Creation of disease model systems to understand and correct genetic diseases through gene or other therapy using iPS cells derived from somatic cells: *the iPS study*
Addendum A: "Informatie voor proefpersonen en bijbehorend toestemmingsformulier voor de vervaardiging en het gebruik van geïnduceerde Pluripotente Stamcellen (iPS cellen) voor patiënten met een cardiovasculaire aandoening."

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 fertilization
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 pre-implantation 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: mouse heart beats at 500-600 times per
minute whilst humans only 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. Control iPSC lines have already been generated
using annonymized waste tissue from the Dept of Dermatology and have been
differentiated into various cell types including cardiomyocytes, vascular
endothelial cells, blood cells, neurons, and smooth muscle cells. Control lines
have also been marked transgenically with lineage reporters. They are shared
with external researchers according to LUMC guidelines for collaboration using
donated annoymized tissue.

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 annonymized by
key-coding. Skin will be obtained by 4 mm punch biopsies; for isolation of
blood cells maximally 80 ml of peripheral blood will be collected. Fat tissue,
oral mucosa and heart muscle tissue can only be obtained in the context of
necessary surgical procedures. For children only non-invasive procedures
(urine, milk teeth) or minimally invasive procedures (peripheral blood with a
maximum volume of 40 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 characterization 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 characterized 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, as
already carried out for healthy control individuals. 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 LUMC 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 LUMC iPSC core facility (and somatic cells if
requested) will be given to the principal investigator who originally requested
the generation iPSCs by the LUMC iPSC core facility.

Frozen backup stocks of somatic cells and hiPSCs will be kept in dedicated
liquid nitrogen tanks of the Department of Anatomy and Embryology (S-05-11,
liquid nitrogen tank 11, T37P2-262S05). All lines derived from LUMC patients
are property of the LUMC and will only be distributed to third parties in
agreement with the principal investigator using a standard LUMC Material
Transfer Agreement (copy attached).

Key-coded (annonymized) patient-specific information will be stored in the
database of the LUMC iPSC core facility, which is only accessible to the staff
of the facility. For LUMC patients, the code will be kept in the accredited
central database of the LUMC in which all patient information is stored. The
database is behind the LUMC firewall and is not accessible from outside the
hospital network. For all other (non-LUMC) patients, their physicians/hospitals
will be responsible for key-coding. 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.