1Giuseppe Noia, 1Alessandro Perillo, 2Luca Pierelli, 1Giuseppina
Bonanno, 3Giovanni Monego, 2Sergio Rutella, 1Anna
Franca Cavaliere, 1Giovanni Scambia, 3Giovanni Zelano, 3Fabrizio
Michetti, 2Giuseppe Leone, 1Salvatore Mancuso
1Department of
Obstetrics and Gynecology,2Department
of Hematology, 3Department
of Anatomy,
The
concept of “stemness” defines cells with self-renewal capacity, long
survival, broad differentiation repertoire and capacity of differentiation in
functionally active cells. Therefore stem cells are able to generate both stem
cells (self renewal) and progenitor cells which progressively loose their
self-renewal capacity and give rise to functionally mature and active cells. On
the other hand, the concept of progenitor identifies
cells with limited survival, poor self-renewal capacity, high proliferative
capacity in response to stress signals, and differentiation capacity in a
limited repertoire of maturative lineages. Non-embryonic stem cells can be
obtained both from adult tissues and cord blood. Hematopoietic stem cells
represent the best-known biological model of non-embryonic stem cells, and show
a characteristic hierarchical organization: it has been described a maturation
from totipotent to pluripotent, multipotent and specialized elements which
progressively loose their self-renewal/differentiation capacity and the CD34
stem/progenitor marker. Moreover, a class of hematopoietic stem cells have been
very recently described which do
not express the CD34 protein
characterizing active hematopoietic progenitor cells. The present clinical
applications of stem/progenitor CD34+ cells consist of both allogeneic (in
acute/chronic leukemias, immunodeficiencies, bone marrow aplasias,
thesaurismosis, hemoglobinopathies) and autologous (in acute leukemias,
myelomas, lymphomas, breast and ovarian cancer, germ cell tumors, autoimmune
diseases) transplantation. Possible sources of human non-embryonic hematopoietic
stem cells are fetal liver and cord blood or adult bone marrow and mobilized
peripheral blood.
Recent
developments in stem cell biology: the concept of “plasticity”
Recent
studies suggest the existence of a so called “interchange” among adult stem
cells residing in different tissues, and this capacity allow stem cells which
are apparently directed to differentiation into specialized tissue-specific
cells, to undertake differentive processes (“trans-differentiation”) giving
rise to cells of other tissues or organs. This concept of “plasticity” of
adult stem cells is based on different biological data from both in vitro and in
vivo (i.e. murine models) experiments, and could also be applied to cord
blood-derived stem cells. Several recent reports suggest that there is far more
plasticity than previously believed in the developmental potential of many
different adult stem cells: rare cells that home to bone marrow can long-term
repopulate primary and secondary recipients. These bone marrow adult stem cells
have tremendous differentiative capacity as they can also differentiate into
epithelial cells of the liver, lung, gastro-intestinal tract, and skin.
Moreover, mesenchymal stem cells, provided with self-renewal capacity,
multilineage mesodermal differentiation ability (i.e. adypogenic and osteogenic)
and high proliferative potential, have also been obtained from different adult
tissues and cord blood. It has also been demonstrated that adult and cord blood
stem cells can be in vitro expanded. All these new biological evidences make
adult and cord blood-derived stem cells more attractive for research projects
than embryonic stem cells. Infact, the use of embryonic stem cells, as compared
to adult and cord blood-derived stem cells, show many problems both from an
ethical and a scientific point of view. Infact, different disadvantages with
embryonic stem cells can be mentioned, such as the immunologic reactions with
heterologous embryonic stem cells, the risk of teratome generation with murine
embryonic stem cells, and the high incidence of aneuploidies in human embryonic
stem cells.
Potential
therapeutic perspectives with stem cells
The
new data on stem cell biology outlined above suggest new clinical applications
of hematopoietic stem cells such as the ex-vivo generation of non-hematopoietic
cells and the possibility of performing a “tissue-repair” therapy for
different degenerative and traumatic diseases (i.e. Alzheimer’s and
Parkinson’s disease, myocardial infarction, spinal cord injures, muscular and
skeletal diseases, etc.). Stem cells could also be used as targets for ex-vivo
cell-based gene therapy strategies, representing in this field an optimal
solution. Infact, stem cells are able to accept and tolerate genes introduced
with different genetic engineering techniques, to replace defective or mutated
genes. The gene transfer into a stem cell could generate “corrected” cells
from blood, skin, liver and brain, in large quantities.
Cord
blood stem cell banking
Transplantation
of hematopoietic progenitor/stem cells (HSC) from bone marrow and mobilized
peripheral blood is a standard of care in a number of malignant and
non-malignant conditions. Despite continous improvement, this therapy still
suffers from important limitations, including the lack of suitable donors fully
matched for the HLA system for approximately one third of candidates, and high
toxicity. Placental/cord blood contains high numbers of HSC. The use of
umbilical cord blood as a source of HSC compared to bone marrow, has significant
advantages:
-
riskless, non-invasive collection procedure
-
virtually unlimited number of potential donors (including ethnic
minorities)
-
ready availability
-
lower number of CMV infections in a newborn donor
-
smaller incidence of severe graft-versus-host disease (GVHD)
and some disadvanteges:
-
limited amount of cord blood available from a single donor
-
diminished graft-versus-leukemia (GVL) effect
The first cord blood transplantation was successfully performed in 1988
at S.Louis Hospital (Paris), in a baby with Fanconi’s Anemia. About 2000 cord
blood transplantations have been performed all over the world. About 35% of
patients with malignant tumors who need a stem cell transplantation, do not have
a HLA-matched related or unrelated bone marrow donor. As compared to adult bone
marrow, currently available data show that cord blood contains a higher
proportion of primitive HSC and that cord blood HSC possess higher proliferation
and expansion potentials. Furthermore, the current evidence indicates that cord
blood HSC engraft and sustain hematopoiesis in vivo and that they are more
adequate than HSC from other sources for genetic manipulation and gene therapy.
Because of the limitations generated by the relatively small number of HSC
present in cord blood, a number of protocols have been developed for the ex-vivo
expansion of cord blood HSC. The differences observed in these studies indicate
that at the present time a universal consensus on the most convenient and
appropriate system for the ex-vivo expansion is lacking and that research on
this topic is still at its infancy. Anyway some expanded products have already
been used for clinical transplantation in humans, although only in
investigational protocols. These initial studies were designed with the main aim
of testing the safety and tolerability of the administration of expanded cells,
which was generally uneventful. Despite the encouraging preliminary results,
additional patient accrual and long-term evaluations of expanded cord blood
recipients will be needed before firm conclusions can be drawn on the clinical
impact of this novel form of transplantation. With regard to current
placental/cord blood banking
programs, the largest cord blood bank is located in the N.Y. Blood Center, where
over 10,000 samples of frozen HLA-typed cord blood are stored. Other large banks
exist in U.S., Europe (Milan, Dusseldorf, Paris, Madrid, etc.) and Japan. The
cord blod banks are coordinated by an international registry through which
searches for certain HLA-types can be carried out. In addition to these public,
worldwide accessible banks for anonimously donated cord blood, some companies
have set up private cord blood banks. In this context,
methods for collection and processing of cord blood and test items for
screening viral infections, etc. differ greatly among banks. The quality of
units stocked worldwide has become an important issue and some major banks are
trying to introduce GMP or ISO system (i.e. the “FAHCT” system) for the
quality assurance of cord blood units. The cord blood banking process includes:
donor selection, cord blood collection, cord blood characterization and
cryopreservation, cord blood unit storage and search/release for
transplantation. Different open questions are involved in the realization of
this complex process:
-
the achievement of specific and more in-depth regulations for cord blood
banking
-
the achievement of financial resources
-
the favourable impact on public health of very-large cord blood banking
programs.
THERAPEUTICAL APPLICATIONS
High-dose
chemotherapy as first-line treatment in ovarian cancer: the Catholic University
of Rome experience
In our Institution we assessed the long-term impact of HDCT as consolidation approach in a large series of advanced chemosensitive ovarian cancer patients. Fifty-five patients with advanced ovarian cancer (stage IIIc or IV, G2-G3 tumors) were enrolled in a phase II study with HDCT and hematopoietic progenitor cell support. These patients were optimally cytoreducted at time of first surgery or at interval debulking surgery (IDS) and received HDCT as front-line chemotherapy. HDCT was administered after the administration of 2-3 courses of cisplatin-based nonmyeloablative chemotherapy and consisted of carboplatin 1,200 mg/mq, etoposide 900 mg/mq and melphalan 100 mg/mq (CEM). Hematopoietic progenitor cell support consisted of autologous bone marrow infusion in 4 patients and circulating progenitor cell infusion in the remaining 51 patients. The median follow-up of the whole series was 48 months (range 8-120). Fifty-five patients received HDCT and 53 patients were evaluable for response and survival assessment due to the occurence of two treatment-related deaths. In the overall population we obtained a pathologically complete response in 34/53 (64%), microscopic partial response in 9/53 (17%), macroscopic partial response in 7/53 (13%) and no-change of disease in 3/53 (6%). In the whole patients’ series the median time to progression (TTP) was 35 months with a 5-year TTP rate of 35% while the median overall survival (OS) was 75 months with a 5-year OS of 59%. In patients who had undergone primary cytoreduction the 5-year TTP rate was 43% and the OS rate was 62%. In patients treated with IDS 5-year TTP rate was 22% and OS rate was 50%. From these data we can conclude that HDCT and hematopoietic support is an effective therapeutic option for advanced ovarian cancer who can benefit both from primary cytoreduction or IDS. However, the great proportion of patients who achieved a complete or microscopic partial response (81%) and the 5-year TTP rate of only 35% observed in these patients suggest that high-dose strategy is unable to produce long-lasting tumor control. Future improvements of intensive treatments should take into account post-transplant immunotherapies or allogeneic hematopoietic progenitor transplant after reduced conditioning.
Fetal
stem cells in maternal peripheral blood
In recent years it has been demonstrated that the fetal/placental unit and the maternal organism communicate mostly through the production of biochemical and hormonal compounds. So all the metabolic, and, let's say, “vital needs” of the fetus are transmitted to the mother through this kind of “dialogue” using hormones and biochemical compounds, produced by the fetus with the internal mediation of the placenta. At present, we know that there is also a “feto-maternal cell trafficking”. Infact, the fetus sends to the mother a number of cells, which are of different kinds:
- Trophoblast cells: these are polynuclear cells characteristic of pregnancy and lacking of specific antigens. These are fragments of trophoblasts, cyto- and the syncytio-trophoblast, which separate from the placental body and go into the maternal circulation and implant themselves in different sites of the maternal organism.
- Lymphoid cells: mononuclear cells able to be present for decades in maternal bone marrow and peripheral blood, remaining from previous pregnancy. These cells have been found even thirty years after the baby was born; they are inside the maternal organism and they continue to produce new daughter cells which are of fetal origin. So the pregnancy doesn't last 40 weeks but many, many years in the maternal organism !
- Erythroid cells: including precursors provided with nucleus (BFU-E, CFU-E), characteristic of pregnancy and expressing different antigens.
And finally:
- Haematopoietic stem cells: these cells are very important in the induction and subsequent maintenance of the immune-tolerance in the maternal organism during pregnancy (we don't forget that 50% of the antigens during pregnancy are of paternal origin; so the maternal organism does not recognize this 50% of external antigens).
Then, it is possible that such microchimerism could contribute to the pathogenesis of selected autoimmune diseases. Diana Bianchi hypothesized that (a couple of years ago), supposing that the genesis of some auto-immune diseases, which are much more present in females than in males, could be dued to the presence and the maintenance in the maternal body of these “foreign cells” of fetal origin. Now, fetal stem cells can get into the maternal circulation and differentiate into a mature thyroid follicle. It has been demonstrated that stem cells from the fetus go inside the thyroid tumor of the mother, with a kind of intent to “treat” the tumor, to provide a sort of “help” to the maternal organism, to “cure” some diseases. And this has been proven also in hepatitis or in myocardial infarction.
The
first allogeneic stem cells transplantation in a human fetus was successfully
performed in 1988 (Lyon). Since then more than 40 similar procedures have been
carried out in 12-48 weeks old fetuses. The procedure outcomes were positive in
50% of the fetuses with immunodeficiencies, achieving the correction of the T
cell defect. On the contrary, in fetuses with hereditary haemoglobinopathies the
results were not so successful. Further researches are necessary to develop this
kind of therapy and to make it suitable for different genetic diseases.
In this context, our group has developed an animal model of prenatal transplantation of human cord blood stem cells into sheep fetuses. Several problems need to be solved to achieve an efficient in utero HSC transplantation. Recent reports pointed out the importance of timing in prenatal stem cell transplantation procedures and showed the advantage of an early HSC injection. An ultrasound-guided intracelomic approach could allow this possibility. In our Institution, the intracelomic route for in utero hematopoietic stem-cell transplantation has been evaluated in pre-immune fetal sheep and engraftment characteristics and fetal loss have been defined.
Nine ovine fetuses (gestational ages: 40-45 days) received intracelomic transplants of human CD3-depleted (50 x106 per lamb) or CD34-selected (1 x105 per lamb) cord-blood hematopoietic stem cells. Engraftment was evaluated from cell suspension of liver, spleen, bone marrow and thymus by flow cytometry and polymerase chain reaction analysis of human b2-microglobulin.
Three fetuses (33%) aborted shortly after intracelomic transplantation and were not evaluable for engraftment. Engraftment was detected in 4 fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degree of engraftment in these four fetuses ranged from 6% to 22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). One fetus obtained after cesarean section at 102 days and one fetus delivered at term, which received CD34-selected cord blood cells had human engraftment with 10% CD45 positive cells in bone marrow. In all fetuses human engraftment was confirmed by PCR analysis for human b2-microglobulin which also identified human cells in brain, spinal cord, heart, lung and skeletal muscle.
Our preliminary data indicates that intracelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and highly effective in terms of engraftment but possibly associated with an increased risk of abortion.