UPDATE FUTURE LIVER CELL.....

[Guest guest]

Group,

The patent application I sent out yesterday was chopped off in paragraph

[0028]. I didn't include the claims in the body of this e-mail because of

length. Also, I have attached the text of the patent application for your

reference. If you want the drawings, you'll need to visit the U.S. Patent

Office web site at uspto.gov.

Once there:

1. Click on Check Status

2. Click on Patent Search

3. Look at yellow box for Patent Application and click on Publication search

number and enter 20020039786.

4. Click on blue underline of 20020039786. (hyper-text).

5. Click on the blue box called images. The directions will set you up for

reading drawings.

FYI,

[0028] The present invention also relates to a method of isolation and

cryopreservation of diploid cells and/or progenitors from human liver which

includes (a)processing human liver tissue to provide a substantially single

cell suspension including diploid adult cells, progenitors and

non-progenitors of one or more cell

lineages found in human liver; ( subjecting the suspension to a debulking

step, which reduces substantially the number of non-progenitors in the

suspension, to provide a debulked suspension enriched in progenitors

exhibiting one or more markers associated with at least one of the cell

lineages; and © selecting from the

debulked suspension those cells, which themselves, their progeny, or more

mature forms thereof express one or more markers associated with several

liver cell lineages; and (d) suspending the cells under conditions optimal

for cryopreservation. More preferably liver progenitors expressing

cytoplasmic proteins such as

alpha-fetoprotein are selected. Processing or debulking steps of this

invention preferably include a density gradient centrifugation of the liver

cell suspension to separate the cells according to their buoyant density and

size which are associated with one or more gradient fractions having a lower

buoyant density.

[0029] Non-progenitors of the liver cell suspension includes mature hepatic,

hemopoietic, and mesenchymal cells. Negative selection of the

non-progenitors includes the use of markers associated with mature hepatic

cells, such as connexin32, markers associated with hemopoietic cells, such

as glycophorin A and CD45, or markers associated with mature mesenchymal

cells, such as retinoids, or von Willebrand Factor.

[0030] A further aspect of this invention provides for liver cell

progenitors of hepatic, hematopoietic, or mesenchymal origin. These cell

lineages, their progenies or their more mature forms are selected by

antigenic markers selected from the group consisting of CD14, CD34, CD38,

CD45, CD117, ICAM, glycophorin A, and/or cytoplasmic markers such as

alpha-fetoprotein-like immunoreactivity, albumin-like immunoreactivity, or

both. Alpha-fetoprotein derives from variant forms of mRNA some of which are

unique to hepatic progenitor cells and some to hemopoietic progenitor cells.

The liver progenitors of this invention can be isolated from the liver of a

fetus, a neonate, an infant, a child, a juvenile, or an adult.

[0031] In accordance with yet a further aspect of this invention, isolated

human liver progenitors, a subpopulation of the diploid cells, are isolated

in a highly

enriched to substantially pure form. Such liver progenitors contain hepatic,

hemopoietic and mesenchymal progenitors. The hepatic progenitors have the

capacity to develop into hepatocytes, biliary cells, or a combination

thereof; the hematopoietic progenitors have the capacity to develop into

macrophages, neutrophils,

granulocytes, lymphocytes, platelets, neutrophils eosinophils, basophils, or

a combination thereof. The mesenchymal progenitors have the capacity to

develop into endothelial cells, stromal cells, hepatic stellate cells (Ito

cells), cartilage cells, bone cells or combinations thereof. The method of

this invention can be used to

select mesenchymal progenitors expressing CD34, osteopontin, bone

sialoprotein, collagen types I, II, or III, or a combination thereof.

[0032] The present inventors overcome many of the above difficulties making

diploid cells, including progenitor cells, ideal for use in cell and gene

therapies and for bioartificial organs. The cells are small, therefore

minimizing the formation of large emboli. Also, the cells have extensive

growth potential meaning that fewer

cells are needed for reconstitution of liver tissue in a patient. Finally,

the progenitors have minimal antigenic markers that might elicit

immunological rejection providing hope that little or no immunosuppressive

drugs might be needed.

[0033] A further aspect of this invention provides for liver progenitors

that harbor exogenous nucleic acid. Such exogenous nucleic acid can encode

one or more polypeptides of interest, or can promote the expression of one

or more polypeptides of interest.

[0034] In accordance with yet a further aspect of this invention, there is

provided a method of alleviating the negative effects of one or more human

disorders or

dysfunctions by administering to an individual suffering from such negative

effects an effective amount of isolated human diploid liver cells and/or

progenitors. The progenitors can be administered parenterally via a vascular

vessel, or administered directly into the liver. The direct administration

can be effected surgically

via portal vein, mesenteric vein, hepatic bile duct, or combinations

thereof. Alternatively, the liver progenitors can be administered into an

ectopic site of the individual, such as spleen.

[0035] The human disorders or dysfunctions that could be alleviated by the

method of this invention include: hepatocholangitis, hepatomalacia,

hepatomegalia,

cirrhosis, fibrosis, hepatitis, acute liver failure, chronic liver failure,

or inborn errors of metabolism, hepatocarcinoma, or hepatoblastoma. Cancer

of the liver could

be a primary site of cancer or one that has metastasized into the liver. The

metastatic tumor could be derived from any number of primary sites

including, intestine, prostate, breast, kidney, pancreas, skin, brain, lung

or a combination thereof. The hepatic disease or dysfunction that can be

treated with this methods

also includes liver disease or dysfunction associated with an impairment in

the mitochondrial compartment of hepatic tissues and can consist of chronic

liver disease, fulminant hepatic failure, viral-induced liver disease,

metabolic liver disease, and hepatic dysfunction associated with sepsis or

liver trauma.

[0036] In accordance with yet a further aspect of the invention, a

bioreactor is provided which includes (i) biological material comprising (a)

isolated progenitors from human liver, their progeny, their maturing or

differentiated descendants, or combinations thereof, ( extracellular

matrix, and © media; (ii) one or more

compartments holding said biological material or the components comprising

said biological material; and (iii) one or more connecting ports. The

biological material of the bioreactor can optionally also include: (d)

hormones, growth factors, or nutritional supplements, or (e) plasma, serum,

lymph, or products derived therefrom.

[0037] The bioreactor is adapted for sustaining said progenitors in a

viable, functional state, and can sustain liver progenitors for a period

ranging from about one week or longer. Specifically, the bioreactor is

adapted for use as an artificial liver, for product manufacturing,

toxicological studies, or metabolic studies, including

studies involving the activity of cytochrome P450, or other types of drug

metabolism.

[0038] In accordance with yet another aspect of this invention, a

composition of isolated human liver progenitors, or a suspension enriched in

progenitors obtained from human liver is provided. The cell suspension is

provided in a pharmaceutically acceptable carrier or diluent and is

administered to a subject in need of

treatment. The composition of this invention includes liver progenitors that

exhibit one or more markers associated with at least one of one or more cell

lineages

found in human liver and are substantially free of mature cells. More

particularly, isolated liver progenitors are derived from one or more liver

cell lineages including hepatic, hematopoietic, or mesenchymal cell lineages

and themselves, their progeny, or more mature forms of the progenitors

thereof express at least

one or more of antigenic markers CD14, CD34, CD38, CD90, or CD117, CD45,

glycophorin A, and cytoplasmic markers of alpha-fetoprotein-like

immunoreactivity, albumin-like immunoreactivity, or both.

[0039] In accordance with yet another embodiment of this invention, a cell

culture of liver progenitors is provided which includes isolated progenitors

from human liver, their progeny, their maturing or differentiated

descendants, or combinations thereof. The cell culture additionally includes

extracellular matrix comprising one

or more collagens, one or more adhesion proteins (laminins, fibronectins),

and other components such as proteoglycans (such as heparan sulfate

proteoglycans); or an individual matrix component. Matrix component includes

fragments of matrix components; matrix mimetics that can be synthetic and/or

biodegradable

materials (i.e. microspheres) coated with one or more of the factors from

one of the classes of extracellular matrices. The cell culture additionally

includes basal media and other nutrients; hormones and/or growth factors,

with or without a biological fluid such as serum, plasma or lymph.

Additionally, the culture media could contain one or more compartments that

holds the biological material such as a culture dish, flask, roller bottle,

transwell or other such container.

[0040] The cultures or bioreactors of this invention could be used to

produce various medically important cell-secreted factors including but not

limited to enzymes, hormones, cytokines, antigens, antibodies, clotting

factors, anti-sense RNA, regulatory proteins, ribozymes, fusion proteins and

the like. The cultures are

suitable to supply a therapeutic protein such as Factor VIII, Factor IX,

Factor VII, erythropoietin, alpha-1-antitrypsin, calcitonin, growth hormone,

insulin, low density lipoprotein, apolipoprotein E, IL-2 receptor and its

antagonists, superoxide dismutase, immune response modifiers, parathyroid

hormone, the interferons

(IFN alpha, beta, or gamma), nerve growth factors, glucocerebrosidase,

colony stimulating factor, interleukins (IL) 1 to 15, granulocyte colony

stimulating factor (G-CSF), granulocyte, macrophage-colony stimulating

factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), fibroblast

growth factor (FGF),

platelet-derived growth factor (PDGF), adenosine deaminase, insulin-like

growth factors (IGF-1 and IGF-2), megakaryocyte promoting ligand MPL,

thrombopoietin, etc.

[0041] As a further embodiment of this invention a pharmaceutical

composition is provided which is useful for treating and preventing a liver

disease. The composition comprises an effective amount of cadaveric liver

progenitor cells and a pharmaceutical carrier. The liver diseases of

interest include acute or chronic

liver disease of toxic, metabolic, genetic, and/or infective origin or of

degenerative nature, or liver damage resulting from the use of drugs or

substances injurious to the liver. Preferably among these conditions and

diseases are inflammation of the liver, viral hepatitis, toxic liver cell

damage, fibrosis of the liver, cirrhosis of the liver, liver congestion,

liver dystrophy, fatty degeneration of liver cells, fatty liver,

disturbances of the detoxification function, disturbances of the excretory

function of the liver, disturbances of the conjugational function of the

liver, disturbances of the synthesizing function of the liver portal

hypertension due to a liver

disease, or a liver failure coma, and intoxication by protein degradation

products of ammonia. More specifically these include but are not limited to

Alagille syndrome, alcoholic liver disease, alpha-1-antitrypsin deficiency,

autoimmune hepatitis, biliary ductopenia, bone marrow failure, Budd-Chiari

syndrome, biliary

atresia, Byler disease, Crigler-Najjar syndrome, Caroli disease, cholestatic

pruritus, cholelithiasis, conjugated hyperbilirubinemia, chronic

graft-versus-host disease, cryptogenic liver disease, diabetes,

Dubin- syndrome, erythrohepatic protoporphyria, extrahepatic bile

duct carcinoma, familial hypercholesterolemia,

galactosemia, Gilbert syndrome, glycogen storage disease, hemangioma,

hemochromatosis, hepatic encephalopathy, hepatocholangitis, hepatomalacia,

hepatomegalia, hepatocarcinoma, hepatoblastoma, hereditary hemochromatosis,

jaundice, intrahepatic cholestasis, liver cysts, liver transplantation,

liver failure

associated with Bacillus cereus, mixed cryoglobulinemia, ornithine

transcarbamylase deficiency, peliosis hepatis, porphyria cutanea tarda,

primary biliary cirrhosis, refractory ascites, Rotor syndrome, sarcoidosis,

sclerosing cholangitis, steatosis, Summerskill syndrome, thrombocytopenia,

tyrosinanemia, variceal bleeding,

venocclusive disease of the liver, disease and combinations thereof.

[0042] Other objects will be made known to the skilled artisan in view of

the following detailed disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0043] FIGS. 1a and 1b illustrate the effect of warm ischemia on the

proportion of isolated cells with small and large nuclei.

[0044] FIGS. 2a and 2b illustrate PCR analysis of truncated

alpha-fetoprotein (AFP) in hemopoietic cells.

[0045] FIG. 3 illustrates the relationship between storage time at

-170.degree. C. and viability of thawed fetal liver cells.

[0046] FIGS. 4a and 4b illustrate typical univariate histograms of fetal

liver cell suspensions analyzed by fluorescence activated cell sorting

(FACS).

[0047] FIG. 5 illustrates percent of cells expressing surface markers CD14,

CD34, CD38, CD45 and Glycophorin A (GA) in unfractionated liver cell

suspensions

[0048] FIG. 6 illustrates percentage of cells in the original cell

suspension expressing alpha-fetoprotein and other antigenic markers

[0049] FIGS. 7a, 7b and 7c illustrate alpha-fetoprotein expression before

and after depletion of red blood cells.

[0050] FIGS. 8a, 8b, 8c, 8d, 8e and 8f illustrates FACS analysis of fetal

liver cell suspension for co-expression of CD14, CD38 and AFP.

[0051] FIG. 9 illustrates CD14 and CD38 enrich for AFP-positive cells.

[0052] FIGS. 10a, 10b, 10c and 10d illustrate fluorescence microscopy of

human hepatic progenitor cells.

[0053] FIGS. 11a, 11b, 11c and 11d illustrate representative cells selected

by expression of AFP.

[0054] FIGS. 12a, 12b and 12c show that there are two AFP positive cells in

this field.

[0055] FIGS. 13a and 13b illustrate cells that are labeled with calcein (A)

to show all cell types.

DETAILED DESCRIPTION OF THE INVENTION

[0056] In the description that follows, a number of terms are used

extensively to describe the invention. In order to provide a clear and

consistent understanding of the specification and claims, the following

definitions are provided.

[0057] Alpha-fetoprotein-like immunoreactivity: Any immune reactions caused

by alpha-fetoprotein. Alpha-fetoprotein derives from variant forms of mRNA

some of which are unique to hepatic progenitor cells and some to hemopoietic

progenitor cells.

[0058] Committed progenitors: Immature cells that have a single fate such as

hepatocytic committed progenitors (giving rise to hepatocytes) or biliary

committed progenitors (giving rise to bile ducts). The commitment process is

not understood on a molecular level. Rather, it is recognized to have

occurred only empirically when the fates of cells have narrowed from that of

a predecessor.

[0059] Hepatic cells: A subpopulation of liver cells, which includes

hepatocytes and biliary cells.

[0060] Liver cells: As used herein, the term " liver cells " refers to all

type of cells present in normal liver, regardless of their origin or fate.

[0061] Stem cells: As used herein, the term " stem cells " refers to immature

cells that can give rise to daughter cells with more than one fate. Some

daughter cells are identical to the parent and some " commit " to a specific

fate. Totipotent stem cells have self-renewal (self-maintaining) capacity,

whereas determined stem cells have questionable self-renewal capacity. Stem

cells can regenerate during a regenerative proliferative process.

[0062] Hepatic progenitors: These cells give rise to hepatocytes and biliary

cells. The hepatic progenitors include three subpopulations: " hepatic stem

cells " , " committed hepatocytic progenitors " , and " committed biliary

progenitors, " the last two being immature cells that are descendants of the

hepatic stem cell and that have a single fate, either hepatocytes or biliary

cells, but not both.

[0063] Hepatic stem cells: A subpopulation of hepatic progenitors.

[0064] Liver progenitors: A cell population from liver, including hepatic

progenitors, hemopoietic progenitors and mesenchymal progenitors.

[0065] Oval cell: a small cell (<15 microns) with oval shaped nuclei

proliferating in animals exposed to oncogenic insults. These cells are

thought to derive from liver progenitors and are partially or completely

transformed.

[0066] The " liver " is a large organ located in the most forward part of the

abdomen, resting against the muscular partition between the abdominal and

chest cavities. The liver is essential for life and performs over 100

important functions, such as detoxifying poisons and drugs, metabolizing

fats, storing carbohydrates, manufacturing bile, plasma proteins and other

substances, and assisting in blood clotting. Liver disease is often

difficult to detect until the illness becomes severe because there is an

overabundance of liver tissue, and the liver can partially regenerate

itself. The signs of liver disease vary with the degree and location of

damage. Various blood tests are necessary to discover the extent and the

nature of liver damage.

[0067] The term " growth factor " as used herein refers to those factors

required to regulate developmental events or required to regulate expression

of genes encoding other secreted proteins that can participate in

intercellular communication and coordination of development and includes,

but is not limited to hepatocyte

growth factor (HGF), insulin-like growth factor-I and II (IGF-I and II),

epidermal growth factor (EGF), type a and type b transforming growth factor

(TGF-alpha and TGF-beta), nerve growth factor (NGF), fibroblast growth

factor (FGF), platelet-derived growth factor (PDGF), sarcoma growth factor

(SGF), granulocyte macrophage colony stimulating growth factor (GM-CSF),

vascular endothelial growth factor (VEGF), prolactin and growth hormone

releasing factor (GHRF) and

various hemopoietic growth factors such as interleukins (IL) IL-1, IL-2,

IL-3, IL-4, IL-5, IL-6, IL-7, L-8, IL-10, IL-11, etc., erythroid

differentiation factor (EDF) or follicle-stimulating hormone releasing

protein (FRP), inhibin, stem cell proliferation factor (SCPF) and active

fragments, subunits, derivatives and combinations

of these proteins among many others known in the art. Generally, as used

hereinafter, the growth factor refers to a secreted protein which is

selected from the group consisting of a cytokine, a lymphokine, an

interleukin, a colony-stimulating factor, a hormone, a chemotactic factor,

an anti-chemotactic factor, a coagulation factor, a thrombolytic protein, a

complement protein, an enzyme, an immunoglobulin, and an antigen.

[0068] Hemopoiesis: Yielding blood cells with cell fates of lymphocytes (B

and T), platelets, macrophages, neutrophils, and granulocytes.

[0069] Mesengenesis: Yielding mesenchymal derivatives with cell fates of

endothelia, fat cells, stromal cells, cartilage, and even bone (the last two

occurring in the liver only under disease conditions).

[0070] Cell Therapy: As used herein, the term " cell therapy " refers to the

in vivo or ex vivo transfer of defined cell populations used as an

autologous or allogenic material and transplanted to, or in the vicinity of,

specific target cells of a patient. Cells can be transplanted in any

suitable media, carrier or diluents, or any type

of drug delivery systems including, microcarriers, beads, microsomes,

microspheres, vesicles and so on.

[0071] Gene Therapy: As used herein, the term " gene therapy " refers to the

in vivo or ex vivo transfer of defined genetic material to specific target

cells for a patient in need thereof, thereby altering the genotype and, in

most situations, altering the phenotype of those target cells for the

ultimate purpose of preventing or

altering a particular disease state. As this definition states, the

underlying premise is that these therapeutic genetic procedures are designed

to ultimately prevent, treat, or alter an overt or covert pathological

condition. In most situations, the ultimate therapeutic goal of gene therapy

procedures is to alter the phenotype of

specific target cell population.

[0072] CD: " Cluster of differentiation " or " common determinant " as used

herein refers to cell surface molecules recognized by monoclonal antibodies.

Expression of some CDs are specific for cells of a particular lineage or

maturational pathway, and the expression of others varies according to the

state of activation, position, or differentiation of the same cells.

[0073] Ploidy: chromosome number within a cell.

[0074] Diploid: two sets of chromosomes per cell.

[0075] Tetraploid: four sets of chromosomes per cell.

[0076] Octaploid: eight sets of chromosomes per cell.

[0077] Polyploid: more than two sets of chromosomes per cell.

[0078] The cells of the normal fetal or neonatal liver are diploid. By the

young adult stage, the liver is a mixture of diploid and polyploid cells. In

rodents, the liver is about 90% polyploid and only about 10% diploid cells.

In humans, the liver of young adults is composed of 50% diploid and 50%

polyploid cells.

[0079] Without limiting to liver, other progenitor cells from various

cadaveric tissues are disclosed and claimed by this invention. As used

hereinafter the term " cadaveric tissue " does not include tissue from dead

fetuses obtained by means such as premature termination of pregnancy by a

surgical procedure. Humans delivered by natural or assisted birth are

considered as neonates or infants but not as fetuses. Accordingly the age of

a human starts at " 0 " at the time of birth or delivery and not from the time

of conception. Thus a neonate dead at the time of birth will be considered

as a cadaver and not as a fetus. Freshly obtained fetal tissues have been

used as a source of some progenitor cells and as such they are excluded from

the breadth of claims of this invention. However, fetal tissue which is

considered unsuitable for further medical use due to the presumed ischemia

effect is still suitable for the purposes of this invention.

[0080] When the terms " one, " " a, " or " an " are used in this disclosure, they

mean " at least one " or " one or more, " unless otherwise indicated.

[0081] FIGS. 2a and 2b illustrate PCR analysis of truncated AFP in

hemopoietic cells. RT-PCR is carried out using primer combination of hAFP1,

hAFP2, hAFP3, and hAFP4. Lanes 1-3 correspond to Hep3B cells; lanes 10-12

correspond to STO cells; lanes 13-15 have no RNA or cDNA. Note, there is a

shared band, a truncated AFP isoform, in lanes 2, 5, and 8. There is a

truncated AFP isoform unique to liver cells noted in lanes 1 and 4. The

complete AFP species is observed in lanes 3 and 6.

[0082] FIG. 3 illustrates the relationship between storage time at

-170.degree. C. and viability of thawed fetal liver cells. Data are

expressed as the percent change in viability measured at the time of

processing versus the time of thawing. These data indicate that the

cryopreservation methods did not significantly affect the viability of the

cells. There was no significant change in viability over a period extending

to 550 days in storage.

[0083] FIGS. 4a and 4b illustrates typical univariate histograms of fetal

liver cell suspensions analyzed by fluorescence activated cell sorting

(FACS). The cell suspension was prepared for immunofluorescence analysis of

alpha-fetoprotein (AFP) using antibodies conjugated to the red dye, Cy5, and

for albumin using

antibodies conjugated to the blue dye (AMCA). Thirty thousand cells were

screened for red (AFP) and blue (albumin) fluorescence. The results show a

clear group of cells positive for each protein. Further analysis shows that

about 80% of the positive populations for each protein are represented by

the same cells (i.e.

co-expression of the two proteins).

[0084] FIG. 5 illustrates the percent of cells expressing surface markers

CD14, CD34, CD38, CD45 and Glycophorin A (GA) in unfractionated liver cell

suspensions. Note that the GA data is plotted on the right axis to preserve

scale.

[0085] FIG. 6 illustrates the percentage of cells in the original cell

suspension expressing alpha-fetoprotein and other antigenic markers.

Mean.+-.SEM for percent of cells positive for alpha-fetoprotein (AFP) and

specific cell surface markers (CD14, 34, 38, 45 and glycophorin A).

[0086] FIGS. 7a, 7b and 7c illustrate alpha-fetoprotein expression before

and after depletion of red blood cells. FIG. 7a illustrates the expression

of alpha-fetoprotein and FIG. 7b illustrates albumin, in suspensions of

fetal liver cells with or without selective depletion of red cells using

Percoll fractionation. FIG. 7c illustrates the proportion of cells

expressing both alpha-fetoprotein and albumin, expressed as a percentage of

AFP or albumin positive cells. Data for cells with red cell depletion are

shown using Percoll fractionation.

[0087] FIGS. 8a, 8b, 8c, 8d, 8e and 8f illustrate FACS analysis of fetal

liver cell suspension for co-expression of CD14, CD38 and AFP. The bivariate

scattergram (8a) shows the distribution of TriColor staining for CD14

(ordinate) versus FITC staining for CD38 (abscissa). Gates were created to

select specific cell groupings according to the CD14 and CD38 signals. These

were then used to display the intensity of AFP staining in each of these

subgroups (FIGS. 8b, 8c,

8d and 8e). The AFP results show that a high level of enrichment for AFP is

produced by selecting cells positive for either CD38 or CD14. The AFP signal

generated from the entire cell suspension (30,000 cells) is shown in FIG.

8f.

[0088] FIG. 9 illustrates CD14 and CD38 enrichment for AFP-positive cells.

The proportion of AFP-positive cells in cell suspensions prepared from fetal

liver can be enhanced dramatically by selecting cells with positive surface

labeling for the markers CD38 and CD14. The combination of the two markers

produces a significantly better enrichment of AFP-containing cells than that

obtained with either marker alone.

[0089] FIGS. 10a, 10b, 10c and 10d illustrate fluorescence microscopy of

human hepatic progenitor cells. Representative hepatic progenitor cells from

the fetal liver stained for AFP content. Cell sizes indicate that both early

progenitors and more advanced hepatic progenitors are present.

[0090] FIGS. 11a, 11b, 11c and 11d illustrate representative cells selected

by expression of AFP. The cells with positive staining for CD14 (11b and

11d) are characteristic of hepatoblasts. The cells with negative staining

for surface markers (FIGS. 11a and 11c) are smaller and consistent in size

and image. FIG. 12b illustrates immunofluorescence with antibody to AFP.

FIG. 12c illustrates overlay (a) and ( indicating the morphology of AFP

positive cells in a group of liver cells

[0091] AFP-positive cells are found to have a similar cell size and

morphology whether isolated from fetal or adult livers.

[0092] FIGS. 13a and 13b illustrates cells that are labeled with calcein (a)

to show all cell types. FIG. 13 ( consist of the same cells co-expressing

AFP and showing that only two cells are AFP-positive. Cell size is not a

factor for AFP positivity.

[0093] The ability of the liver to regenerate is widely acknowledged, and

this usually is accomplished by the entry of normally proliferatively

quiescent hepatocytes into the cell cycle. However, when hepatocyte

regeneration is impaired, small bile ducts proliferate and invade into the

adjacent hepatocyte parenchyma. In humans and experimental animals these

ductal cells are referred to as oval cells, and their association with

defective regeneration has led to the belief that they are transformed stem

or progenitor cells. These cells are of great biological interest since

their normal counterparts, the hepatic progenitors can be used as

alternative to liver transplants and they can also be useful vehicles for

gene therapy for the correction of inborn errors of metabolism. While the

ability of

progenitors to differentiate into hepatocytes has been demonstrated

unequivocally the demand for said cells has not met the desired supply due

to the paucity of donor liver tissue.

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