Cellular compositions and methods of making and using them

• US 20050074435A1
• Filed: 12/20/2002
• Published: 04/07/2005
• Est. Priority Date: 12/21/2001
• Status: Active Grant

• Current Assignee: Mount Sinai Hospital (Sinai Health System)
• Original Assignee: Mount Sinai Hospital (Sinai Health System)
• Inventors: Casper, Robert, Rogers, Ian
• Granted Patent: US 7,625,752 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/51 Umbilical cord; Umbilical cord blood; Umbilical stem cells
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 25/32 Alcohol-abuse
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/90 : Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 : Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 : Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 : Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/12 : Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/125 : Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/13 : Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/145 : Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/165 : Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/22 : Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 : Interleukins [IL]
• CPC Class Codes: C12N 2501/26 : Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/385 : of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/42 : Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2502/13 : connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2503/02 : Drug screening
• CPC Class Codes: C12N 2506/11 : from blood or immune system cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0607 : Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: G01N 33/5073 : Stem cells
• CPC Class Codes: G01N 33/56966 : Animal cells

Abstract

The invention relates to cellular compositions comprising hematopoietic cells with the potential or increased potential to form non-hematopoietic cells; methods for producing such cellular compositions; methods for differeentitation of cells of cellular compositions of the invention into cells that exhibit morphological, physiological, functional, and/or immunological features of non-hematopoietic cells; and uses of the cellular compositions. The invention also relates to a method for the expansion of hemtopoietic stem and progenitor cells.

Claims

1. A method for producing an isolated cellular composition comprising or comprising essentially cells with potential or increased potential to form different types of non-hematopoietic cells comprising:

(a) obtaining hematopoietic cells;

(b) enriching the hematopoietic ells for hematopoietic stem cells and progenitor cells by positive or negative selection to obtain an enriched hematopoietic cell preparation.

(c) culturing the enriched hematopoietic stem cells and progenitor cells under proliferation conditions so that cells in the preparation develop the potential or an increased potential to form different types of non-hematopoietic cells.

2. A method as claimed in claim 1 wherein the hematopoietic cells in step (a) are obtained from umbilical cord blood.

3. A method as claimed in claim 1 wherein the proliferation conditions in step (c) comprise culturing the cells in the presence of one or more positive growth factors for a sufficient time to enable cells in the enriched hematopoietic cell preparation to complete sufficient cell cycles to obtain cells with the potential or increased potential to form non-hematopoietic cells and tissues.

4. A method as claimed in claim 3 wherein the positive growth factor is a member of the fibroblast growth factor (FGF) family including FGF-4 and FGF-2, epidermal growth factor (EGF), stem cell factor (SCF), thrombopoietin (TPO), FLT-3 ligand, interleukin-3 (11-3), interleukin-6 (IL-6), neural growth factor (NGF), VEGF, Granulocyte-Macrophage Growth Factor (GM-CSF), HGF, or Notch.

5. A method as claimed in claim 4 wherein the positive growth factors are one or more of FGF-4, FGF-3, IL-3, SCF, FLT3 ligand, TPO, GM-CSF, and NGF.

6. A method as claimed in claim 1 wherein the proliferation conditions in step (c) comprise culturing the cells in the presence of

(i) FGF-2 or FGF-4, FLT3ligand, and SCF;

(ii) TPO, FLT-3ligand, and SCF;

or

(iii) NGF, SCF and FLT-3ligand,

for a sufficient time to enable cells in the enriched hematopoietic cell preparation to complete sufficient cell cycles to obtain cells with the potential or increased potential to form non-hematopoietic cells.

7. A method as claimed in claim 1 wherein the enriched hematopoietic cell preparation comprises essentially CD45⁺HLA-ABC⁺Lin.

8. A method as claimed in claim 1 wherein the non-hematopoietic cells are endothelial cells, osteoclasts, adipocytes, muscle cells, astocytes, renal cells, pancreatic cells, hepatocytes, retinal cells, corneal cells, connective tissue cells, or neural cells.

9. A method as claimed in claim 1 which further comprises inducing cells with potential or increased potential to form non-hematopoietic cells to differentiate into cells and tissues of non-hematopoietic lineages in vitro or in vivo.

10. A cellular composition produced by a method of claim 1.

11. A cellullar composition as claimed in claim 10 wherein the cells wherein the cells in the cellular composition have the potential to differentiate into cells that exhibit morphological, physiological and/or immunological features of non-hematopoietic cells.

12. A cellular composition as claimed in claim 10 wherein the cells in the cellular composition express embryonic or early non-hematopoietic tissue markers.

15. A cellular composition of claim 1 wherein the cells in the cellular composition have been genetically modified.

17. A method of using a cellular composition as claimed in claim 1 in cell therapies and gene therapies aimed at alleviating disorders and diseases involving hematopoietic or immune system cells.

18. A method of using a cellular composition as claimed in claim 1 in cell therapies and gene therapies aimed at alleviating disorders and diseases involving non-hematopoietic cells.

19. A method of treating a patient with a condition involving a non-hematopoietic cell comprising transferring a cellular composition of claim 1 comprising cells with the potential to form non-hematopoietic cells into the patient, wherein the cell differentiates into the non-hematopoietic cells.

20. A method of using a cellular composition as claimed in claim 1 to screen for potential therapeutics that modulate development or activity of cells with the potential to form non-hematopoietic cells, or cells differentiated therefrom.

21. A method of using a cellular composition of claim 1 to prepare model systems of disease or to produce growth factors or hormones.

28. A pharaceutical composition comprising a cellular composition of claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

33. A kit for producing a cellular composition in claim 1 for for carrying out a method of an isolated and purified cellular composition comprising cells characterized by the following:

(a) CD45⁺HLA-ABC⁺;

(b) Capable of differentiating into hematopoietic cells or progenitor cells;

(c) Capable of differentiating into one or more, two or more, three or more, four or more, five or more, or six or more, different non-hematopoietic cell types;

(d) round shape and non-adherent growth requirements; and

(e) stem cell factor receptor (KIT)⁺.

13. An isolated and purified cellular composition comprising cells characterized by the following:

(a) CD45⁺HLA-ABC⁺;

(b) Capable of differentiating into hematopoietic cells or progenitor cells;

(c) Capable of differentiating into one or more, two or more, three or more, four or more, five or more, or six or more, different non-hematopoietic cell types;

(d) round shape and non-adherent growth requirements; and

(e) stem cell factor receptor (KIT)⁺.

27. A method of identifying the presence of cells of a cellular composition as claimed in claim 13 in a mixed cell population comprising:

exposing the cell population to an antibody or fragment thereof immunogenetically specific for markers (a) or (e) in claim 13, the occurrence of the markers being indicative of the presence of the cells in the cell population.

14. An isolated and purified cellular composition comprising cells characterized by the following:

(a) DC45⁺HLA-ABC⁺;

(b) Capable of differentiating into hematopoietic cells or progenitor cells;

(c) Capable of differentiating into one or more, two or more, three or more, four or more, five or more, or six or more, different non-hematopoietic cell types;

(d) round shape and non-adherent growth requirements;

(e) stem cell factor receptor (KIT)⁺

(f) FLT3ligand receptor⁺;

(g) FGF receptor⁺;

(h) express embryonic stem cell proteins;

(i) HoxB4⁺;

(j) Flk-1⁺;

(k) CD34±

;

(l) non-tumorigenic;

(m) CD38^±

; and

(n) derived from umbilical cord blood.

16. A method of using a cellular composition as claimed in for replacing body tissues, organs, components or structures which are missing or damaged due to trauma, age, metabolic or toxic injury, disease, or idiopathic loss.

22. A method for expanding hematopoietic stem cells and progenitor cells from umbilical cord blood comprising (a) obtaining hematopoietic stem cells and progenitor cells from umbilical cord blood;

(b) culturing the hematopoietic stem cells and progenitor cells under proliferation conditions; and

(c) isolating increased numbers of hematopoietic stem cells and progenitor cells.

23. A method as claimed in claim 22 wherein the number of hematopoietic stem cells and progenitor cells is increased by at least about 2-fold.

24. A method as claimed in claim 22 wherein the hematopoietic stem cells and progenitor cellsare CD45⁺HLA-ABC⁺, preferably CD45⁺HLA-ABC⁺Lin⁻

.

25. A method as claimed in claim 22 wherein the resulting cultured cells in (b) are characterized as follows:

(a) CD45⁺HLA-ABC⁺;

(b) Capable of differentiating into hematopoietic cells or progenitor cells;

(c) Capable of differentiating into one or more, two or more, three or more, four or more, five or more, or six or more, different non-hematopoietic cell types;

(d) round shape and non-adherent growth requirements; and

(e) stem cell factor receptor (KIT)+.

26. A method as claimed in claim 22 which further comprises administering the expanded hematopoietic stem cells and progenitor cells to a patient to alleviate a disorder or disease involving hematopoietic or immune system cells.

29. A method for obtaining non-hematopoietic cells for autologous transplantation from a patient'"'"'s own hematopoietic cells comprising (a) obtaining a sample comprising hematopoietic cells from the patient;

(b) separating out an enriched cell preparation comprising hematopoietic stem cells and hematopoietic progenitor cells, preferably CD45⁺HLA-ABC⁺ cells, more preferably CD45⁺HLA-ABC⁺Lin⁻

cells;

(b) culturing the cells under proliferation conditions to produce a cellular composition comprising cells with the potential or increased potential to form non-hematopoietic cells.

32. A method as claimed in claim 29 wherein the cellular composition is as defined in an isolated and purified cellular composition comprising cells characterized by the following:

(a) CD45⁺HLA-ABC⁺;

(b) Capable of differentiating into hematopoietic cells or progenitor cells;

(c) Capable of differentiating into one or more, two or more, three or more, four or more, five or more, or six or more, different non-hematopoietic cell types;

(d) round shape and non-adherent growth requirements; and

(e) stem cell factor receptor (KIT)⁺.

30. A method for assaying the activity of a test substance comprising the steps of:

(a) culturing cells in an enriched hematopoietic cell preparation comprising hematopoietic stem cells and progenitor cells under proliferation conditions to obtain a cellular composition comprising cells which have potential or increased potential to form non-hematopoietic cells;

(b) optionally culturing the cells which have potential or increased potential to form non-hematopoietic cells under differentiation conditions in vitro;

(c) exposing the cultured cells in step (a) or (b) to test a substance; and

(d) detecting the presence of absence of an effect of the test substance on the survival of the cells or on a morphological, functional, or physiological characteristic and/or molecular biological property of the cells, whereby an effect altering cell survival a morphological, functional, or physiological characteristic and/or a molecular biological property of the cells indicates the activity of the test substance.

31. A method to screen a potential new drug to treat a disorder involving non-hematopoietic cells comprising the steps of:

(a) obtaining hematopoietic cells from a sample from a patient with a disorder involving non-hematopoietic cells;

(b) preparing from the hematopoietic cells an enriched hematopoietic cell preparation comprising hematopoietic stem cells and progenitor cells;

(c) culturing the enriched hematopoietic cell preparation under proliferation conditions to obtain a cellular composition comprising cells with potential or increased potential to form the non-hematopoietic cells;

(d) optionally culturing the cells with potential or increased potential to form the non-hematopoietic cells under differentiation conditions in vitro;

(e) exposing the cultured cells in (c) or (d) to a potential new drug; and

(f) detecting the presence or absence of an effect of the potential new drug on the survival of the cells or on a morphological, functional, or physiological characteristic and/or molecular biological property of said cells, whereby an effect altering a cell survival, a morphological, functional or physiological characteristic and/or a molecular biological property of the cells indicates the activity of the potential new drug.