Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2000-09-18
2002-11-26
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S325000, C435S366000, C435S378000, C435S395000
Reexamination Certificate
active
06485971
ABSTRACT:
This invention relates to a method of enriching for and isolating subpopulations of epithelia cells, isolation of keratinocyte stem cells, to keratinocyte stem cells and uses for keratinocyte stem cells.
BACKGROUND OF THE INVENTION
In common with other rapidly renewing tissues such as the haemopoietic system and the intestinal epithelia, the human epidermis is in a process of constant regeneration. Terminally differentiated cells lost continuously from the skin surface, are replaced by an intricate and highly regulated proliferative process within the basal layer of the epidermis. Stem cells in these rapidly renewing tissues are the earliest progenitors of a hierarchy of proliferative cells which are ultimately responsible for the generation of all mature cells for the lifetime of an individual (Lajtha, 1979). In murine epidermis, this process is achieved by two kinetically distinct subpopulations: (a) keratinocyte stem cells (KSC) which represent a minor subpopulation of relatively quiescent cells, defined by their great proliferative potential and an unlimited capacity for self renewal, identified as slow-cycling,
3
H-Tdr label-retaining cells; and (b) transit amplifying (TA) cells—the progeny of the stem cells, with a limited proliferative capacity identified as a pool of rapidly proliferating cells that are lest from the basal layer to terminal differentiation within 4-5 days (Potten, 1983: Morris et al, 1985; MacKenzie & Bickenbach, 1985; Potten, 1986; Bickenbach et al, 1986). In addition, a third Subpopulation of basal keratinocytes representing post-mitotic differentiating cells in the early stages of keralinisation can also be identified (Potten, 1983; Morris et al, 1985; MacKenzie & Bickenbach, 1985; Potten, 1986; Bickenbach et al, 1986; Christophers, 1971; Allen & Potten, 1974). Human epidermis has similar populations.
Given that all proliferative activity in the human epidermis is restricted to the basal layer, this is presumably where the stem cells and TA cells reside. It has also been established that the hair follicle can act as an important reservoir of epidermal stem cells, and that cells within the bulge region have extensive proliferative potential. Physiological cell renewal in interfollicular epidermis however, is most likely to be achieved by stem cells and TA cells within the basal layer. However there are no molecular markers that distinguish between basal keratinocytes that have made a commitment to differentiate (TA cells) and immature stem cells.
In the haemopoietic system, multilineage reconstituting stem cells can be physically separated from committed progenitor cells (analogous to the TA cells of the epidermis). based upon differences in their expression of cell surface markers (Civin et at, 1984; Spangrude et al, 1988; Berenson et al, 1991; Terstappen et al, 1991; Baum et al, 1992). Clearly the availability of appropriate cell surface markers on basal epidermal cells would greatly facilitate the isolation and characterisation of human KSCs. However, the cell surface antigenic phenotype of these cells remains relatively poorly defined.
One of the best studied classes of cell surface molecules expressed by keratinocytes are the integrin superfamily of cell adhesion receptors. Integrins are heterodimeric cell surface glycoproteins that primarily mediate the attachment of basal keratinocytes to extracellular matrix proteins found in the basement membrane, but can also mediate intercellular adhesion. In vivo, basal keratinocytes express the &bgr;
1
integrins &agr;
3
&bgr;
1
and as well as the integrin &agr;
6
&bgr;
4
(Peltonen et at, 1989; Carter et al, 1990a Carter et al, 1990b). Important evidence for proliferative heterogeneity in human basal keratinocytes has been provided by recent work using a fluorescence activated cell sorting (FACS) approach, demonstrating that both cultured and primary human foreskin keratinocytes could be separated into cells with high levels of &bgr;
1
integrin (&bgr;
1
bright) which had a high plating efficiency assayed after two weeks in culture, compared to those keratinocytes with low levels of this integrin (Jones & Watt, 1993; Jones et al, 1995). Furthermore, bright keratinocytes were shown to be capable of generating an epithelial sheet when grafted onto mice, suggesting that this fraction of the basal layer contain KSCs (Jones et al, 1995).
In vivo studies suggest that epidermal stem cells constitute between 1%-10% of the basal layer depending on the methodology used (Morris et al, 1985; MacKenzie & Bickenbach, 1985; Bickenbach et al, 1986; Potten & Hendry, 1973; Morris & Potten, 1994). Since approximately 40% of the basal layer in human foreskin exhibits high levels of &bgr;
1
integrin in vim (Jones et at, 1995) it is highly likely that basal keratinocytes with this phenotype contain both the KSC population and a significant number of TA cells and therefore there are drawbacks in the use of cells enriched for high level expression of &bgr;
1
.
OBJECT OF THE INVENTION
An object of one aspect of the invention is to generate a more purified population of keratinocyte stem cells than has been achieved by prior art methods. An object of a further aspect of the present invention is to provide methods for purifying subpopulations of epithelial cells.
SUMMARY OF THE INVENTION
A strategy for distinguishing between the TA cells and the KSCs of the epidermis based on the use of two cell surface antigens has been shown to be effective. In view of functional data demonstrating the role of integrin &agr;
6
&bgr;
4
in mediating adhesion of basal keratinocytes to the basement membrane via hemi-desmosomes (Sonnenberg et al, 1991; Dowling et at, 1996; Georges-Labouessee et al, 1996; Van-der-Neut et al, 1996) it was hoped that this integrin may provide a suitable marker for epidermal stem cells since these cells are permanently anchored to the basement membrane.
It is now shown that while basal keratinocytes expressing low levels of &agr;
6
&bgr;
4
represent a subpopulation of post-mitotic, differentiating keratinocytes, this integrin is expressed at high levels on both the KSC and TA cells. Thus this cell surface marker alone, cannot be used to separate KSCs from TA cells to a high degree of purity but can do so to a degree of purity higher than where &bgr;
1
integrin is used.
It is the finding of the inventors that enrichment for human KSCs to a high degree of purity can be successfully achieved on the basis of a second cell surface component whose expression is proliferation-related in conjunction with &agr;
6
&bgr;
4
integrin. The experiments conducted to date have used transferrin receptor as the cell surface component that is proliferation related. It is also suggested that sufficient purification should be achievable where another marker capable of identifying KSC and TA cells (and perhaps also cells that have been differentiated further) is used in place of &agr;
6
&bgr;
4
in the above two step process and that other marker might be another integrin such as &agr;
2
&bgr;
1
or &agr;
3
&bgr;
1
.
In a first aspect the invention could be said to reside in a method of enriching a viable population of KSCs from a population of epidermal cells comprising,
a) a first enriching step of enriching for cells carrying a high level of cell surface integrin from the population of epidermal cells to form a partially enriched pool, and
b) a second enriching step of removing cells that carry high level expression of a marker associated with proliferation from the partially enriched pool.
Conversely TA cells might be purified from KSCs whereby a proportion of cells with low expression of a marker associated with proliferation are removed from the partially enriched pool.
The epidermal cell population might be derived (torn a tissue sample of the skin. This method normally involves the separation of epidermis from the skin sample, before the enrichment. One particularly good source of KSC cells is from the basal layer of the epidermis. The proportion of these cells that are KSCs will depend upon the type of skin, and the age of
Kaur Pritinder
Simmons Paul J.
Davis Katharine F
Peter MacCallum Cancer Institute
Yucel Remy
LandOfFree
Keratinocyte stem cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Keratinocyte stem cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Keratinocyte stem cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2955675