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The Consiglio Nazionale delle Ricerche (CNR, National Research Council) is
the main Public Research Agency funded by the Italian Government. It is made up of several
Institutes devoted to various fields. The Istituto di Tecnologie Biomediche (ITB) focuses on
biological themes which include human and mouse genetics. Historically, the Human Genome Section of
ITB has worked on genetics of human diseases, contributing to the identification of the genes
responsible for primary immunodeficiency such as X-linked thrombocytopenia, JAK3-dependent SCID,
Omenn syndrome, as well as osteopetrosis and X-linked Cornelia de Lange syndrome. A second line of
pursued research has been the field of models transgenesis, whose technology was especially applied
to the study of experimental models for human diseases, including cancer.
In September 2007, an Agreement between the ITB/CNR and the Istituto Clinico
Humanitas (ICH) came into effect and portions of the ITB have been transferred to the Humanitas
Research Centre in Rozzano. Two Research Units have been established there: the first, denominated “
Human Genome”, is led by Anna Villa, while the second, “Medical Biotechnologies” is coordinated by
Paolo Vezzoni. These two Units, whose researchers shared a common origin, while still closely
collaborating, started to diversify in the last couple of years and now have projects of their own.
Therefore, while some achievements in the past are common to both Units, the future projects tend
to investigate related but different topics.
MAIN RESULTS
In the last three years, while still working in collaboration with the other (Anna
Villa’s) group, this Unit has focused on stem cell therapy and regenerative medicine. We will
briefly review here the achievements of the last three years and will explain the further evolution
of the research.
1. Genome stability and related issue, including the discovery of SMC1 cohesin subunity as
the gene responsible for X-linked Cornelia de Lange syndrome.
Genome integrity is now recognized as fundamental in cell physiology and in cancer
prevention. Building on our work on the role of the cohesin complex and other
centromere-interacting proteins during mitosis, we decided to investigate the role of SMC1 (a
cohesin subunit) in patients affected by Cornelia de Lange syndrome (CdLS). The identification of
the involvement of this gene in this developmental defect has open up a new perspective on the role
of cohesin complex in transcriptional regulation.
2. Stem cells for early treatment of severe genetic defects already present at
birth.
Autosomal recessive osteopetrosis (ARO) is a severe genetic disease of the bone
which has been extensively studied by our group. Severe bone defects are already present at birth,
conditioning pancytopenia, blindness, deafness and, ultimately, death. The only available treatment
is BMT, which when performed postnatally usually does not rescue all the bone defects. ARO
therefore is the prototype of a number of genetic diseases which must be treated before birth in
order to prevent all the stigmata of the disease and really cure it. In addition to collaborating
with the other CNR/ICH Unit in the molecular basis of ARO, we have recently used the in utero stem
cell injection approach to show that both adult bone marrow and fetal liver cells can completely
rescue the phenotype and produce models indistinguishable from wild type ones.
3. Fusion in liver.
Investigation of adult stem cell potential for regenerative diseases have raised
the possibility of a great plasticity of tissue-specific adult stem cells such as neural or
hematopoietic stem cells. There is now a general consensus that this is not the case, and a set of
data have been interpreted to be the result of cell fusion. This seems to be especially true for
the liver, in which cell fusion with exogenous cells has been proposed to be of potential interest,
even leading to the cure of degenerative liver diseases. We tried to understand the cellular basis
of this phenomenon and set up a series of experiments to investigate whether cell fusion could
occur also in normal liver. Our data point to cell fusion as being a normal event in mouse liver.
This conclusion has been reached by work on chimeric models in which we were able to detect single
hepatocytes bearing markers of two different genotypes. In fact, single polyploid hepatocytes
containing both GFP and b-gal were detected in chimeric GFP/lacZ models; likewise, binucleated
hepatocytes with an XX chromosomal content in one nucleus and an XY pattern in the other were also
detected in XX/XY (female/male) chimeric models suggesting, again, a fusion event. We believe that,
if confirmed, these results are of both biological and medical interest, since they challenge the
old hypothesis which attribute liver polyploidy to endoduplication followed by aborted cytokinesis,
and raises the possibility of fusion between defective endogenous and exogenously provided normal
hepatocytes in degenerative liver diseases.
MAIN OBJECTIVES AND RESE
ARCH LINES
The Unit will further focus on regenerative medicine, trying to develop reagents
and methods for the preclinical treatment of bone and immune system diseases.
1. Development of iPS
The therapeutic application of stem cells depends on the availability of
self-renewing and pluripotent cells, the use of which is not limited by technical, ethical or
immunological considerations. An approach that satisfies all these stringent requirements has been
recently described as genetic reprogramming, a technology by which adult somatic cells can be
reprogrammed to pluripotent cells, coined iPS, by ectopically expressing a set of genes encoding
for transcription factors expressed in Embryonic Stem cells (ESCs). iPS cells are indistinguishable
from ESCs in gene expression profile, chromatin state and developmental potential. Application of
this approach to human cells would have enormous potential and could generate patient-specific
pluripotent stem cells to study and ameliorate human diseases. Steps towards this scenario have
already been accomplished by showing the feasibility of deriving human iPS cells that display
similar features to those of human ESCs from adult dermal fibroblasts and other tissues. However,
several limitations are still present in the efficiency and safety of the procedure that represent
major hurdles towards its future application in clinical settings. Among these hurdles are the
genotoxic effects of the retroviral insertions used to ectopically express the reprogramming genes,
while the oncogenic potential associated with constitutive expression of myc has been already
solved by producing iPS cells without this transgene. Several cell lines have been produced in
various laboratories. We plan to produce iPS cells from the
oc/oc mouse strain. We will use standard techniques for producing these cells in
collaboration with Luigi Naldini’s group at TIGET, since we do not plan to focus on improvement of
this step; since it is a field which is in rapid evolution, we simply plan to use either the
3-transgenes technology or any improvement which may become available in the next few months. We
will focus instead on the differentiation step leading from iPS to hematopoietic stem cells which
will be used for treatment of the
oc/oc mouse.
2. Stem cells for the in utero treatment of bone genetic diseases
The
in utero approach will be further pursued in the mouse model of ARO with stem cells of
different origin, in particular, fetal liver cells. In addition, the approach will be used to
investigate the possibility of treating the Osteogenesis Imeprfecta (O.I.) disorder. OI is a
genetic bone disorder, usually transmitted as an autosomal dominant trait, characterized mainly by
skeletal fragility and deformity, due to interference of the abnormal protein in the assembling of
the normal collagen structure. The molecular defect is predominantly mutations in the genes, COL1A1
and COL1A2, coding for the alpha chains of type I collagen, the major structural component of
bone.
No definitive cure is available for OI. The pharmacological treatments partially
ameliorated OI patients’ bone density and fracture rate, but they have a temporary effect and do
not cure the disease, since they do not eliminate the basic genetic defect.
Since mosaic carriers of mutant alleles have been described for OI and have a
mild or normal phenotype even in the presence of high percentage of abnormal cells, cell therapy
seems a promising treatment for this disease. Stem cell transplantation will provide precursor
cells that will originate normal osteoblasts and generate a mosaic situation.
For our preclinical study, we will use the only knock-in experimental model for
OI, BrtlIV, which accurately duplicates the type of mutation, type of transmission and clinical
outcome of human OI patients. Whole bone marrow from experimental adult models transgenic for the
green fluorescent protein (GFP), mesenchymal stem cells from bone marrow obtained by plastic
adhesion in culture and mesenchymal stem cells obtained from other fetal sources of GFP models will
be used as the source of cells for transplantation. We will perform
in utero transplantation since the stigmata of the disease are already present during
fetal life. The GFP will be used as a tracer to precisely evaluate the donor cells engraftment in
bone marrow and bone. Confocal microscopy analysis of bone cryosection will be used to evaluate the
donor cells distribution in vivo in bone tissue. Normal and mutant type I collagen expression at
transcript and protein level will be determined respectively by allele specific real time PCR and
gel electrophoresis following CNBr digestion of fluorescent labelled type I collagen extracted from
bone; two techniques available in the framework of a collaboration with A. Forlino of the Pavia
University. Bone histology and bone mineral density will also be measured in normal and BrtlIV
treated and untreated models.
In summary, the overall goal of this study will be to use a knock-in experimental
model to evaluate the feasibility of in utero cell therapy for Osteogenesis Imperfecta (OI),
analysing the outcome at molecular, biochemical, histological and bone density levels. |
Istituto Clinico Humanitas