared. Which tissue source is chosen for
reprogramming depends on what is most conveniently available as part of routine
patient treatment. Secondary use (studies on cell types not related to the
primary disease of the patient, beyond the duration of the project,
implementing emerging and established research methods) is part of the consent
procedure. Generation of iPSC For the generation of iPS cells the transcription
factors, OCT4, SOX2, KLF4 and occasionally cMyc will be overexpressed with the
help of integrating or non-integrating DNA or RNA agents. As part of the basic
characterisation iPSCs will be induced to differentiate into derivatives of all
three primary germ layers to confirm pluripotency. After generation of the
tissue-specific cell types from the iPSCs, the differentiated cells will be
characterised using appropriate molecular, biochemical and functional assays,
aimed to precisely delineate the signature of the affected cells in the context
of the disease. This will take place in the iPSC core facility. Suitable
candidate cellular pathways will then be targeted by genetic and pharmacologic
methods in either the undifferentiated cells or in the differentiated
derivatives of interest aiming to repair the cellular defect. This will take
place in the department responsible for the investigator initiated research
after transfer of the iPSCs under key-code. We intend to generate multiple
(3-5) iPS cell lines from each patient. Multiple lines are needed since it is
unclear how much variability there will be in differentiation potential and
phenotype. This forms the baseline for determining how many patients with
different diseases will be included. At present, data is being published in
excellent peer reviewed journals with 2-3 patient iPS cell lines with similar
functional mutations. This will likely be sufficient for each particular
disease in first instance. The present capacity of the EMC iPSC core facility
is approximately 60 new lines per year, which corresponds to approximately the
same number of patients. Storage of data and material hiPSC lines generated by
the EMC iPSC core facility (and somatic cells if requested) will be given to
the principal investigator who originally requested the generation of iPSCs by
the EMC iPSC core facility. Frozen backup stocks of somatic cells and hiPSCs
will be kept in dedicated liquid nitrogen tanks of the Department of
Developmental Biology (lab nr. Ee909, liquid nitrogen tank 6 and labnr. Ee1029,
liquid nitrogen tank 4). All lines derived from EMC patients are property of
the EMC and will only be distributed to third parties in agreement with the
principal investigator using a standard EMC Material Transfer Agreement
(available on EMC website). Key-coded (anonymised) patient-specific information
will be stored in the database of the EMC iPSC core facility, which is only
accessible to the staff of the facility. For EMC patients, the code will be
kept in the accredited central database of the EMC in which all patient
information is stored. The database is behind the EMC firewall and is not
accessible from outside the hospital network. For all other (non-EMC) patients;
their physicians/hospitals will be responsible for keycoding. Somatic cells and
hiPSCs will only be used and accessible by researchers after removal of all
information allowing patient identification and coding. This conforms with the
Personal Data Protection Act (Wbp) of the Netherlands.

Insights into the mechanism of human disease facilitate the development of new
treatment modalities that either reduce the rate of development of even reverse
disease symptoms. hiPSC lines can be created from every individual that
captures both normal and disease genotypes. The collection of tissues or body
fluids to collect somatic cells is minimally or non-invasive. The benefits for
gaining insights into disease and creating new treatments outweigh the risk of
collecting the tissues and cells.