1. A method of selecting a population of ex-vivo expanded hematopoietic stem cells suitable for transplantation, the method comprising:
(a) determining prior to administration in a candidate population of expanded hematopoietic cells at least one of the following parameters:
(i) proportion of CD34+ cells in said population;
(ii) fold expansion of total cells in said population;
(iii) viability of said cells in said population; and
(iv) total number of viable cells; and
(b) selecting or excluding said candidate population according to predetermined values of at least one of said parameters,
thereby selecting a population of ex-vivo expanded hematopoietic stem cells suitable for transplantation.
2. The method of claim 1, wherein said proportion of said CD34+ cells is at least about 4 percent of total cells.
3-4. (canceled)
5. The method of claim 1, wherein said fold expansion of total cells of said population is at least about 10 times.
6-7. (canceled)
8. The method of claim 1, wherein said viability of total cells in said population is at least about 65 percent at 21 days culture.
9-10. (canceled)
11. The method of claim 1, wherein said total number of viable cells of said expanded population is at least about 15\xd7106 viable cells.
12-13. (canceled)
14. The method of claim 1, wherein said selecting is determined according to the values of at least two of said parameters.
15. The method of claim 1, wherein said selecting is according to the values of at least three of said parameters.
16. The method of claim 1, wherein said selecting is according to the values of all four of said parameters.
17. The method of claim 1, wherein said selecting is according to the following values of said parameters:
(i) said proportion of said CD34+ cells being about 4 percent of total cells;
(ii) said fold expansion of total cells of said population is about 50 times;
(iii) said viability of total cells in said population is about 85 percent at 21 days culture; and
(iv) said total number of viable cells of said expanded population is about 23\xd7106 viable cells.
18. The method of claim 1, wherein said hematopoietic stem cells are expanded by propagation ex-vivo by culturing hematopoietic cells in the presence of cytokines and a copper chelator.
19. The method of claim 18, wherein said cytokines are early acting cytokines.
20-22. (canceled)
23. The method of claim 18, wherein said copper chelator is tetrethylenepentamine (TEPA).
24. (canceled)
25. The method of claim 1, wherein said expanded hematopoietic stem cells have been cultured for 21 days.
26. (canceled)
27. The method of claim 1, wherein said hematopoietic cell population is selected from a source consisting of umbilical cord blood, peripheral blood and bone marrow.
28-30. (canceled)
31. The method of claim 27, wherein said hematopoietic cell population has been enriched for CD34+ or CD133+ cells prior to expansion.
32. An expanded hematopoietic stem cell population selected suitable for transplantation according to the method of claim 1.
33. An expanded hematopoietic stem cell population selected suitable for transplantation according to the method of claim 17.
34. A method of treating a hematological disease or condition in a subject in need thereof, the method comprising administering to a subject in need thereof the population of expanded hematopoietic stem cells selected suitable for transplantation of claim 1.
35. A method of treating a hematological disease or condition in a subject in need thereof, the method comprising administering to a subject in need thereof the population of expanded hematopoietic stem cells selected suitable for transplantation of claim 17.
36-43. (canceled)
44. The method of claim 34, wherein said hematological disease is selected from the group consisting of acute myelocytic leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CLL), Hodgkins lymphoma (HL), non-Hodgkins lymphoma (NHL) and myelodysplastic syndrome (MDS).
45-51. (canceled)
52. An article of manufacture comprising a packaging material and a selected ex-vivo expanded hematopoietic cell population, said hematopoietic cell population selected suitable for transplantation by:
(a) determining prior to administration in a candidate population of expanded hematopoietic cells at least one of the following parameters:
(i) proportion of CD34+ cells in said population;
(ii) fold expansion of total cells in said population;
(iii) viability of said cells in said population; and
(iv) total number of viable cells; and
(b) selecting or excluding said candidate population according to predetermined values of at least one of said parameters,
and wherein said packaging material comprises a label or package insert indicating that said hematopoietic cell population is for treating a hematological disease or condition in a subject in need thereof.
53. The article of manufacture of claim 52, wherein said selecting is according to the following values of said parameters:
(i) said proportion of said CD34+ cells being about 4 percent of total cells;
(ii) said fold expansion of total cells of said population is about 50 times;
(iii) said viability of total cells in said population is about 85 percent at 21 days culture; and
(iv)said total number of viable cells of said expanded population is about 23\xd7106 viable cells.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. An isolated nucleic acid molecule comprising a synthetic nucleotide sequence encoding a firefly luciferase comprising a fragment of at least 300 nucleotides having 80% or less nucleic acid sequence identity to a parent nucleic acid sequence having SEQ ID NO:43 or 85% or less nucleic acid sequence identity to a parent nucleic acid sequence having SEQ ID NO:14 and having 99% or more nucleic acid sequence identity to SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23 or the complement thereof, wherein the decreased sequence identity is a result of different codons in the synthetic nucleotide sequence relative to the codons in the parent nucleic acid sequence, wherein the synthetic nucleotide sequence encodes a firefly luciferase which has at least 85% amino acid sequence identity to the corresponding luciferase encoded by the parent nucleic acid sequence, and wherein the synthetic nucleotide sequence has a reduced number of regulatory sequences relative to the parent nucleic acid sequence.
2. The isolated nucleic acid molecule of claim 1 wherein the regulatory sequences include transcription factor binding sequences, intron splice sites, poly(A) sites, promoter modules, andor promoter sequences.
3. The isolated nucleic acid molecule of claim 1 wherein a majority of the codons of the synthetic nucleotide sequence which differ from the corresponding codons of the parent nucleic acid sequence are ones that are preferred codons of a desired host cell andor are not low-usage codons in that host cell.
4. The isolated nucleic acid molecule of claim 3 wherein the majority of the codons of the synthetic nucleotide sequence which differ from the corresponding codons of the parent nucleic acid sequence are those which are employed more frequently in mammals.
5. The isolated nucleic acid molecule of claim 3 wherein the majority of the codons of the synthetic nucleotide sequence which differ from the corresponding codons of the parent nucleic acid sequence are those which are preferred codons in humans.
6. The isolated nucleic acid molecule of claim 3 wherein the majority of codons which differ are the codons CGC, CTG, AGC, ACC, CCC, GCC, GGC, GTG, ATC, AAG, AAC, GAG, CAC, GAC, TAC, TGC and TTC.
7. The isolated nucleic acid molecule of claim 1 wherein the synthetic nucleic acid molecule is expressed in a mammalian host cell at a level which is greater than that of the parent nucleic acid sequence.
8. The isolated nucleic acid molecule of claim 1 wherein the synthetic nucleic acid molecule has an increased number of AGC serine-encoding codons, an increased number of CCC proline-encoding codons, an increased number of ATC isoleucine-encoding codons andor an increased number of ACC threonine-encoding codons relative to the number of these codons in the parent nucleic acid sequence.
9. The isolated acid molecule of claim 1 wherein the synthetic nucleotide sequence has at least 10% fewer transcription regulatory sequences relative to the parent nucleic acid sequence.
10. The isolated nucleic acid molecule of claim 1 wherein the codons in the synthetic nucleotide sequence which differ from the corresponding codons of the parent nucleic acid sequence encode the same amino acids as the corresponding codons in the parent nucleic acid sequence.
11. The isolated nucleic acid molecule of claim 1 wherein the nucleic acid molecule encodes a fusion of the luciferase with one or more other peptides or polypeptides, wherein at least the luciferase is encoded by the synthetic nucleic acid sequence.
12. The isolated nucleic acid molecule of claim 1 wherein one or more other peptides are peptides having protein destabilization sequences.
13. A plasmid comprising the nucleic acid molecule of claim 1.
14. The plasmid of claim 13 which further comprises a multiple cloning region.
15. The plasmid of claim 13 which further comprises a promoter operatively linked to the synthetic nucleotide sequence.
16. An expression vector comprising the nucleic acid molecule of claim 1 linked to a promoter functional in a cell.
17. The expression vector of claim 16 wherein the promoter is functional in a eukaryotic cell.
18. The expression vector of claim 16 wherein the expression vector further comprises a multiple cloning site.
19. The expression vector of claim 16 wherein the promoter is functional in a mammalian cell.
20. The expression vector of claim 16 wherein the synthetic nucleotide sequence is operatively linked to a Kozak consensus sequence.
21. An isolated host cell comprising the expression cassette of claim 16.
22. An isolated host cell comprising the plasmid of claim 13.
23. A kit comprising, in suitable container means, the plasmid of claim 13.