U.S. patent application number 10/147925 was filed with the patent office on 2003-01-30 for differentiation of specialized dermal and epidermal cells into neuronal cells.
Invention is credited to Broaddus, William C., Fillmore, Helen, Gillies, George, Hoover, Shelley.
Application Number | 20030022369 10/147925 |
Document ID | / |
Family ID | 26845341 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022369 |
Kind Code |
A1 |
Fillmore, Helen ; et
al. |
January 30, 2003 |
Differentiation of specialized dermal and epidermal cells into
neuronal cells
Abstract
Cells are generated from skin biopsies for use in cell
implantation by identifying a source of skin cells that have a
surface concentration of at least one cell selected from the group
consisting of keritinocytes and/or melanocytes; taking a sample of
tissue from the surface area; mechanically disaggregating the
tissue samples; collecting the disaggregated cells; washing the
disaggregated cells; filtering the washed disaggregated cells;
providing a cell suspension with filtered and washed keritinocytes
and/or melanocytes; and suspending the cell suspension in a medium.
In greater particularity, the method may comprise a) identifying a
source of skin cells that have a surface concentration of at least
400 cells per square millimeter of surface area of at least one
cell selected from the group consisting of keritinocytes and/or
melanocytes; b) taking a sample of those skin cells and culturing
them to enrich for cells selected from the group consisting of
keritinocytes and melanocytes to form cultured cells for
transplantation purposes c) concentrating the cells selected from
the group consisting of keritinocytes and/or melanocytes from the
cultured mass of cells; and d) differentiating the concentrated
cells selected from the group consisting of keritinocytes an/or
melanocytes.
Inventors: |
Fillmore, Helen; (US)
; Hoover, Shelley; (Richmond, VA) ; Broaddus,
William C.; (Midlothian, VA) ; Gillies, George;
(Charlottesville, VA) |
Correspondence
Address: |
WHITHAM, CURTIS & CHRISTOFFERSON, P.C.
11491 SUNSET HILLS ROAD
SUITE 340
RESTON
VA
20190
US
|
Family ID: |
26845341 |
Appl. No.: |
10/147925 |
Filed: |
May 20, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60292176 |
May 18, 2001 |
|
|
|
Current U.S.
Class: |
435/371 ;
424/93.7 |
Current CPC
Class: |
C12N 2506/091 20130101;
C12N 5/0618 20130101; C12N 5/0626 20130101; C12N 5/0629 20130101;
C12N 2510/00 20130101 |
Class at
Publication: |
435/371 ;
424/93.7 |
International
Class: |
C12N 005/08 |
Claims
What is claimed:
1. A method for generating cells for use in cell implantation
comprising: a) identifying a source of skin cells that have a
surface concentration of at least 400 cells per square millimeter
of surface area of at least one cell selected from the group
consisting of keritinocytes and melanocytes; b) taking a sample of
those skin cells and culturing them to increase the surface
concentration of cells selected from the group consisting of
keritinocytes and melanocytes to form a cultured mass of cells; c)
concentrating the cells selected from the group consisting of
keritinocytes and melanocytes from the cultured mass of cells; and
d) differentiating the concentrated cells selected from the group
consisting of keritinocytes and melanocytes.
2. The method of claim 1 wherein the differentiated cells are
implanted into a patient.
3. The method of claim 1 wherein the identified cells comprise
melanocytes.
4. The method of claim 1 wherein the identified cells are
keritinocytes.
5. The method of claim 3 wherein the surface concentration of
melanocytes is at least 500 melanocytes per square millimeter of
skin.
6. The method of claim 3 wherein the surface concentration of
melanocytes is at least 700 melanocytes per square millimeter of
skin.
7. The method of claim 3 wherein the surface concentration of
melanocytes is at least 800 melanocytes per square millimeter of
skin.
8. The method of claim 4 wherein the surface concentration of
keritinocytes is at least 500 melanocytes per square millimeter of
skin.
9. The method of claim 1 further comprising the step of
concentrating the cells selected from the group consisting of
keritinocytes and melanocytes
10. The method of claim 1 wherein said step of differentiating in
carried out by a technique selected from the group consisting of
genetic manipulation, the use of growth factors or inhibitors, or
transplantation into a host.
11. The method of claim 1 wherein said step of differentiating in
carried out by the technique of genetic manipulation, wherein one
or more genes which are inserted are under the control of an
inducible promoter system.
12. The method of claim 11 wherein said inducible promoter system
is selected from the group consisting of tetracycline induced and
magnetic resonance imaging induced.
13. The method of claim 11 wherein said one or more gene is
selected from the group consisting of EGFP and tyrosine
hydroxylase.
14. The method of claim 1 wherein said source of skin cells is
selected from the group consisting of skin biopsy tissue, mole
biopsy tissue, and fetal foreskin.
15. A method for generating cells from skin biopsies for use in
cell implantation comprising: identifying a source of skin cells
that have a surface concentration of at least one cell type
selected from the group consisting of keritinocytes and
melanocytes; taking a sample of tissue from the surface area;
chemically or mechanically disaggregating the tissue sample;
collecting the disaggregated cells; washing the disaggregated
cells; filtering the washed, disaggregated cells; providing a cell
suspension with filtered and washed keritinocytes or melanocytes;
suspending the cell suspension in a medium.
16. The method of claim 15 wherein the source of skin cells has a
surface concentration of keritinocytes and melanocytes of at least
400 cells per square millimeter of surface area of skin.
17. The method of claim 15 wherein the medium in which the cell
suspension is suspended comprises a nutritional medium.
18. The method of claim 17 wherein the nutritional medium is
selected from the group consisting of DMEM/F12+N2 supplement, bFGF,
EGF containing less than 10% fetal calf serum, and Melanocyte basal
medium.
19. The method of claim 15 wherein said step of providing further
includes the step of separating said cell type by the use of
specific surface antibodies.
20. The method of claim 15 wherein said source of skin cells is
selected from the group consisting of skin biopsy tissue, mole
biopsy tissue, and fetal foreskin.
21. A method for the implantation of cells generated from skin
biopsies, comprising the steps of a) identifying a source of skin
cells that have a surface concentration of at least 400 cells per
square millimeter of surface area of at least one cell selected
from the group consisting of keritinocytes and melanocytes; b)
taking a sample of said skin cells and culturing them to increase
the surface concentration of cells selected from the group
consisting of keritinocytes and melanocytes to form a cultured mass
of cells; c) concentrating the cells selected from the group
consisting of keritinocytes and melanocytes from the cultured mass
of cells; d) differentiating the concentrated cells selected from
the group consisting of keritinocytes and melanocytes; and e)
transplanting said differentiated cells into a host.
22. A method for producing cells for cell implantation or cell
transplantation therapies, comprising the steps of: obtaining a
sample of skin cells which includes at least one cell selected from
the group consisting of keritinocytes and melanocytes at a surface
concentration of at least 400 cells per square millimeter;
culturing said sample of skin cells; concentrating cells selected
from the group consisting of keritinocytes and melanocytes from a
cultured mass of cells generated in said culturing step; and
isolating whole differentiated implantable or transplantable cells
from a concentration of cells generated in said concentrating
step.
23. The method of claim 22 wherein said obtaining step is performed
by skin biopsy.
24. The method of claim 22 wherein undifferentiated and
differentiated cells remain together during the process until said
isolating step.
25. The method of claim 22 wherein said obtaining step includes the
steps of: disaggregating said sample to produce disaggregated
cells; washing and filtering said disaggregated cells; and
suspending said disaggregated cells in a suspension medium.
26. The method of claim 25 wherein said suspension medium includes
nutrients for said disaggregated cells.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the field of cellular
biology, particularly to the field of cell implantation therapy,
more particularly to the development of a source of cells that may
be used directly or for the cloning of cells for use in cell
implantation therapy, and more particularly to the in vivo
dedifferentiation of certain skin cells as implantable cells for
the production of cells for transplantation or implantation cell
therapies.
[0003] 2. Background of the Art
[0004] Cell implantation requires significant levels of control in
the introduction of cells. The cells must be capable of performing
the specific functional replacement or addition therapy desired in
the transplantation or implantation, and the cells must be
compatible with the receiving organism. Present techniques
emphasize the use of same species differentiated cells or the use
of compatible undifferentiated cells for use in the implantation
therapy. Conservative protocol has generally motivated
investigators to prefer same species undifferentiated cells or
specialized same species differentiated cells for use in such
procedures. This considered preference has limited the source of
cells that can be used for implantation therapy. For example,
viable, mature, differentiated cells are not readily available, and
the moral implications of fetal tissue collection of
undifferentiated cells has limited the sources of such cells, as
well as the natural quantity limitation on the source itself.
[0005] Cell implantation using tissue-derived nuclear transfer
embryos has been performed, with attendant nuclei replacement to
provide the targeted specialty cells for implantation. Bovine skin
fibroblast cells were transferred into enucleated bovine oocytes,
resulting in a fetus. The ventral mesoncephalon was isolated from
the bovine fetus. When transplanted, this tissue successfully
ameliorated symptoms in the Parkonsonian rat (Zawada et al., 1998,
Nature Medicine, 4:569-574). Skin fibroblast cells from cows,
sheep, pigs, monkeys, and rats have been used as sources of donor
nuclei, as well as nuclei from human sources.
[0006] The clinical management of numerous neurological disorders
has been frustrated by the progressive nature of degenerative,
traumatic or destructive neurological diseases and the limited
efficacy and the serious side-effects of available pharmacological
agents. Because many such diseases involve destruction of specific
"neuronal clusters" or brain regions, it has been hoped that
grafting of neural cells or neuron-like cells directly into the
affected brain region might provide therapeutic benefit. Cell
transplant approaches have taken on a major emphasis in current
Parkinson's disease research, and may prove useful in promoting
recovery from other debilitating diseases of the nervous system
including Huntington's disease, Alzheimer's disease, severe seizure
disorders including epilepsy, familial dysautonomia, as well as
injury or trauma to the nervous system. In addition, the
characterization of factors which influence neurotransmitter
phenotypic expression in cells placed into the brain may lead to a
better understanding of normal processes and indicate means by
which birth defects resulting from aberrant phenotypic expression
can be therapeutically prevented or corrected. Neurons or
neuronal-like cells can be grafted into the central nervous system
(CNS), in particular, into the brain, either as solid tissue blocks
or as dispersed cells. However, to date, a number of problems of
both a technical and ethical nature have plagued the development of
clinically feasible grafting procedures.
[0007] Parkinson's disease results from a selective loss of
dopaminergic nigrostriatal neurons, resulting in a loss of input
from the substantia nigra to the striatum. Solid grafts of tissues
potentially capable of producing dopamine, such as adult adrenal
medulla and embryonic substantia nigra (SN), have been used
extensively for experimental grafting in rats and primates treated
with 6-hydroxydopamine (6-OHDA) to destroy dopaminergic cells
(Dunnett, S. B. et al., Brain Res. 215: 147-161 (1981); ibid. 229:
457-470 (1981); Morisha, J. M. et al., Exp. Neurol. 84: 643-654
(1984); Perlow, M. J. et al., Science 204: 643-647 (1979)). Grafts
of embryonic SN have also been used as therapy for primates
lesioned with the neurotoxin 1 -methyl-4-phenyl-
1,2,3,4-tetrahydropyridine (MPTP), which produces a
Parkinson's-like disease (Redmond, D. E. et al., Lancet 8490:
112-27 (1986)).
[0008] The methods of the present invention are useful for treating
a number of human neurological disease. Parkinson's Disease can be
treated according to the present invention by implanting
dopamine-producing cells in the recipient's striatum. Alzheimer's
disease involves a deficit in cholinergic cells in the nucleus
basalis. Thus, according to the invention, a subject having
Alzheimer's disease or at risk therefore may be implanted with
cells producing acetylcholine.
[0009] Huntington's disease involves a gross wasting of the head of
the caudate nucleus and putamen, usually accompanied by moderate
disease of the gyrus. A subject suffering from Huntington's disease
can be treated by implanting cells producing the neurotransmitters
gamma amino butyric acid (GABA), acetylcholine, or a mixture
thereof. According to the present invention, the support matrix
material to which such cells are attached is preferably implanted
into the caudate and putamen.
[0010] Epilepsy is not truly a single disease but rather is a
symptom produced by an underlying abnormality. One skilled in the
art will appreciate that each epileptic subject will have damage or
epileptic foci which are unique for the individual. Such foci can
be localized using a combination of diagnostic methods well-known
in the art, including electroencephalography, computerized axial
tomography and magnetic resonance imaging. A patient suffering from
epilepsy can be treated according to the present invention by
implanting the support matrix material to which GABA-producing
cells are attached into the affected site. Since blockers of
glutamate receptors and NMDA receptors in the brain have been used
to control experimental epilepsy, cells producing molecules which
block excitatory amino acid pathways may be used according to the
invention. Thus implantation of cells which have been modified as
described herein to produce polyamines, such as spermidine, in
larger than normal quantities may be useful for treating
epilepsy.
[0011] The methods of the present invention are intended for use
with any mammal that may experience the beneficial effects of the
methods of the invention. Foremost among such mammals are humans,
although the invention is not intended to be so limited, and is
also applicable to veterinary uses.
[0012] Thus, while the feasibility of the transplant approach has
been established experimentally, this approach is severely limited
by the need for the use of fetal tissue or specifically
differentiated cells from the same organ of the organism, which is
of limited availability and potentially of great political
consequence. In essence, transplantation of human fetal tissue from
aborted pregnancies has been prohibitive in the United States. It
would thus be of great benefit if simple, routine and safe methods
for the successful transplantation of socially acceptable and
available tissue into the brain were available for the treatment of
debilitating neurological disease.
[0013] One potential approach to this problem has been the
implantation of adult cells, attempted by Aebischer and his
colleagues, who have successfully implanted into the brain
selectively permeable biocompatible polymer capsules encapsulating
fragments of neural tissue which appeared to survive in this
environment (Aebischer, P. et al., Brain Res. 448: 364-368 (1988);
Winn, S. R. et al., J. Biomed Mater Res. 23: 31-44 (1989). The
polymer capsules, consisting of a permselective polyvinyl chloride
acrylic copolymer XM-50, completely prevented the invasion of the
encapsulated tissue by host cells. Based on the permeability,
antibodies and viruses would be expected to be excluded as well.
When dopamine-releasing polymer rods were encapsulated into such a
permselective polymer and implanted into denervated striatum in
rats, alleviation of experimentally-induced Parkinson disease
symptoms was achieved (Winn S. R. et al., Exp. Neurol. 105: 244-50
(1989). Furthermore, U.S. Pat. No. 4,892,538 (Aebischer et al.,
issued Jan. 9, 1990) discloses a cell culture device for
implantation in a subject for delivery of a neurotransmitter
comprising secreting cells within a semipermeable membrane that
permits diffusion of the neurotransmitter while excluding viruses,
antibodies and other detrimental agents present in the external
environment. The semipermeable membrane is of an acrylic copolymer,
polyvinylidene fluoride, polyurethane, polyalginate, cellulose
acetal, polysulphone, polyvinyl alcohol, polyacrylonitrile, or
their derivatives or mixtures and permits diffusion of solute of up
to 50 kD molecular weight. This device was said to be useful in
treatment of neurotransmitter-deficient conditions, such as
Parkinson's disease, by sustained, local delivery of
neurotransmitters, precursors, agonists, fragments, etc., to a
target area, especially the brain. The device may be made
retrievable so that the contents may be renewed or supplemented,
and the cells are protected against immunological response and
viral infection.
[0014] By the term "neural or paraneural origin" is intended a cell
which is derived from the embryonic neural crest. A preferred
example of a cell of paraneural origin is an adrenal medullary
chromaffin cell. The precursor cells to the mammalian adrenal
medulla are of neural crest origin and possess the potential to
develop along either neuronal or endocrine lines of differentiation
(Bohn, M. C. et al., 1981, supra, Devel. Biol. 89: 299-308 (1982);
Unsicker, K., Develop. Biol. 108: 259-268 (1985)). Chromaffin cells
from the rat, monkey, and human adrenal medulla, when removed from
adrenal cortical influences and exposed to nerve growth factor
(NGF), change from an endocrine to a neuronal phenotype (Notter, M.
F. et al., Cell Tiss. Res. 244: 69-70 (1986); Stromberg, I. et al.,
Exp. Brain Res. 60: 335-349 (1985); Unsicker, K. et al., 1978,
supra). When co-grafted with cerebral cortical or hippocampal
tissue into the anterior chamber of the rat eye, adrenal chromaffin
cells form nerve fibers which innervate the adjacent co-grafted
brain tissue (Olson, L. A. et al., Exp. Neurol. 70414-426 (1980)).
Another paraneural cell type is a retinal pigment epithelium cell
(Song, M-K et al., J. Cell. Physiol. 148: 196-203 (1990)).
[0015] U.S. Pat. No. 5,958,767 discloses that clones of human NSCs
(neural stem cells) --unambiguously affirmed by the presence of a
common retroviral insertion site and propagated by either
epigenetic or genetic means --can participate in normal CNS
development in vivo and respond to normal microenvironmental cues,
including migration from various germinal zones along
well-established migratory routes to widely disseminated regions. A
single NSC is capable of giving rise to progeny in all 3
fundamental neural lineages--neurons (of various types),
oligodendroglia, and astroglia (hence, multipotency)--as well as
giving rise to new NSCs with similar potential (i.e.,
self-renewal). In vivo, following transplantation into mouse hosts,
a given human NSC clone is sufficiently plastic to differentiate
into neural cells of region- and developmental stage-appropriate
lineages along the length of the neural axis: into neurons where
neurogenesis normally persists, and into glia where gliogenesis
predominates, emulating patterns well-established for endogenous
murine progenitors, with which they intermingle seamlessly. Thus,
for example, they will give rise to neurons following migration
into the Olfactory Bulb (OB) at one end of the neuraxis and into
granule neurons in the cerebellum at the other, yet also yield
astroglia and oligodendroglia, the appropriate cell types born in
the postnatal neocortex, subcortical white matter, and striatum. Of
additional significance, as might be expected of a true stem cell,
many of the neuronal types into which these NSCs could
differentiate, are born not at the developmental stage from which
the cells were initially obtained (e.g. midgestation), but rather
at the stage and region of NSC implantation, thus affirming
appropriate temporal (in addition to regional) developmental
responsiveness.
[0016] Neural stem cells were thought to have limited
differentiation capabilities within the species. However, adult
mouse NSC were transferred into early mouse and chick embryos and
contributed to all three germ layers in the developing mouse and
chick embryo. This demonstrated that these particular NSC cells
were capable of differentiating into multiple cell types (Clarke et
al., 2000 Science vol 288:1660). The mechanism was not stated to be
effective for other than the chosen organisms, there was no
specific mechanism described such as transdifferentiaion or
mutation, and there was no indication that determined cells could
actually reverse their direction of differentiating and
differentiate in a different direction in human neural stems cells,
which are quite advanced compared to the cells selected in this
analysis.
[0017] Stenevi et al. (Brain Res. 114: 1-20 (1976) found that the
best results were obtained with fetal CNS neurons which were placed
next to a rich vascular supply. In fact, a review of the literature
reveals that tissue from almost every area of the fetal brain can
be successfully transplanted if care is taken with procedural
details (see, for example, Olson, L. A. et al., In: Neural
Transplants: Development and Function, Sladek, J. R. et al., eds,
Plenum Press, New York, 1984, pp. 125-165).
[0018] Embryonic tissue provides an excellent source of cells that
will differentiate in a foreign environment and become integrated
with the host tissue. For example, grafts of embryonic SN into
6-OHDA treated rats have been shown to produce dopamine, to reduce
apomorphine- or amphetamine-induced rotation, to alleviate sensory
deficits and to make synapses in the host striatum (Dunnett et al.,
Morisha et al., Perlow et al., supra). Grafted neurons are also
spontaneously active, thus mimicking normal adult SN neurons
(Wuerthele, S. M. et al., In: Catecholamines, Part B, (E. Usdin et
al., eds.), A. R. Liss, Inc., New York, pp. 333-341).
[0019] In contrast to successful grafting of fetal neural tissue,
mature CNS neurons have never been found to survive in transplants
(Stenevi, U. et al., Brain Res. 114: 1-20 (1976)). The reason fetal
CNS neurons survive grafting procedures, while adult neurons do not
is uncertain, but probably related to several factors. First, fetal
neurons are less affected by low oxygen levels than mature neurons
(Jilek, L., In: Developmental Neurobiology, Himwich, W. A., ed., C.
C. Thomas Publisher, Springfield, Ill., 1970, pp. 331-369), and
grafting procedures necessarily involve periods of anoxia until an
adequate blood supply to the transplant is established. Secondly,
fetal neurons seem to survive best when they are taken during a
rapid growth phase and before connections are established with
target tissues (Boer, G. J. et al., Neuroscience 15: 1087-1109,
(1985)). Also, fetal tissue may be especially responsive to growth
(or "survival") factors that are known to be present in the milieu
of the damaged host brain (Nieto-Sampedro, M. et al., Science 217:
860-861 (1982); Proc. Natl. Acad. Sci. USA 81: 6250-6254
(1984)).
[0020] In further human studies (Lieberman, supra; Lindvall, O., J.
Neurol. Neurosurg. Psychiat., 1989, Special Supplement, pp. 39-54;
Bakay, R. A. E., Neurosurg. Clin. N. Amer. 1: 881-895 (1990)),
autologous grafts have been attempted to replace the need for fetal
material. In this procedure the patients first underwent initial
abdominal surgery for the removal of a healthy adrenal gland. The
patient then was subjected to similar neurosurgery as that for the
fetal adrenal transplant. The surgical morbidity-mortality for the
combined adrenalectomy/neurosurgery was expectedly high. The
ultimate therapeutic result was claimed to be as high as 30% but
may have been as low as one patient in the series of six. There was
no evidence that the adrenal material transplanted into these
patients survived.
[0021] However, despite the promise of fetal tissue and cell
transplants, the art has turned to alternate sources of donor
tissues for transplantation because of the ethical, moral, and
legal problems attendant to utilizing fetal tissue in human
medicine. These sources include neural and paraneural cells from
organ donors and cultured cell lines. (See, for example: Gash, D.
M. et al., In: Neural Grafting in the Mammalian CNS, Bjorklund, A.
et al., eds, Elsevier, Amsterdam, 1985, pp. 595-603; Gash, D. M. et
al., Science 233: 1420-22 (1986)).
[0022] There are suggestions in the literature that there may be an
additional advantage of grafting dissociated cells compared to
blocks of tissue in that the cells can be precultured with various
substances such as growth factors prior to grafting or they can be
co-grafted with other cells or substances which promote specific
parameters of differentiation. Furthermore, glial cells may have
specific regional effects and produce neuronal growth factors
(Barbin, G. et al., Devel. Neurosci. 7: 296-307 (1985);
Schurch-Rathgeb, Y. et al., Nature 273: 308-309 (1978); Unsicker,
K. et al. Proc. Natl. Acad. Sci. USA 81: 2242-2246 (1984);
Whitaker-Azmitia, P. M. et al., Brain Res. 497: 80-85 (1989)). This
suggests that co-transplanting cells providing the desired
neurotransmitters along with specific types of glia that produce
glial-derived factors, may promote neuronal growth and the desired
differentiation of grafted cells.
[0023] Although early clinical experiments using the grafting
approach did not result in long-lasting effects, an initial report
of one study appeared more promising (Madrazo et al., Soc.
Neurosci. Abstr. 12: 563 (1986); for an overview, see: Lieberman,
A. et al., Adv. Tech. Stand. Neurosurg. 17: 65-76 (1990), which is
hereby incorporated by reference). However, the surgical procedure
used required craniotomy or full "open brain" surgery in which a
portion of healthy striatum was removed and replaced with "chunks"
of fetal adrenal gland. The therapeutic results obtained were
somewhat controversial. However, both the need for serious
neurosurgery in an already debilitated population and the need to
use fetal tissue makes this approach undesirable.
[0024] Transdetermination has been observed in lower orders such as
Drosophila, where a sample of cultured imaginal cells sometime
differentiate into a structure appropriate to an imaginal disc
other than that from which the culture was derived.
Transdetermination represent a switch from one heritable state to
another and so resembles the consequence of genetic mutation. (see
Bruce Alberts, et al., Molecular Biology of the Cell, 1983, Garland
Publishing Co., New York, N.Y., Ch. 15, pages 838-839). This
phenomenon has been reported as occurring with groups of cells, but
with cells of both the mutant and normal genotypes present.
[0025] Neural progenitor/stem cells obtained from fetal tissue or
non-human tissue have been shown to be effective for cell
replacement therapy for neurodegenerative disorders, head trauma,
stroke and spinal cord injuries, and have been extrapolated to
predict similar efficacy in repair of any type of nerve cell or
brain cell damage. It has been recently found that neural
progenitor/stem cells exist in the adult human brain, and that when
these cells are cultured, the cells repeatedly divide and can
(under the appropriate influences well defined in the art)
differentiate into neurons, astorcytes and oligodendroglia.
[0026] In addition, PCT application WO 98/07841 discloses that one
human cross species embryo was produced using human oral cavity
epithelium as the donor nucleus. One NT unit developed to what was
asserted to be a blastocyst stage embryo. This was placed on a
feeder layer of cells. A cell mass appeared on the plate. However,
there was no report of any information to suggest that the cell
line was of human origin.
[0027] U.S. Pat. No. 6,087,168 (Levesque et al.) describes a method
of converting, or transdifferentiating the epidermal cells into
viable neurons useful in both cell therapy and gene therapy
treatment methodologies. The method of transdifferentiating
epidermal cells into neuronal cells comprises the following steps:
obtaining skin cells from a patient; dedifferentiating these cells
with an appropriate medium, neurotrophin or cytokine; transfecting
the skin cells with one or more expression vector(s) encoding at
least one neurogenic transcription factor or active fragments
thereof; expressing at least one of the neurogenic transcription
factors; growing the transfected cells in an appropriate medium;
and adding to the medium one or more antisense oligonucleotide(s)
corresponding to at least one negative regulator of neuronal
differentiation, whereby the epidermal cells are
transdifferentiated into neuronal cells. The Experimental Basis of
that Invention is described as a transdifferentiation process
involving the following basic steps of:
[0028] 1. Isolation of proliferating epidermal basal cells from the
skin of a patient in need;
[0029] 2. Dedifferentiation of epidermal basal cells in calcium
free growth media;
[0030] 3. Expression of neurogenic basic-Helix-Loop-Helix (NeuroD1,
NeuroD2, ASH1) and/or Zn-finger (Zic3, MyT1) transcription factors
with simultaneous suppression of the expression of homeobox genes
MSX1 and bHLH transcription factor HES1 in epidermal basal cells;
and
[0031] 4. Growing cells resulting from step 3 (cells which
over-express neurogenic transcription factors and have suppressed
expression of MXS1 and HES1) in the presence of low concentrations
of all-trans retinoic acid and various neurotrophins, such as,
BDNF, NGF, NT-3, and NT-4.
[0032] In the first step of that invention, epidermal or skin cells
are obtained from a patient in need. These epidermal cells are
obtained or isolated via any type of surgical procedure.
Preferably, these isolated cells are epidermal basal cells obtained
from the skin of a patient. However, epithelial, or any other type
of basal cell or proliferating cell population, can be used for the
conversion of these cells into neurons.
[0033] In the second step of that process, preferentially epidermal
basal cells are dedifferentiated in a calcium free growth medium.
This step involves treatment of the cells obtained in step one so
that the cells lose the majority of differentiation specific gene
expression to become dedifferentiated, that is, more primitive or
developmentally less advanced. The dedifferentiation process is
significant in that it allows for reprogramming of the neuronal
development pathway. Since calcium ions are required to support
development of keratinocytes (skin cells) from basal cells, removal
of calcium results in dedifferentiation of basal cells. In other
proliferating cell types, however, calcium may not be necessary to
support development of any particular developmental pathway that is
being deregulated. Other means to achieve the desired end of
dedifferentiation involve treating the cells with specific growth
factor or cytokines. Also, altering the specific gene expression
pathway that is responsible for differentiation of epidermal cells
by genetic manipulation may be used instead of eliminating calcium
in the growth media. Moreover, elimination of calcium may not be
required if other than proliferating epidermal basal cells are
used.
[0034] In the third step, the process of that invention utilizes
molecular manipulation techniques to alter the cell differentiation
pathway of epidermal cells. This alteration is accomplished by
allowing for the expression of neurogenic transcription factors,
such as the basic-Helix-Loop-Helix factors, Neuro D1, Neuro D2, or
ASH1, and/or zinc-finger transcription factors, such as Zic3 or
MyT1, while simultaneously, or near simultaneously, suppressing the
expression of genes responsible for suppression of the neuronal
development pathway, such as the basic-Helix-Loop-Helix factor HES1
and/or the homeobox factor MSX1. In addition to these genes, any
other set of neurogenic and anti-neurogenic genes can be
manipulated so as to achieve the desired end of
transdifferentiation of epidermal cells or other proliferating cell
types. Manipulations that can be used in this step of the inventive
process include the use of variety of gene transfer protocols, such
as microinjection of expression constructs, and a variety of DNA
transfection techniques (such as, lipofections, liposomes,
coprecipitation techniques, and different carriers), and viruses.
Also protein transfer methods can be used to transiently express
neurogenic transcription factors in the proliferating
dediffernentiated cells. Finally, in the fourth step of that
invention, the transdifferentiated cells are preferably grown in
the presence of a retinoid, such as all trans retinoic acid or
vitamin A derivatives. In addition, neurotrophins or cytokines,
such as BDNF, NGF, NT-3, NT-4, IL-6, can be used to obtain a
substantial population of transdifferentiated neuronal cells. This
step is optional in that it is not required for
transdifferentiation. However, treatment with a retinoid and at
least one neurotrophin increases the number of cells obtained.
SUMMARY
[0035] A method for generating cells from skin biopsies for use in
cell implantation comprises: identifying a source of skin cells
that have a surface concentration of at least one cell selected
from the group consisting of keritinocytes and/or melanocytes;
taking a sample of tissue from the surface area; mechanically
disaggregating the tissue samples; collecting the disaggregated
cells; washing the disaggregated cells; filtering the washed
disaggregated cells; providing a cell suspension with filtered and
washed keritinocytes or melanocytes; and suspending the cell
suspension in a medium. In greater particularity, the method may
comprise a) identifying a source of skin cells that have a surface
concentration of at least 400 cells per square millimeter of
surface area of at least one cell selected from the group
consisting of keritinocytes and melanocytes; b) taking a sample of
those skin cells and culturing them to increase the surface
concentration of cells selected from the group consisting of
keritinocytes and/or melanocytes to form a cultured mass of cells;
c) concentrating the cells selected from the group consisting of
keritinocytes and melanocytes from the cultured mass of cells; and
d) differentiating the concentrated cells selected from the group
consisting of keritinocytes and melanocytes. The differentiated
cells may then be implanted into a patient. A preferred surface
concentration of melanocytes is at least 500, at least 700, and at
least 800 melanocytes per square millimeter of skin. A preferred
surface concentration of keritinocytes is at least 500
keritinocytes per square millimeter of skin, or at least 700
keritinocytes per square millimeter. The method may use a medium in
which the cell suspension is suspended comprises a nutritional
medium. Such a nutritional medium may, for example, be selected
from the group consisting of DMEM/F12+N2 supplement, bFGF, EGF
containing less than 10% fetal calf serum, and Melanocyte basal
medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1A-F. Transplantation of melanocytes into 6-OHDA
lesioned rats. Intracranial injection of 6-OHDA into the substantia
nigra is a well characterized model for Parkinson's Disease. It is
well known that rats with lesions treated with amphetamine will
display circling behavior that can be measured in rotations. In the
controls FIGS. 1E and F), the rats were lesioned with 6-OHDA,
however there were no cells transplanted. Rats that have no lesion
and where treated with amphetamine will not circle (data not
shown). In experimental rats (FIGS. 1A-D), melanocytes were
injected into the substantia nigra using sterotactic coordinates.
Note the differences in scale of the six figures.
[0037] FIGS. 2A and B. Tyrosine hydroxylase positive
immunohistochemistry of 6-OHDA lesioned rats transplanted with
melanocytes. Arrows indicate tyrosine hydroxylase positive cells.
Control lesioned rats not transplanted with melanocytes do not show
positive cells (data not shown).
DETAILED DESCRIPTION OF THE INVENTION
[0038] Two specific types of cells that can be sourced from skin
biopsies have been identified as providing the potential for
enhanced capability in the generation of adult stem cells. In
particular, melanocytes and pericytes have been identified as being
capable of being differentiated into other cells when exposed to
appropriate differentiating factors. These cells may be collected
from regions of their naturally high concentration in the skin by
biopsy and/or concentrated from biopsies taken with more moderate
levels of these particular cells being present. By selecting,
focusing upon, and concentrating cells that predominate with
melanocytes and pericytes, a large source of cells is provided for
efficient differentiation into specific cell types.
[0039] To appreciate this invention, the difference between the
selection of source cells used in the practice of the present
invention and the source of cells used by Levesque (U.S. Pat. No.
6,087,168) should be appreciated. Levesque takes a complete biopsy
of a dermis. The biopsy includes only epidermis cells, and then
selects only epidermal basal cells for further differentiation.
Levesque shows that basal cells will differentiate into transient
amplification cells, including keritinocytes. Any type of
proliferating cells can be used according to Levesque. That
reference, in fact, uses a whole gamish of cells in the original
biopsy sampling, and doesn't distinguish among the cells in the
practice of the invention.
[0040] In the present invention, biopsies rich in melanocytes and
pericytes are used. Fetal foreskin is a particularly rich source of
these cells. It is also feasible to isolate these classes of cells
from an adult, with or without procedures to concentrate these
cells from the sample. The pericytes and melanocytes would then be
differentiated in their rich sample, preferably with different
approaches for each type of cell (i.e., using different
differentiating procedures and materials).
[0041] One particularly desirable method for providing these cells
is to collect a sample that has a modest or relatively rich content
of these cells and then to increase that specific type of cell from
source. For example, a melanocyte is not an epidermal basal cell,
but is a transient cell derived from a neural crest cell. During
embryonic development they develop along the spine. The original
rich source of the cells may be obtained by first seeking an
initial source (e.g., by using cell surface markers to separate
them).
[0042] The present invention comprises a process of generating
cells from skin biopsies for use in cell implantation
comprising:
[0043] a) identifying a source of skin cells that have a surface
concentration of at least 400 cells per square millimeter of
surface area of at least one cell selected from the group
consisting of keritinocytes and melanocytes;
[0044] b) taking a sample of those skin cells and culturing them to
increase the surface concentration of cells selected from the group
consisting of keritinocytes and melanocytes to form a cultured mass
of cells; and
[0045] c) concentrating the cells selected from the group
consisting of keritinocytes and melanocytes from the cultured mass
of cells;
[0046] d) differentiating the concentrated cells selected from the
group consisting of keritinocytes and melanocytes.
[0047] The method may then have the differentiated cells implanted
into a patient. The method may select the identified cells to
comprise melanocytes or keritinocytes. The surface area
concentration of the cells may be, for example, at least 500 cells
(e.g., melanocytes) per square millimeter of skin, at least 700
melanocytes per square millimeter of skin, or at least 800
melanocytes per square millimeter of skin.
[0048] Information on Melanocytes
[0049] Melanocytes, which are derived from the neural crest, are
localized in the retinal pigment epithelium (RPE) and uveal tract
of the eye, the leptomeninges, mucous membranes, hair matrix, skin
dermal-epidermal interface, and dermis. In the skin and hair
follicles, the melanoctyes are considered secretory melanocytes (as
opposed to continent melanocytes) and in the skin the melanocytes
are located in the basal layer and project dendrites into the
malpighian layer of the epidermis. The number of melanocytes varies
depending on location and range between 1000-2000 cells per square
millimeter (Table 1.) Melanocytes located in the RPE (these are
derived from optic cup not neural crest), mucous membrane, and
leptomeninges are considered continent melanocytes. The distinction
between secretory and continent pertains to the synthesis and
release of melanin pigments.
[0050] The present invention arose in part from an investigation of
the effects of LIF on cells of the neural crest. The neural crest
is a population of precursor cells that arises from the dorsal lip
of the neural tube during embryogenesis and migrates through the
embryo along a complex series of pathways. After migration, the
crest cells give rise to a great variety of cell types including
the neurons and Schwann cells of the sensory and autonomic ganglia,
the enteric nervous system, adrenal medulla, melanocytes of the
skin and facial mesenchyme. When studied at the population level,
the crest appears to be a multipotent collection of stem cells. The
extensive transplantation experiments of Le Douarin and colleagues,
whereby quail neural crest were grafted into chick embryos, showed
that the developmental fate of the crest cells was determined by
the location of this graft in the chick embryo. This not only
indicated that the full developmental repertoire of the crest is
contained in the different subpopulations of grafted crest cells,
but also that environmental factors play a major role in the final
differentiated phenotype of the cells.
[0051] In the last decade it has become increasingly clear that the
neural crest contains subpopulations of cells that are already
committed to particular developmental pathways (2. Ziller, C.,
Fauquet, M., Kalcheim, C., Smith, J., & Le Douarin, N. M. Dev.
Biol. 120: 101-111, 1987; Anderson, D. J. Neuron 3: 1-12,
1989).
[0052] A number of soluble trophic factors have been shown to act
as survival agents for neural crest derived neurons, but none of
these have been shown to act directly on the neuronal precursor
cells within the neural crest. These factors include nerve growth
factor (NGF; Levi-Montalcini, R. Annu. Rev. Neurosci. 5: 341-362,
1982).
[0053] However, it is also clear that the differentiation of these
cells is determined by environmental factors, such as brain-derived
neurotrophic factor (BDNF; Barde, Y. Neuron. 2: 1525-1534, 1989),
and ciliary neurotrophic factor (CNTF; Barbin, G., Manthorpe, M.,
& Varon, S. J. Neurochem. 43: 1468-1478, 1984) and the
fibroblast growth factors (FGF's; see Barde, supra).
[0054] Levesque describes the use of "epidermal basal cells" in the
practice of that invention. This is a very broad category of cells
and does not distinguish among the many available types of cells.
The specific cell types that Levesque selects are within the basal
layer. Basal cells are themselves derived from pericytes, and as
transient amplifying cells become keratinocytes. Levesque, in fact,
suggests they are trying to get rid of keratinocytes and then
differentiate the residue (the basal cells) second generation cells
from original stem cells.
[0055] Epidermal stem/Pericytes are cells that are often described
as being located at the bottom of hills on the juncture of cells,
at the junction between the dermis and epidermis. Upon removal of
biopsies rich in pericytes, according to the invention one would
then amplify the concentration of pericytes. Germinative/stem cells
are located mostly in the stratum basalis but also at the tips of
epidermal rete ridges. The epidermal stem/pericytes have low
metabolic activity. Sourcing of melanocytes could be done by taking
a biopsy of normal skin and amplifying the melanocytes or taking
out a mole (nevus) with melanocytes that are in the dermis and or
epidermis (e.g., neva). One may remove the basal cells by panning
them out to concentrate the melanocytes. This is in stark contrast
to Levesque. Antibodies, cell surface antibodies, binding agents,
may be used to conjugate the targeted cells to assist in their
initial concentration from suspensions. Epidermal stem/Pericytes
may be treated in the same way to concentrate them. This tends to
provide a mass of cells that approaches foreskin model in quality.
This rich supply of conjugated cells may then be separated from the
different skin cells in the mass.
[0056] The conjunctiva is the tissue that lines the eyelids, and
covers the anterior portion of the globe of the eye (except for the
cornea). The conjunctiva consists of a delicate membrane composed
of an epithelium, and a substantia propria, that overlie the tough
outer portion of the eyeball known as the sclera. The epithelium is
stratified into multiple cell layers, and provides a barrier to the
penetration of compounds into the eye. There are desmosomal
junctions connecting the epithelial cells to one another, and tight
junctions between the surface cells that prevent the penetration of
small ions. Other cells that can be found in the normal
conjunctival epithelium include: goblet cells, which are
specialized cells that secrete mucin; Langerhans cells;
melanocytes; a small population of immune cells (lymphocytes and
neutrophils); and an interspersed neuronal component. Beneath the
epithelium, the substantia propria contains stromal cells
interspersed in a layer of connective tissue. The substantia
propria also contains a microvasculature, lymphatics, immune cells,
and neurons. Mast cells are not found in the normal conjunctival
epithelium, but tissue type mast cells reside in the substantia
propria. The conjunctiva responds to eye irritants by mounting an
inflammatory response. The conjunctival response is assessed in the
Draize rabbit eye test as redness, chemosis, and discharge.
[0057] It is also of significance to note as another
distinguishinging feature from Levesque, that even though that
reference selects a mass (dermis) with a large number of different
types of cells, melanocytes are not technically included included
as cells that originate in the dermis. Rather, they migrate to that
locations and therefore only incidentally reside at this site (both
in the epidermal layer and the dermal layer).
[0058] Levesque also refers to dedifferentiating cells by supplying
the cells with molecular factors for transdifferentiation. Again,
this is a broad and general statement. In fact, it appears that the
process supplies the cells with molecular factors for
transdifferentiation, which may properly be considered to acting on
the cells with transfection methods on their cDNA constructs.
[0059] Therefore, it appears that in that portion of the text,
Levesque is using a large amount of a chemical to block certain
genes and insert certain genes for genetic expression. Levesque is
adding desired genes and blocking others that exist in the cells.
It is also not clear what resultant cell types are in the
exemplified process of Levesque, even in specific examples. For
example, in Example I, it isn't clearly apparent as to what cells
are being obtained.
[0060] Collection and propagation/culturing of cells in the
practice of the present invention can be described in fairly
general terms that are supported by common practices in the field
that are tailored to the practice of the present invention. First,
previous work with skin keritinocytes in tissue culture suggested
that cells with stem cell-like characteristics were enriched in
cell culture when grown in conditions that induced premature
terminal differentiation (Rogers et al., J. Cell Biol. 110:
1767-1777, 1990; Parkinson et al., Carcinogenesis 3, 525-531,
1982). Second, retinoic acid (RA) has been demonstrated to be an
effective differentiating agent in embryonal stem cells in tissue
culture (Humes and Cielinski, supra; Rogers et al., supra). Third,
Epidermal Growth Factors (EGF) is potent (Humes et al., Lab Invest.
64: 538-545, 1991; Parkinson, supra). Thus, the combination of a
potent growth promoter, EGF, and a differentiating agent, RA, would
provide positive selection pressure for cells which have a high
capacity for replication and negative selection pressure for cells
which are terminally differentiating. Although serial passage of
specifically differentiated cells from skin cells have been
difficult to achieve previously, these growth conditions with RA
and EGF are examples of conditions than can result in an ability to
grow these cells. The use of both RA and EGF are desirable for
consistent passage of these cells. The ability of these particular
classes of epidermal cells to morphologically differentiate and
form into the desirable cell structures can be easily demonstrated
in primary culture followed by growth under selection pressure with
RA and EGF for several passages. Resultant cells grown under this
selection condition can then be dispersed to prepare a single cell
preparation, suspended in three-dimensional collagen gels and grown
in serum-free, hormonally defined culture media supplemented with
RA and EGF for 7 to 14 days (suitable procedure are described in
Yang et al., Proc. Natl. Acad. Sci. U.S.A., 76: 3401-3405, 1979;
Bennett, Nature 285: 657-659, 1980; Montesano et al., Cell 42:
469-477, 1985, the texts of which are incorporated herein by
reference). Within several days, cells grown under these conditions
in collagen gels form luminal tubular structures as evidenced by
phase contrast microscopy. Progressive passage of cells promoted
increasingly more defined structures. Semi-thin sections of the
collagen preparation can confirm the nature of the cell clusters.
In either EGF, RA, or both EGF and RA in combination are omitted
from the culture media, structures within the collagen gel are not
as likely to form in satisfactory quantities. Thus, both EGF and RA
may be present in the growth media for preferred methods of
cultivation of cells to form into the appropriate phenotype in
vitro.
[0061] A filtration device may be used to concentrate either the
source cells and/or the resultant cells. Such a device has been
used for purifying blood and suitably comprises either a single
semipermeable hollow fiber or a collection of semipermeable hollow
fibers in which are coated, either externally or internally, with a
layer of extracellular matrix (ECM) upon which either may or may
not be grown a confluent monolayer of epithelial and/or endothelial
cells. Alternatively, the cells or matrix may be incorporated
directly within or on the polymeric structure of the semipermeable
hollow fiber during manufacture.
[0062] A filtration device promotes ultrafiltration of blood via
convective transport of water and solutes out of the blood or
supporting liquid and across the wall of a semipermeable hollow
fiber with high hydraulic permeability. Filtration of blood by a
convective process has several distinct advantages: it imitates the
glomerular process of toxin removal with increased clearance of
higher molecular weight solutes and removes all solutes up to a
selected molecular weight cutoff at the same rate. Convective
transport occurs independently of the existing concentration
gradient and depends predominantly on the hydraulic pressure
gradient across the membrane.
[0063] With respect to the use of a de-differentiating
process/procedure followed by differentiation, those of skill in
the art will recognize that is not absolutely necessary to
genetically modify the makeup of cells used in implantation. The
addition of certain growth factors can also be used, so that the
expression of genes is changed but not the genetic makeup of cells,
as is the case when transfecting DNA is utilized. For example,
different growth factors may be added, e.g., growth factors that
differentiate cells into dopaminergic neuron cells. In this manner,
one may take melanocytes, culture them, grow them, and replace
them. A first general process involves harvesting or collecting
determined but not differentiated donor cells from an organism, and
then growing the collected cells by either maturing the cell,
propagating the cell, culturing the cell, or implanting the cell.
This characterization of determination versus differentiation is
measured or detected by an ability to detect differentiated cells
or determined cells in vitro via membrane surface markers,
different gene expression patterns and/or morphological markers.
Previous teachings emphasizes collection and the direct
implantation of determined but not differentiated cells in vivo to
assert dedifferentiation followed by targeted differentiation.
Alternatively, the determined but not differentiated cells may be
placed into an in vitro environment with a differentiating
environment established by the presence of differentiating cells
and/or differentiating factors present in the in vitro environment,
with subsequent implantation.
[0064] Almost any cell implantation procedure sourcing in vitro
generated or propagated cells and tissue must use a cell growth
technique. Cell growth can be performed, for example, according to
the known techniques of U.S. Pat. Nos. 5,945,577 and 5,096,822,
which are incorporated herein by reference for this applicable
disclosure.
[0065] In a preferred embodiment, the donor cells are provided from
a human source. The process is carried out by propagating the donor
cell in appropriate media or directly introducing the determined
but not differentiated cells into a host and inducing
dedifferentiation and/or differentiation into an implantable cell
for the target species. In the case of providing cells for neural
cell therapy, it is most desirable to select cells that are already
determined but not differentiated towards the appropriate neural
cell or select other determined but undifferentiated cell that will
differentiate within the in vivo host implantation environment or
by in vitro propagation of the cell in an environment that has been
stocked with the appropriate differentiating factors.
[0066] The cells from the species from which the cells are
collected may be chosen from among any determined but
undifferentiated cell, such as being selected from germ cells or
stem cells, such as primordial germ cells, oogonia, neural stem
cells, trophoblast, slightly differentiated embryonic stem cells
(primitive ectoderm) or other less determined but still
undifferentiated cells. The selected determined but not
differentiated, cells act substantially the same as native young,
differentiating but undifferentiated cells that can flourish in the
growth to harvesting stage (or maturation stage in vivo). The fact
that these essentially `whole` differentiated implantable cells are
grown from determined but undifferentiated plastic cells (whether
in vitro or in vivo) and then implanted, assures that local
differentiating factors at the implantation site will assure or at
least assist their proper growth to the desired functionality. This
practice is substantially different from the common practices
today, where essentially undetermined cells from living organisms
are grown in vitro in the presence of selected differentiation
factors. That method may use both or either local and natural
differentiating factors to direct the functional growth of the
collected cells.
[0067] Material and Methods for the Isolation of Melanocytes and
Skin Stem Cell/pericytes.
[0068] In the performance of a biopsy, it is necessary to discuss
risk/benefits and obtain patient consent. The area must be cleaned
(e.g., with betadine solution) to create a sterile environment. A
local anesthesia may be given (injection of 1% lidocaine with
epinephrine). The surgeon would then take a 4 mm punch biopsy
obtained by cutting through the epidermis and dermis subcutaneous
tissue. The punched sample of tissue is placed in a betadine
solution for transport to the laboratory. The lesioned area is
closed with a 4-0 nylon suture. It is then typical to apply a
topical antibiotic and wound dressing, following written and verbal
instructions on wound care. Sutures are removed 10-14 days
following biopsy. Tissue dissociation and preparation of single
cell suspension follows.
[0069] In the present application, by "genetic manipulation of the
cells" we mean any means of adding or inhibiting gene expression in
the cells e.g. the addition of new genetic material by DNA
transfection, viral transduction, anti-sense RNA addition, etc.
Examples include but are not limited to: insertion of a gene
encoding the enzyme responsible for dopamine synthesis (tyrosine
hydroxylase); and inhibition of tyrosinase by the addition of
anti-sense RNA or DNA. By "the use of growth factors and/or
inhibitors" we mean the addition of those factors either during
culturing of the cells or after transplantation of the cells into a
host. Alternatively, such factors may be added to the cells (or to
their environment) at both of those junctures. Inhibitors may be
either chemical (e.g. a chemical inhibitor of tyrosinase) or
genetic (as described above).
[0070] In particular, when the cells are melanocytes, it may be
preferable to both inhibit one precess while stimulating another,
e.g. inhibiting tyrosinase and inducing tyrosine hydroxylase
expression, the end result being the production of dopamine.
[0071] The following examples are to be considered as exemplary of
various aspects of the present invention and are no intended to be
limiting with respect to the practice of the invention. Those of
ordinary skill in the art will appreciate that alternative
materials, conditions, and procedures may be varied and remain
within the skill of the ordinarily skilled artisan without
departing from the general scope of the invention as taught in the
specification.
EXAMPLES
Example One
[0072] This example relates to a method for obtaining single cell
suspensions from human tissue from biopsy tissue. Single cell
suspensions are obtained from the skin and or nevus by conventional
punch or other surgical means. Single cell suspensions can be
prepared from biopsy either by chemical or mechanical dissociation
techniques which are standard techniques known in the field. Single
cell suspensions were prepared using the DAKO Medimachine.TM. and
accessories (Carpinteria, Calif.). For each tissue sample, the
tissues were placed on a pre-wetted medicon (paper) filter and
inserted into the Medimachine.TM. to mechanically disaggregate the
tissue samples for 30 seconds to 1 min. Following the collection of
dissociated cells, the medicon filter was washed with 1 ml ice-cold
PBS and run through the Medimachine.TM. once again. Following the
second collection, the cells were combined and pipetted (ten times)
using a glass pipette and then filtered twice over a filcon filter
(30 micrometer, DAKO). Separate and sterile medicon filters and
filcon filters were used for each sample. The cell suspension was
centrifuged and washed with ice-cold PBS, centrifuged again, then
resuspended in media. The media is either DMEM/F12+N2 supplement,
bFGF, EGF (20 ng) containing no or 10% fetal calf serum or
Melanocyte basal medium and supplements purchased from Clonetics
(BioWhittaker Inc.).
[0073] Growth factors that were used or could be used include, but
are not limited to, the protein family members of, Interlukein 1,
CNTF, PDGF, NT, retinoic acid, NGF, scatter factor, BMP, GDNF, LIF,
and Nurr. In addition, in some cases the cells will be incubated
with human neurons hTN in the presence or absence of substrate.
Example Two
[0074] This prophetic example shows the potential use of cells for
the stable transfection of the tyrosine hydroxylase gene and the
dopa decarboxylase gene under an inducible promoter. The tyrosine
hydroxylase gene and other genes that would act as co-factors for
the synthesis of dopamine would be cloned into inducible systems.
The inducible systems could include, but are not limited to the
tetracycline inducible system, or an irradiation inducible system.
The inducible systems can be genetically engineered to be activated
with the addition of drug, stimulus or removal of drug or stimulus.
The tyrosine hydroxylase gene and other genes which could act as
co-factors for the synthesis of dopamine would be inserted into the
collected cells. According to procedures understood in the art, the
cells would be rendered into a tetracycline inducible system, an
irradiation inducible system or other inducible system.
Example Three
[0075] This prophetic example anticipates the use of cells for the
inhibition of the tyrosinase gene and protein product by either
genetic engineering of an antisense DNA construct or the use of
chemical inhibition or growth factor inhibition.
Example Four
[0076] Another prophetic use of the cells would be for the
induction of dopa decarboxylase protein expression by growth factor
stimulation.
Example Five
[0077] Another prophetic use of cells would be for the stable
transfection of the EGFP gene and the EGFP gene co-expressed with
other genes of interest for the visualization of cells in vivo. TA
marker gene would also be subcloned into an inducible system
including the tetracycline, irradiation or MR induced construct.
The cells stably transfected with a marker gene under the control
of for example an MRI inducible system would allow for the
detection of cells transplant into host tissue.
Example Six
[0078] This prophetic example shows the potential use of these
cells to immunopurify specific cell types by using antibodies such
as c-Kit (CD117) conjugated to superparamagnetic (magnetic only in
a magnetic field) microparticles (sized in the micron range) that
are synthesized by polymerizing polystyrene or polyacrolein in the
presence of a magnetite ferrofluid or by formation of an
agglomerate by silanation of a ferrofluid. With both of these
preparations antibodies can be covalently coupled by the resultant
surface character of the particles. Magnetic microparticles have
been used in cell separation, immunoassays, isolation,
identification and genetic analysis of specific nucleic acid
sequences, and for isolation of DNA binding proteins. The present
inventors have examined the possibility of using similar magnetic
microparticles as a solid support for immunoabsorption of intact
plastids from whole cell lysates because their relatively high
magnetic moment allows ease of separation using simple rare earth
magnets. Another class of particle preparations can be described as
magnetic nanoparticles (ferrofluid derivatives sized in the
nanometer range) consisting of ferric oxide crystals encapsulated
by dextran (Molday, R., Yen, S., and Rembaum, A., slowly in
conventional ferromagnetic fields. Consequently, organelles labeled
with magnetic nanoparticles must be separated in a magnetic
affinity column (Miltenyi et al., supra). Disclosed herein is the
use of antibodies specific to exposed epitopes of proteins on
chloroplast outer envelopes coupled to magnetic nanoparticles to
immuno-isolate various plastid subtypes from whole cell
lysates.
[0079] Table 1. This Table provides information upon which the
present invention is based, with the document source content of the
number of melanocytes per millimeter square skin has been reported.
Numbers are taken from Jimbow et al., (1993) and represent a median
age of 16-92 years. The number of melanocytes per millimeter square
decrease with increasing age. The idea place for skin pericytes is
the fetal foreskin.
1 Location Numbers of melanocytes/mm.sup.2 Scalp 1025-1060 Face
1010-1194 Neck 920-926 Chest 687-918 Arm 717-908 Back 865-880
Abdomen 578-605 Buttock 405-565 Genitalia 1047-1228 Thigh 771-917
Lower Leg 812-814
[0080] Reference Biology of Melanocytes. Jimbow K., Quevedo WC.,
Fitzpatrick TB., and Szabo G. (1993) pp 261-289. In Dermatology in
General Medicine Vol I. 4th edition. Eds. Fitzpatrick, T. B.,
Eisen, A. Z., Wolff K., Freedberg, I. M., Austen K. F. McGraw-Hill
Inc. New York.
Example Seven
[0081] This prophetic example shows the potential use of cells for
transplantation. An example is demonstrated in FIGS. 1 and 2.
According to procedures understood in the art, the cells
(melanocytes, keritinocytes, or pericytes) would be transplanted
into CNS tissue and monitored for de-differentiation either without
growth factor or genetic manipulation or with growth factor and
genetic manipulation. As seen in FIGS. 1-2, melanocytes were
implanted into the brain of 6-OHDA rats. Those skilled in the art
will recognize, this well-characterized Parkinson's Disease model.
In this example, melanocytes are transplanted without genetic or
growth factor manipulation to determine if the environment or
"niche" can influence de-differentiation. In two of the
experimental animals (FIGS. A and B) rotational behavior was
improved suggesting an effect of cell transplantation. (Note the
difference in scale of the various figures). To determine if the
transplantation survived and de-differentiated into dopamine
producing cells, immunohistochemistry was conducted as described in
FIG. 2.
* * * * *