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Reprogramming Cells for Therapeutic
Applications: A Novel Avenue?
Institute of Medical Biochemistry, University of Oslo, Oslo, Norway.
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Ways of directly turning a somatic cell into another would alleviate
difficulties associated with current nuclear transplantation procedures
and be beneficial for producing replacement cells for therapeutic
purposes. In addition to active research on the reprogramming potential
of (adult) stem cells, new transdifferentiation strategies are being
developed. Recent progress from the laboratory of Dr Philippe Collas
at the Institute of Medical Biochemistry of the University of Oslo,
suggests that it is possible to reprogram somatic cells in the test
tube. In vitro cell reprogramming may create possibilities for producing
isogenic replacement cells.
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Methods for directly and efficiently reprogramming a somatic
cell into another somatic cell type, a process referred to as transdifferentiation,
would circumvent difficulties associated with nuclear transplantation
and be beneficial for producing isogenic (patient's own) replacement cells
for therapeutic applications. Reports on the transdifferentiation potential
of adult stem cells or somatic cells have broadened our view on the plasticity
of committed or differentiated cells. In this communication, we briefly
review the takes on the transdifferentiation potential of adult stem cells
and somatic cells. We also present a novel somatic cell reprogramming
strategy recently developed in our laboratory and discuss implications
of these findings.
Transdifferentiation of adult stem cells
Tissue-specific stem cells reside in adult tissues including blood, skin,
central nervous system, liver, gastro-intestinal tract or skeletal muscle
(Blau et al., 2001). Adult stem cells are responsible for regenerating
damaged tissue and maintaining tissue homeostasis, such as replenishment
of skin and blood cells. In contrast to the pluripotent embryonic stem
cells, the differentiation and regenerative potential of adult stem cells
has traditionally been thought to be restricted to the tissues in which
they reside.
It turns out, however, that adult stem cells have a surprisingly broad
differentiation ability (Blau et al., 2001). For example, bone marrow-derived
cells not only replenish blood but also contribute to heart, brain, liver,
muscle, skin and vascular endothelium. Other reports claim stem cell movement
in the other direction, with for example, central nervous system-derived
cells giving rise to blood. Similarly, neural stem cells can turn into
skeletal muscle and hepatic stem cells into pancreas. Thus, it appears
that in some situations, adult stem cells exhibit sufficient plasticity
to differentiate into cell types outside their predicted developmental
lineage.
Somatic cells placed into a new environment can alter
their fate
Somatic cells can change their fate when placed into a new environment.
This may be achieved through engraftment into ectopic regions of the body.
Transdifferentiation into various cell types has been largely exemplified
in fruit flies, tadpoles or chick embryos. In certain situations, this
also holds true in mammals: injection of endothelial cells into damaged
heart tissue has been shown to cause their trans-differentiation into
beating cardiomyocytes (Condorelli et al., 2001). Several laboratories
have also achieved forms of transdifferentiation of mammalian somatic
cells by manipulation of cell culture conditions. For example, myo-blasts
have been shown to transdifferentiate into adipocytes (Hu et al., 1995),
pancreatic cells have been turned into hepatocytes (Shen et al., 2000),
osteoblasts into adipocytes (Schiller et al., 2001), or umbilical vein
endothelial cells into beating cardiomyocytes (Condorelli et al., 2001).
It seems, therefore, that the microenvironment can play a key role in
redirecting cell fate.
Reprogramming fibroblast function in a somatic cell
extract
We have recently developed a procedure to reprogram cells in vitro (Håkelien
et al., 2002; Landsverk et al., 2002). The strategy is illustrated in
Figure 1. The plasma membrane of 'donor' cells (e.g., fibroblasts) is
reversibly permeabilized with the bacterial toxin, Streptolysin O. Fibroblast
function is reprogrammed by incubation of the permeabilized cells in a
nuclear and cytoplasmic extract (or 'soup') prepared from a different
somatic cell type (in our study, cells of the immune system or T cells).
The extract provides regulatory components that mediate alterations in
the gene expression profile of the target genome. At the end of incubation,
the cells are resealed and cultured.
Figure 1. A novel strategy for reprogramming
cells. Healthy cells (e.g., fibroblasts) are reversibly permeabilized,
reprogrammed in a test tube containing an extract from the cell type one
wishes to obtain (e.g., insulin-producing
cells, neurons, cells of the immune system, cardiomyo-cytes, etc), resealed
and cultured for analysis.
This technology may represent an attractive alternative to transdifferentiating
isogenic (patient's own)
cells for therapeutic applications.
Cell reprogramming was characterized by:
- Uptake of transcriptional regulators present in the
extract by the fibroblast nuclei.
- Alterations in the structure of the chromatin in the
reprogrammed cells: in specific promoter regions, the chromatin acquired
a conformation similar to that found in the target T cell type.
- Changes in growth characteristics of the reprogrammed
cells.
- Changes in gene expression profile: genes coding for
a variety of hematopoietic cell-specific proteins, such as hormones
and growth factors or cell surface receptors were activated.
- Expression of hematopoietic cell-specific surface receptor
proteins.
- Induction of a complex T cell-specific regulatory function
after stimulation of receptors on the surface of the reprogrammed cells.
- Extension of neurite-like outgrowths and expression
of a neurofilament protein, after reprogramming fibroblasts into an
extract of neuronal precursor cells.
- Expression of a reprogrammed phenotype over at least
several weeks in culture, in 20-80% of the cells, depending on the target
cell type obtained.
These observations predict that it may be possible to
induce other complex (for example, secretory) functions in fibroblasts
reprogrammed into other cell types. This functional aspect will be of
crucial importance in further development of cell reprogramming for therapeutic
applications.
Innovative aspects of reprogramming cells in vitro
If in vitro cell reprogramming proves to be functional on a large scale,
it would present innovative technological, commercial and societal benefits.
(i) Current strategies for cell reprogramming or controlled differentiation
(i.e., directed differentiation of stem cells) are based on a receptor-mediated
approach. In vitro reprogramming uses permeabilized cells, thus 'reprogramming
factors' access the cellular interior directly. This may not only render
the strategy more effective, but has the advantage of being useful without
having a great deal of prior knowledge of regulatory mechanisms controlling
cell function. (ii) In vitro reprogramming may be applied to many cell
types and thus, has potential for treating many diseases. (iii) It may
also allow for the production of isogenic (patient's own) cells for transplantation.
(iv) Unlike reprogramming by nuclear transplantation into eggs, somatic
cells are used as a source of reprogramming material. Unlike eggs, these
can be grown in large numbers to produce a consistent supply of reprogramming
material. (v) Finally, ethical and legal issues regarding human cloning
and the production of embryonic stem cells from human embryos would be
avoided.
Yet, it would be biased to say that everything is perfect. Our reprogramming
assay involves extensive cell manipulation. Thus, application of this
technology to produce replacement cells requires significant developments
and a large body of data is needed before this system can be applied to
the generation of cells used for therapy in human patients.
A therapeutic cell?
Our studies indicate that genome reprogramming under current conditions
is not complete. For example, only a subset of genes is up- or down-regulated
and, to date, the reprogrammed cells do not express a pure T cell-specific
phenotype. Nevertheless, for cell replacement therapy, one may not need
to produce a fully reprogrammed cell. For instance, for treating Type
1 diabetes, it may sufficient to engineer a cell that secretes regulated
levels of insulin in response to glucose stimulation, very much like a
pancreatic b cell, however the replacement cell may not need to be a b
cell stricto sensu. This may even present advantages to overcome the autoimmunity
that characterizes Type 1 diabetes. This is, however, a highly speculative
hypothesis and testing it will require extensive genetic and functional
investigations. Nevertheless, I propose a concept whereby a partially
reprogrammed cell that displays the necessary therapeutic properties,
a therapeutic cell, may be what it takes to treat certain diseases.
While research is being actively pursued to develop novel cell-based therapies,
the reprogramming assay described here may provide a powerful tool for
analyzing nuclear reprogramming processes. A clearer understanding of
nuclear reprogramming will not only lead to a better appreciation of (de)differentiation,
cloning or stem cell biology, but also will be relevant to the aberrant
programming that occurs in cancer and other pathological situations.
Acknowledgements
Work in the Collas laboratory is supported by the grants from Research
Council of Norway, the Norwegian Cancer Society, the Human Frontiers Science
Program, Nucleotech LLC and the European Union Marie Curie Training Program.
Selected references
Blau, H.M., Brazelton, T.R., and Weimann, J.M. (2001). The evolving concept
of a stem cell: entity or function? Cell 105, 829-841.
Condorelli, G., Borello, U., De Angelis, L. et al. (2001). Cardiomyocytes
induce endothelial cells to trans-differentiate into cardiac muscle: implications
for myocardium regeneration. Proc. Natl. Acad. Sci. U.S.A. 98, 10733-10738.
Håkelien, A.M., Landsverk, H.B., Robl, J.M. et al. (2002). Reprogramming
fibroblasts to express T-cell functions using cell extracts. Nat. Biotechnol.
20, 460-466.
Hu, E., Tontonoz, P., and Spiegelman, B.M. (1995). Transdifferentiation
of myoblasts by the adipogenic transcription factors PPAR gamma and C/EBP
alpha. Proc. Natl. Acad. Sci. U.S.A. 92, 9856-9860.
Landsverk, H.B., Håkelien, A.M., Küntziger, T. et al. (2002).
Reprogrammed gene expression in a somatic cell-free extract. EMBO Rep.
3, 384-389.
Schiller, P.C., D'Ippolito, G., Brambilla, R. et al. (2001). Inhibition
of gap-junctional communication induces the trans-differentiation of osteoblasts
to an adipocytic phenotype in vitro. J. Biol. Chem. 276, 14133-14138.
Shen, C.N., Slack, J.M., and Tosh, D. (2000). Molecular basis of transdifferentiation
of pancreas to liver. Nat. Cell Biol. 2, 879-887.
Dr Philippe Collas
Institute of Medical Biochemistry
University of Oslo
PO Box 1112 Blindern
0317 Oslo, Norway
Tel: 0047 2285 1066
Fax: 0047 2285 1058
Email: philippe.collas@basalmed.uio.no
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