血液幹細胞和眼角膜幹細胞關係之研究(3/3)
Date Issued
2004
Date
2004
Author(s)
胡芳蓉
DOI
922314B002157
Abstract
The cornea comprises three major cellular layers: an outermost stratified, non-keratinized
squamous epithelium, a stroma with keratocytes, and an innermost monolayer of specialized
endothelial cells. The structure of the cornea allows it to serve as a barrier to the outside
environment and as a major element in the optical pathway of the eye. The cornea is
transparent, avascular, and immunologically privileged, making it an excellent candidate for
tissue engineering for transplantation. The corneal epithelium is maintained by stem cells,
which reside in the basal layer of the limbus. Depletion of the limbal stem-cell pool results in
an abnormal corneal surface, which cannot be normalized without the introduction of a new
source of stem cells. Corneal diseases as a result of endothelial cell dysfunction may cause
epithelial and stromal edema and penetrating keratoplasty is the surgical choice to improve
vision. However, due to the shortage of cornea donor, especially with the universal of
refractive surgery, to investigate the potential of adult stem cell therapy in corneal diseases is
imperative.
Adult stem cells have several advantages as compared with embryonic stem cells (ES
cells) which have ethical burden and immunologic concerns. Recent data suggest that adult
stem cells generate differentiated cells beyond their own tissue boundaries, a process termed
“developmental plasticity. This finding has made the adult stem cells being a practical source
of cell therapy for tissue regeneration after trauma, disease, or aging. Not restricted in in-vitro
studies, both preclinical and clinical trials of various adult stem cells for tissue repair or
replacement have been applied in many disease categories. At present, the hempatopoietic
stem cells (HSCs), and the stem-like cells for nonhematopoietic tissues which are currently
referred to as mesenchymal stem cells (MSCs), are the most potential sources of adult stem
cells. Their multipotentiality to differentiate into various cells, such as bone, cartilage,
adipocytes and myocyte are proved by many researchers. Strategy using human MSCs for the
treatment of the children with osteogenesis imperfecta had aroused us to investigate the
possibility of using similar strategy on treating corneal diseases. In our present study, we
focused on the MSCs due to encouraging initial results.
The purposes of our project are threefolds:
(1) To culture and propagate the human mesenchymal stem cells (hMSC) in vitro, to maintain
the undifferentiated status of these cells, and identified the cells by various surface markers
proving by flow cytometry.
(2)To induce the transdifferentiation of cultured human mesenchymal stem cells(hMSC) in
the co-culture system with adding different growth factors and cytokines, to simulate the
microenvironment, or so-called niche, of stem cell differentiation.
(3)To prove the transdifferentiated phenotype of these induced human mesenchymal stem
cells by RT-PCR on the RNA level and by immunofluorescent staining on the protein level.
In our study, we had successfully isolated, cultured and propagated the hMSC and kept
the undifferentiated status in a prolonged period. We had also induced the transdifferentiation
of hMSC into cells with the morphology similar to the normal human corneal endothelial cells
in a co-culture system with adding various growth factors and cytokines such as human
leukemia inhibitory factor (hLIF), TGF-β1, TGFβ2, human epidermal growth factor (hEGF),
and human fibroblast growth factor (hFGF) at different time point. We also induced the
expression of a relative specific marker, type VIII collagen, of corneal endothelial cells and
proved the gene expression by RT-PCR on the RNA level and by immunofluorescent staining
study on the protein level. However, due to the highly variable co-culture condition and
possible heterogeneity of the hMSC, our results cannot be reproduced in every repeated
experiment. Besides, the key factor to determine this transdifferentiation is still not defined
from our study due to the highly complexity of co-culture system.
In the future, further effort has to be made to define the key factor that determines the
transdifferentiation, to induce other specific functional marker like Na+-K+ ATPase of corneal
endothelial cells to make the clinical application feasible. Besides, to culture a mesenchymal
stem cell clone with incorporated marker such as EGFP (enhanced green fluorescent protein)
will make the establishment of the in vivo model of MSC transplantation easier.
Subjects
Human mesenchymal stem cells
adult stem cells
corneal endothelial cells
regenerative medicine
SDGs
Publisher
臺北市：國立臺灣大學醫學院眼科
Type
journal article
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