Stem cells: back to the future

Stem-cell researchers around the world face both religious and secular concerns about the therapeutic use of stem cells from surplus embryos or aborted foetuses.

But finding another source of pluripotent stem cells is an imperative, simply because the limited supply of embryonic stem cells will make it difficult to find cells to genetically match all patients requiring grafts.

As an immunologically privileged environment, the central nervous system (CSN), comprising the brain and spinal cord, will accept "non-self" stem-cell grafts, but other therapeutic applications will require autologous grafts to avoid immune rejection.

Formidable technical challenges confront efforts to create "self" stem cells via somatic cell nuclear transfer (SCNT). In SCNT, a nucleus from one of the patient's own somatic cells is transplanted into an enucleated donor ovum, then stimulated to divide and form a blastocyst, the source of ES cells.

When Professor Freda Miller's research group at Canada's Hospital for Sick Children in Toronto set out to find an alternative to embryonic stem cells to repair damaged brains and spinal cords, it went only skin deep.

Miller told ALS that the skin looked a promising place to search because it contains sensory receptors to detect physical pressure. "There was data suggesting that the dermis contains some sort of precursor cells that regenerates the nerve cells that sense pressure," she says.

"We used the protocols used to isolate stem cells from the brain to identify a population of similar stem cells in the dermis. We succeeded in finding them in rodent skin, so we then turned to human skin.

"Since then, we've gone on to determine what these cells are, where they occur in the dermis, and whether they can be used therapeutically."

The big surprise for Miller and her colleagues was that the stem cells turned out to be multipotent - not only could they form neural cells, they were able to form other mesoderm cell types. In the embryo, mesoderm stem cells form muscles, major organs and the digestive tract.

Grown in vitro, the skin stem cells differentiated into Schwann cells - the specialised cells that wrap neurons in the brain and spinal cord in an insulating myelin sheath.

Transplanted into animal models of the demyelinating disorder multiple sclerosis, they produced new Schwann cells - "They do make the real thing," Miller says.

"In retrospect, it shouldn't have been a surprise," she says. "We believe they are basically neural crest precursor cells that persist in the body after embryonic development.

"The neural crest makes the entire peripheral nervous system, including the myelinated Schwann cells that sheath neurons in the brain and spinal cord.

"Schwann cells not only make myelin, they produce nerve growth factors, so they create a good environment for nerve growth."

That made the neural precursor cells in skin prime candidates for repairing paralysing spinal cord injuries. In subsequent, unpublished research, Miller's group has shown that the stem cells cells not only form new neurons to bridge damaged regions of spinal cords, they remyelinate axons exposed by the apoptotic death of oligodendrocytes, the myelinating CNS cells, after the original injury.

Miller says rodents with paralysing spinal cord injuries show some functional recovery within months of receiving stem cell grafts.

The Canadian researchers found their stem cells in the dermal papillae, at the base of hair follicles. "It made perfect biological sense, because the dermal papillae have been postulated to act as a reservoir for the replacement of different dermal cell types," Miller says.

"When we put them into dermis tissues, they grew new hair follicles, and were involved in wound repair.

"We're now trying to determine what makes them home to their preferred niches in the body, and differentiate into different cell types. When we put them into dermis, they reconstitute hair follicles. When we put them into neural tissue, they form Schwann cells.

Miller says the precursor cells' capacity to produce new hair follicles has been a source of humour around her laboratory - any cell that can produce new hair follicles would find a ready mass market among the tonsorially challenged.

Reprogramming somatic cells

At Kyoto University Medical School, Professor Shinya Yamanaka's research team took a very different route in its quest of an alternative source of ES cells.

Its aim is to take differentiated somatic cells from patients needing stem-cell therapy, and reprogram them return them to a pluripotent, ES-like state in which they would proliferate rapidly, creating an abundant supply of "self" cells for autologous grafting.

Its strategy involved using gene-expression arrays and real-time PCR (polymerase chain reaction) to identify a sub-set of genes that are highly expressed in ES cells, but not in somatic cells, on the assumption that they would be involved in maintaining stem cells in their self-renewing, pluripotent state.

If so, re-activating or over-expressing these genes in somatic cells could induce them to revert to an ES-like state.

In a paper in the international journal Cell last August, Yamanaka's group reported success, using mouse skin fibroblast cells.

Their initial comparative screening identified 24 candidate genes that were either exclusively or highly expressed in early embryonic cells, but not in fibroblasts.

By linking these genes to a high-expression promoter and using retroviruses to transfect them into fibroblasts, they were able to narrow the selection to just four genes, all encoding transcription factors: the well known oncogene c-Myc, and Oct3/4, Sox2, and Klf4.

Transcription factors modify cell behaviour and identity by coordinating the activity of hundreds of other genes.

Two of the four "stem-inducing" genes, c-Myc and Klf4m, are also expressed in mature fibroblasts, but at a much lower level than in stem cells.

Inserted beneath the skin of mice, the induced pluripotent stem (iPS) cells they formed teratomas - cancer-like masses containing various differentiated tissues that normally derive from the three primary layers of tissue in newly formed embryos - ectoderm, mesoderm and endoderm.

When the Kyoto University researchers inserted iPS cells into the core regions of blastocysts, they contributed to normal embryonic development.

Although these findings indicate that iPS cells are pluripotent, and self-renewing like ES cells, Yamanaka and his colleagues are claiming only that they are "very similar".

They will now attempt to achieve similar results with human fibroblast cells, using the homologous human genes.

Yamanaka says the use of retroviruses to integrate the cell-transforming genes into the chromosomes of somatic cells involves a risk that the transgenes may switch on neighbouring genes that are normally inactivated, and turn the cell cancerous.

His group is now experimenting -so far, unsuccessfully - with adenoviruses that don't permanently integrate the transgenes into the host cell, or to use RNA interference (RNAi) to selectively de-repress the cell's own genes, or increase expression levels.

Yamanaka says there is intense competition between research teams in other parts of the world, most notably at Harvard (US) and Cambridge (UK) to develop a reliable way of inducing fibroblasts to revert to ES-like cells.

"It would be much better if we can make ES-like cells from human somatic cells, because our systems do not require any embryos or oocytes," Yamanaka says.

Yamanaka says it's not clear that the project's success would satisfy current religious or ethical objections to using embryonic stem cells from surplus embryos or aborted foetuses - we'll just have to wait and see.