Changing the Cell Source in Cell Therapy?
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《新英格兰医药杂志》
One dream of scientists is to be able to rebuild, in vitro, "spare parts" to replace injured or diseased tissues — a notion that was once relegated to the realm of science fiction. The concept that stem cells that are ostensibly site-specific can give rise to (and maintain) mature tissues has made adult stem cells the focus of intense research designed to explore their promise for the treatment of a variety of human diseases.
Adult stem cells are defined as clonogenic, self-renewing progenitor cells that can generate one or more specialized types of cells. However, major obstacles in this field have been a lack of molecular markers to identify stem cells (most available markers identify proliferating cells rather than stem cells) and uncertainty regarding the precise location of the putative stem cells in vivo. It is widely believed that the maintenance of stem cells is controlled by their particular microenvironments (or "niches"), which are best thought of as clusters of environmental cues affecting the state and behavior of the cell.
Visual function requires an intact ocular surface. The integrity of this surface is maintained in humans by two highly specialized epithelia — the conjunctival epithelium and the limbal–corneal epithelium. A population of keratinocyte stem cells in defined locations governs the renewal of these stratified epithelia. These stem cells generate transient amplifying cells that terminally differentiate after a discrete number of cell divisions. Corneal stem cells are segregated in the basal layer of the limbus, the transitional zone between the cornea (the transparent part of the ocular surface) and the bulbar conjunctiva (which covers the white part of the ocular surface).
Corneal transplantation, which entails the replacement of the central part of the cornea, in its full thickness, with the same part from a donor cornea, is widely used to treat diseases such as keratoconus, as well as to repair damage due to chemical burns or trauma. After a corneal transplant has been grafted in a recipient, its outer surface will eventually be covered by a host-derived corneal epithelium that migrates in from the limbus, having been generated by limbal stem cells (see upper panel of Figure). However, if the patient has a total deficiency of limbal stem cells, the donor cornea will be rejected and will be covered by cells migrating from the conjunctival epithelium. The results include scarring, opacification, and neovascularization of the stroma, with loss of visual acuity as well as severe discomfort (see lower panel of Figure).
Figure. Corneal Transplantation in a Patient without Limbal Stem-Cell Deficiency and in a Patient with Anatomical Limbal Stem-Cell Deficiency.
The only way to prevent this conjunctival invasion of the corneal surface is to restore the limbus.1 In 1997, the discovery that cultured limbal cells include stem cells, detectable as holoclones (clones that are derived from human epithelial stem cells and that have high proliferative potential), permitted the development of limbal cultures for the treatment of patients with a partial deficiency of limbal stem cells.2 Limbal epithelial cells were obtained from a small biopsy specimen from a healthy area of the patient's cornea; after culturing, these cells developed into corneal epithelium, which was successfully transplanted back into the patient. This treatment represented a substantial advance in ocular-surface reconstruction. Currently, cell culture for the reconstruction of epithelium is performed on various cell substrates that can be sutured, are suitable for surgery, and are easy to handle, but these substrates have not routinely been tested for their capacity to maintain stem cells in culture. Several hundred patients with a deficiency of limbal stem cells have now been treated with this method, although the success rate has varied according to the cause of the deficiency.
Genetic diseases (such as aniridia) represent one group of causes that cannot be corrected by transplantation alone, with or without cultured limbal cells. For limbal stem-cell deficiency due to systemic diseases (such as the Stevens–Johnson syndrome) or other conditions (such as trauma, infections, or burns), the transplantation of cultured stem cells has often been useful, but the results have varied.
Ocular burns or damage due to infection or trauma, for example, can be treated successfully with the transplantation of cultured autologous stem cells in nearly 80 percent of patients. Of course, success rates among patients with deficiencies from any cause depend on several factors, including the availability of a small healthy area on the patient's ocular surface, the maintenance of stem cells in culture (particularly on novel, tissue-engineered culture surfaces), and the maintenance of critical structures such as the fornix and the tear film.
This technology cannot be used in patients with a total deficiency of limbal stem cells, since autologous stem cells are unavailable for transplantation. In cases of bilateral damage, limbal cells have been obtained from donor biopsy specimens. This procedure requires continuous immunosuppression, however, with associated side effects and risks.
In this issue of the Journal, Nishida and colleagues (pages 1187–1196) report the transplantation of epithelium cultured from autologous oral mucosal cells in four patients with the Stevens–Johnson syndrome who had a total deficiency of limbal stem cells. Follow-up over a mean period of 14 months revealed the recovery of visual function and the restoration of a normal-looking epithelium and good corneal transparency.
Do these results suggest that stem cells from the oral mucosa can differentiate into corneal epithelium? Not necessarily. It is not actually possible to confirm this hypothesis, since the cells express similar markers. Much previous research has suggested that adult epithelial cells retain their differentiation program even when transplanted at different sites in the body. An alternative explanation for the positive clinical results should therefore be considered. Certainly, if no stem cells are present on the corneal surface (i.e., if there is what I refer to as "anatomical" deficiency of limbal stem cells), the restoration of the corneal surface requires engraftment of autologous or donor stem cells that are able to restore the limbal reservoir. However, in some cases, residual stem cells are present on the ocular surface, but the number of cells is too small for them to cover the surface on their own or the cells cannot be stimulated by wound-healing signals (i.e., there may be a "functional" deficiency of limbal stem cells). When undamaged cultured epithelial cells are transplanted onto the ocular surface, they induce modifications of the microenvironment that make the ocular surface suitable for cell survival; this may result in the stimulation and proliferation of the recipient's own stem cells. Supporting this hypothesis are some reported cases of successful treatment of total limbal stem-cell deficiency with allogeneic stem cells, in which long-term follow-up revealed the presence of autologous cells3 that had not been active in promoting epithelialization before transplantation.
The human ocular surface, with its well-defined locations for different types of cells, provides a useful model for cell therapy. Additional experience with limbal stem-cell transplantation may pave the way for stem-cell therapy for other tissues and organs.
Source Information
From the Veneto Eye Bank Foundation, Epithelial Stem Cell Research Center, Ospedale Civile di Venezia, Venice, Italy.
References
Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology 1989;96:709-722.
Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997;349:990-993.
Henderson TR, Coster DJ, Williams KA. The long term outcome of limbal allografts: the search for surviving cells. Br J Ophthalmol 2001;85:604-609.(Graziella Pellegrini, Ph.)
Adult stem cells are defined as clonogenic, self-renewing progenitor cells that can generate one or more specialized types of cells. However, major obstacles in this field have been a lack of molecular markers to identify stem cells (most available markers identify proliferating cells rather than stem cells) and uncertainty regarding the precise location of the putative stem cells in vivo. It is widely believed that the maintenance of stem cells is controlled by their particular microenvironments (or "niches"), which are best thought of as clusters of environmental cues affecting the state and behavior of the cell.
Visual function requires an intact ocular surface. The integrity of this surface is maintained in humans by two highly specialized epithelia — the conjunctival epithelium and the limbal–corneal epithelium. A population of keratinocyte stem cells in defined locations governs the renewal of these stratified epithelia. These stem cells generate transient amplifying cells that terminally differentiate after a discrete number of cell divisions. Corneal stem cells are segregated in the basal layer of the limbus, the transitional zone between the cornea (the transparent part of the ocular surface) and the bulbar conjunctiva (which covers the white part of the ocular surface).
Corneal transplantation, which entails the replacement of the central part of the cornea, in its full thickness, with the same part from a donor cornea, is widely used to treat diseases such as keratoconus, as well as to repair damage due to chemical burns or trauma. After a corneal transplant has been grafted in a recipient, its outer surface will eventually be covered by a host-derived corneal epithelium that migrates in from the limbus, having been generated by limbal stem cells (see upper panel of Figure). However, if the patient has a total deficiency of limbal stem cells, the donor cornea will be rejected and will be covered by cells migrating from the conjunctival epithelium. The results include scarring, opacification, and neovascularization of the stroma, with loss of visual acuity as well as severe discomfort (see lower panel of Figure).
Figure. Corneal Transplantation in a Patient without Limbal Stem-Cell Deficiency and in a Patient with Anatomical Limbal Stem-Cell Deficiency.
The only way to prevent this conjunctival invasion of the corneal surface is to restore the limbus.1 In 1997, the discovery that cultured limbal cells include stem cells, detectable as holoclones (clones that are derived from human epithelial stem cells and that have high proliferative potential), permitted the development of limbal cultures for the treatment of patients with a partial deficiency of limbal stem cells.2 Limbal epithelial cells were obtained from a small biopsy specimen from a healthy area of the patient's cornea; after culturing, these cells developed into corneal epithelium, which was successfully transplanted back into the patient. This treatment represented a substantial advance in ocular-surface reconstruction. Currently, cell culture for the reconstruction of epithelium is performed on various cell substrates that can be sutured, are suitable for surgery, and are easy to handle, but these substrates have not routinely been tested for their capacity to maintain stem cells in culture. Several hundred patients with a deficiency of limbal stem cells have now been treated with this method, although the success rate has varied according to the cause of the deficiency.
Genetic diseases (such as aniridia) represent one group of causes that cannot be corrected by transplantation alone, with or without cultured limbal cells. For limbal stem-cell deficiency due to systemic diseases (such as the Stevens–Johnson syndrome) or other conditions (such as trauma, infections, or burns), the transplantation of cultured stem cells has often been useful, but the results have varied.
Ocular burns or damage due to infection or trauma, for example, can be treated successfully with the transplantation of cultured autologous stem cells in nearly 80 percent of patients. Of course, success rates among patients with deficiencies from any cause depend on several factors, including the availability of a small healthy area on the patient's ocular surface, the maintenance of stem cells in culture (particularly on novel, tissue-engineered culture surfaces), and the maintenance of critical structures such as the fornix and the tear film.
This technology cannot be used in patients with a total deficiency of limbal stem cells, since autologous stem cells are unavailable for transplantation. In cases of bilateral damage, limbal cells have been obtained from donor biopsy specimens. This procedure requires continuous immunosuppression, however, with associated side effects and risks.
In this issue of the Journal, Nishida and colleagues (pages 1187–1196) report the transplantation of epithelium cultured from autologous oral mucosal cells in four patients with the Stevens–Johnson syndrome who had a total deficiency of limbal stem cells. Follow-up over a mean period of 14 months revealed the recovery of visual function and the restoration of a normal-looking epithelium and good corneal transparency.
Do these results suggest that stem cells from the oral mucosa can differentiate into corneal epithelium? Not necessarily. It is not actually possible to confirm this hypothesis, since the cells express similar markers. Much previous research has suggested that adult epithelial cells retain their differentiation program even when transplanted at different sites in the body. An alternative explanation for the positive clinical results should therefore be considered. Certainly, if no stem cells are present on the corneal surface (i.e., if there is what I refer to as "anatomical" deficiency of limbal stem cells), the restoration of the corneal surface requires engraftment of autologous or donor stem cells that are able to restore the limbal reservoir. However, in some cases, residual stem cells are present on the ocular surface, but the number of cells is too small for them to cover the surface on their own or the cells cannot be stimulated by wound-healing signals (i.e., there may be a "functional" deficiency of limbal stem cells). When undamaged cultured epithelial cells are transplanted onto the ocular surface, they induce modifications of the microenvironment that make the ocular surface suitable for cell survival; this may result in the stimulation and proliferation of the recipient's own stem cells. Supporting this hypothesis are some reported cases of successful treatment of total limbal stem-cell deficiency with allogeneic stem cells, in which long-term follow-up revealed the presence of autologous cells3 that had not been active in promoting epithelialization before transplantation.
The human ocular surface, with its well-defined locations for different types of cells, provides a useful model for cell therapy. Additional experience with limbal stem-cell transplantation may pave the way for stem-cell therapy for other tissues and organs.
Source Information
From the Veneto Eye Bank Foundation, Epithelial Stem Cell Research Center, Ospedale Civile di Venezia, Venice, Italy.
References
Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology 1989;96:709-722.
Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997;349:990-993.
Henderson TR, Coster DJ, Williams KA. The long term outcome of limbal allografts: the search for surviving cells. Br J Ophthalmol 2001;85:604-609.(Graziella Pellegrini, Ph.)