22 January 1999
THE USE OF HUMAN CNS STEM CELLS FOR TRANSPLANTATION
INTO THE CHRONICALLY INJURED, SPINAL CORD.
Angelo L. Vescovi.
Laboratory of Neuropharmacology
National Neurological Institute
Milan Italy
Introduction.
Intraspinal transplantation of exogenous cells or tissue has been proposed as
a possible way to produce partial reconstruction in the damaged spinal cord.
While implantation of cells could function by serving as a bridge within the
lesion, by providing chemical and/or mechanical guidance for host neurons to
grow across the gap, by providing factors capable of rescuing dying neurons
and/or modulate neural networks, the replacement of lost neurons and the
restoration of spinal cord damaged circuitry through the formation of local
micro-relay represents one of the essential requirement for the therapy of
chronic SCI. In this perspective, while a variety of tissues and cells have
been used in the attempt to restore/regenerate lesions of the cord, the
engraftment of fetal CHS human material has long be viewed as a necessary step
in spinal cord transplantation.
This research attempts to obviate to the ethical and moral issues inherent to
the use of fetal human CNS tissue, and to provide effective control upon some
essential transplantation parameters such as cell composition, age, storage,
viability and contamination. In this perspective, we have aimed at
establishing some novel types of hu man brain cell preparations to be used as
donor materials in intraspinal transplantation. These novel "cell prosthetics
will be established from peculiar cells of the human brain, that have the
capacity to self-reproduce and expand in vitro in an unlimited fashion and
that are called stem cells. Knowing that the use of human CNS stem cells
enables us to generate human nerve cells without the need of continuously
using primary fetal tissue, we are testi ng a series of protocols based on
biochemical manipulations that modulate the capacity of stem cells to generate
the neuronal cells and the glia (ie; those cells that normally nurture and
support the neurons) to be used for intra spinal transplantation. Thus, cell
preparations are being established that are enriched, alternatively, for their
neuronal or glial contents. The maturation of the two cell types is also being
studied. Following this initial step, these cell preparations will be injected
into the spinal cord of adult rats bearing chronic post-traumatic lesion, and
the functional parameters that pertain directly to graft-induced improvement
of function in SCI injury, such as the survival of grafted cells, their
capacity to filI cavern and cavities and the generation of novel neurons and
neuronal circuitry at the site of lesion will be monitored.
The study described here is being carried out using exclusively cells of human
origin that are already available in our "Human CNS Stem Cell Bank". Hence,
any positive results that will be obtained at the end of the two years of
investigation could be directly translated into pre-clinical/ clinical
applications for therapy of chronical spinal cord injury and will be of
immediate use in order to request permission to the competent agencies to
begin clinical trials in humans. In the first six months of this project we
have pursued the following objectives.
Aim # 1: To Set Up Continuous Cultures Of Stem Cells From The Human Spinal
Cord, Cortex And Diencephalon.
Aim #2: To Obtain Cell Preparations That Are Enriched For Their Neuronal
Content.
Aim # 3 To Obtain Cell Preparation Enriched For Their Glial Content.
PROGRESS AIM 1. The first aim of this proposal was the establishment of
continuous non-transformed cell lines from the human spinal cord, cortex and
diencephalon that would provide a renewable source of neural cells to be
differentiated into transplantable neurons and glia. This goal has been
accomplished. As indicated in our original project, we have used stem cells
that were already available in our "Human CNS Stem Cells Bank". Thus, cells
stored in cryogenic vials have been quickly thawed up and plated in plastic
dishes in the presence of specific nurturing medium that prevents cells from
differentiating into mature brain cells and forces them to grow and expand in
number for months. Thus, in the first 4 months of this project (mid-July
through mid-November), we have cultured and expanded human spinal cord,
cortical and diencephalic stem cells and have established a supply of cells
that will suffice for the next 6-8 month of experiments. Slight differences
exist in the rate of expansion of the various cell lines, with the
diencephalic cells doubling in number every 7 days, as compared to the 10 and
12 days necessary for the cortical and spinal cord cells, respectively.
Nevertheless, the routine characterization carried out in these first 4 months
has shown that all these cells would spontaneously differentiate, in a similar
fashion, into neurons as well as astrocytes and oligodendrocytes (two gli= al
cell types) when triggered to do so by removal of some specific proteins
contained in the growth medium called "growth factors".
PROGRESS AIM 2. It is known that when mouse neural stem cells are triggered
to differentiate by removal of the growth factors used as mitotic agents, the
relative numbers of neurons, and oligodendrocytes that they generate can be
modified by the presence specific proteins such as neurotrophic factors and
cytokines. From the beginning of November, we have screened 5 of these
cytokines for their capacity to induce the formation of a high number of
neuronal cells among the stem cell differentiated progeny. The achievement of
this goal is our highest priority because, as discussed above, it is likely
that the transplantation of material enriched in neuronal cells represent one
of the key elements for successful intraspinal transplantation. The results
have been somewhat surprising, and encouraging at the same time. In fact,
while a cytokine called platelet derived growth factor (PDGF ?BB) known to
enhance neuronal differentiation in mouse stem cells failed in doing the same
in human cells, two other molecule called, respectively, ciliary neurotrophic
factor (CNT= F), and Leukemia Inhibitor Factor (LIF), were shown to almost
double and triple the number of neurons generated by human stem cells. This
happened regardless of whether cells were derived from the spinal cord, cortex
or Diencephalon. We also confirmed the capacity of fibroblast Growth factor 2
(FGF2) to increase the production of neurons, while the keratinocyte growth
factor (KGF) had only a small effect. Fig. 1 shows this phenomenon as
determined in cells derived from the presumptive Diencephalon.
While the aim # 2 can be considered partially accomplished, we are now
extending our investigation to other numerous cytokines, also testing them in
various combinations. Preliminary data show that up to 55% of the human stem
cell progeny can be differentiated into neurons, but a more detailed analysis
will be presented in the next progress report. The final aim is to obtain
culture of pure neurons.
PROGRESS AIM 3. Glial cells May play a very important role in the context of
spinal cord transplantation, as they can not only nurture the transplanted
neurons, but because they can also provide a permissive substrate that may
allow for and the re-extension of regenerating host nerves, survival of
transplanted neurons and provide the ground for the formation on local
neuronal relays. With this view in mind, it is important to be able to
generate pure glial cultures that, when mixed with pure neuronal cultures,
will allow for the establishment of grafting cell suspension in which the
neuronal/glia ratio is pre-determined in order to evaluate the effect of cell
type composition on the effectiveness of the transplantation. We have achieved
the goal of obtaining pure (100%) astroglial cultures, by removing the
mitogens and allowing stem cell to differentiate in the presence of low
concentrations of fetal bovine serum (0.5-1%) with the simultaneous exposure
to FGF2. In this medium pure astroglial cells are generated for up to two
months. Notably, these astroglial cells remain rather immature under these
conditions, as determined by the lack of expression of markers like the glial
fibrillary acidic protein (GFAP). This is a very important and positive fact,
as it is known that young and immature astrocytes are more permissive for the
regrowth of regenerating nerve fibers. Thus AIM 3 has been completed.
CONCLUSION AND FUTURE DEVELOPMENTS. The results described above represent the
outcome of the first 6 months of research funded by the SCS. The data are very
encouraging as we already have all the necessary human cells available and can
control their differentiation to obtain pure astroglial cultures as well as
preparations highly enriched in neurons. We can safely predict that in the
coming six months this line of research will proceed even more expeditiously,
as most of the time consuming technical tasks have been carried out. Thus,
the achievement of pure neuronal cultures, as well as the establishment of
grafting suspension with cells pre-set at progressive stages of maturation to
be tested in intraspinal transplantation should be achieved very shortly.
Importantly, we have decide to anticipate our attempt to transplant the human
CNS stem cell differentiated progeny into the spinal cord of experimental
animals. So, while we pursue the objectives listed above, we shall also,
simultaneously, test the engrafting capacity into the cord of the cell
suspensions enriched in neurons or glia described in the present progress
report. This will also allow us to obtain preliminary data on human CNS
intraspinal transplantation enabling us to approach the second phase of this
project -- systematic transplantation of multiple types of human CNS stem
cell-derived grafting suspensions into the normal and then chronically injured
spinal cord -- with a more proficient and rapid strategy that will
conclusively establish how efficient the transplantation of these cells can be
in inducing neurological improvement in chronic spinal cord injury.