Compositions and methods of making expanded hematopoietic stem cells using derivatives of fluorene
This invention is directed to, inter alia, compounds, methods, systems, and compositions for the maintenance, enhancement, and expansion of hematopoietic stem cells derived from one or more sources of CD34+ cells. Sources of CD34+ cells include bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood. Also provided herein are compounds of Formula I which are useful in maintaining, enhancing, and expanding of hematopoietic stem cells.
1 . A method for expanding hematopoietic stem cells in culture, the method comprising contacting a source of CD34+ cells in culture with an effective amount of a compound of Formula I, thereby expanding hematopoietic stem cells in the culture, wherein the compound of Formula I is
or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein
A is a fused cyclic moiety selected from the group consisting of a phenyl, C 3-6 cycloalkyl, heterocycloalkyl, and heteroaryl, or is absent,
wherein the heterocycloalkyl comprises from 3 to 6 ring members having 1 to 3 nitrogen atom ring members, and
the heteroaryl comprises 5 to 6 ring members having 1 to 3 nitrogen atom ring members;
R 1 is selected from the group consisting of —C(O)—NR b —R 1a , —NR b —C(O)—R 1a , —NR b —C(O)—R 1b , —NR b —X 1 —C(O)—R 1a , —C(O)—X 1 —NR b —R 1a , —X 1 —C(O)—NR b —R 1a , —X 1 —NR b —C(O)—R 1a , —NR b —C(O)—X 1 —C(O)—R 1b , C(O)—NR b —X 1 —C(O)—R 1b , —NR b —C(O)—O—R 1a , —O—C(O)—NR b —R 1a , —X 1 —NR b —C(O)—O—R 1a , —X 1 —O—C(O)—NR b —R 1a , —NR b —R 1a , —C(O)—R 1a , —O—C(O)—R 1a , halo, and —NO 2 ;
R 1a is selected from the group consisting of H, C 1-10 alkyl, and C 1-10 haloalkyl;
R 1b is selected from the group consisting of —OR a , —NR a R b , heterocycloalkyl, and phenyl,
wherein the heterocycloalkyl comprises from 5 to 6 ring members having 1 to 3 heteroatom ring members selected from the group consisting of nitrogen, oxygen, and sulfur, and
the heterocycloalkyl and the phenyl is unsubstituted or substituted with one to four C 1-4 alkyl, —OH, and halo;
each R 2 is independently selected from the group consisting of halogen, —CN, —C 1-8 alkyl, —C 2-8 alkenyl, C 2-8 alkynyl, C 1-8 haloalkyl, —C 1-8 alkoxy, —X 1 —C 1-8 alkoxy, —C(O)—R 2a , —NR b —C(O)—R 2a , —SR a , —X 1 —SR a , —OR a , —X 1 —OR a , —NR a R b , —X 1 —NR a R b , —S(O) 2 R a , —S(O) 2 NR a R b , —X 1 —S(O) 2 R a , —X 1 —S(O) 2 NR a R b , and —O—C(O)—R a ;
each R 3 is independently selected from the group consisting of halogen, CN, —C 1-8 alkyl, —C 2-8 alkenyl, C 2-8 alkynyl, C 1-8 haloalkyl, —C 1-8 alkoxy, —X 1 —C 1-8 alkoxy, —C(O)—R 3a , —SR a , —X 1 —SR a , —OR a , —X 1 —OR a , —NR a R b , —X 1 —NR a R b , —S(O) 2 R a , —S(O) 2 NR a R b , —X 1 —S(O) 2 R a , and —X 1 —S(O) 2 NR a R b ;
each R 2a and R 3a is independently selected from the group consisting of H, C 1-10 alkyl, C 1-10 haloalkyl, —OR a , —X 1 —OR a , —NR a R b , and —X 1 —NR a R b ;
R 4a is selected from the group consisting of —OR a , —NR a R b , —O—C(O)—R a , and cyano;
R 4b is H; or R 4a and R 4b are combined to form an oxo or an oxime moiety;
each R a and R b is independently selected from the group consisting of H and C 1-4 alkyl;
each X 1 is C 1-4 alkylene;
the subscript n is an integer from 0 to 3; and
the subscript m is an integer from 0 to 2.
2 . The method of claim 1 , wherein the source of CD34+ cells is selected from the group consisting of bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood.
3 . The method of claim 1 , wherein the source of CD34+ cells is mobilized peripheral blood.
4 . The method of claim 1 , wherein the source of CD34+ cells is cord blood.
5 . The method of claim 1 , wherein the source of CD34+ cells is bone marrow.
6 . The method of claim 1 , wherein the source of CD34+ cells is non-mobilized peripheral blood.
7 . The method of claim 2 , wherein the source of CD34+ cells comprises one or more of (a) CD34+ hematopoietic progenitors; (b) CD34+ early hematopoietic progenitors and/or stem cells; (c) CD133+ early hematopoietic progenitors and/or stem cells; and/or (d) CD90+ early hematopoietic progenitors and/or stem cells.
8 . The method of claim 2 , wherein the source of CD34+ cells comprises one or more of (a) CD34+ hematopoietic progenitors; (b) CD34+ early hematopoietic progenitors and/or stem cells; (c) CD133+ early hematopoietic progenitors and/or stem cells; (d) CD90+ early hematopoietic progenitors and/or stem cells; (e) CD45RA− early hematopoietic progenitors and/or stem cells; and/or (f) CD38 low/− early hematopoietic progenitors and/or stem cells.
9 . The method of claim 1 , wherein the method further comprises contacting the cells with a retinoic acid receptor (RAR) inhibitor or modulator.
10 . The method of claim 9 , wherein the retinoic acid receptor (RAR) inhibitor or modulator is ER50891.
11 . The method of claim 1 , wherein the method further comprises culturing the cells under atmospheric oxygen conditions.
12 . The method of claim 11 , wherein atmospheric oxygen conditions comprise an atmosphere containing about 20% oxygen.
13 . The method of claim 1 , wherein the method further comprises culturing the cells under low oxygen conditions.
14 . The method of claim 13 , wherein low oxygen conditions comprise an atmosphere containing about 5% oxygen or less.
15 . The method of claim 1 , wherein the method further comprises contacting the cells with one or more agents selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), hepatocyte growth factor (HGF), p38 MAPK inhibitor, epidermal growth factor (EGF), JAK/STAT inhibitors, interleukin 3 (IL-3), interleukin 6 (IL-6), human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), VEGF-C and ALK5/SMAD modulators or inhibitors.
16 . The method of claim 1 , wherein the method further comprises contacting the cells with thrombopoietin (TPO), stem cell factor (SCF), and fms-related tyrosine kinase 3 ligand (FLT3L).
17 . The method of claim 1 , wherein the method further comprises contacting the cells with thrombopoietin (TPO), stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (FLT3L), and interleukin 6 (IL-6).
18 . The method of claim 1 , wherein the method further comprises contacting the cells with thrombopoietin (TPO) and stem cell factor (SCF).
19 . The method of claim 1 , wherein said method stabilizes the hematopoietic stem cell phenotype.
20 . The method of claim 19 , wherein the hematopoietic stem cell phenotype comprises CD45+, CD34+, CD133+, CD90+, CD45RA−, and CD38 low/−, and is negative for major hematopoietic lineage markers selected from the group consisting of CD2, CD3, CD4, CD5, CD8, CD14, CD16, CD19, CD20, and CD56.
21 . The method of claim 1 , wherein the cells exhibit at least about two times the number of CD133+ and/or CD90+ positive cells compared to cells in culture that are not contacted with a compound of Formula I after 7 days in culture.
22 . The method of claim 1 , wherein the source of the CD34+ cells is a human being.
23 . The method of claim 1 , wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein
A is phenyl, C 3-6 cycloalkyl, or absent;
R 1 is selected from the group consisting of —NR b —C(O)—R 1a , —NR b —C(O)—R 1b , —NR b —R 1a , —O—C(O)—R 1a , halo, and —NO 2 ;
R 1a is selected from the group consisting of H, C 1-10 alkyl, and C 1-10 haloalkyl;
R 1b is selected from the group consisting of —NR a R b , heterocycloalkyl, and phenyl,
wherein the heterocycloalkyl comprises from 5 to 6 ring members having 1 to 3 heteroatom ring members selected from the group consisting of nitrogen, oxygen, and sulfur, and
the heterocycloalkyl and the phenyl is unsubstituted or substituted with one to four C 1-4 alkyl, —OH, or halo;
each R 2 is independently selected from the group consisting of halogen, C 1-8 alkyl, C 1-8 haloalkyl, —NR b —C(O)—R 2a , —OR a , —NR a R b , and —O—C(O)—R a ;
each R 3 is independently selected from the group consisting of halogen, C 1-8 alkyl, C 1-8 haloalkyl, —OR a , and —NR a R b ;
each R 2a and R 3a is independently selected from the group consisting of H, C 1-10 alkyl, C 1-10 haloalkyl, and —NR a R b ;
R 4a is selected from the group consisting of —OR a , —NR a R b , —O—C(O)—R a , and cyano;
R 4b is H; or R 4a and R 4b are combined to form an oxo or an oxime moiety;
each R a and R b is independently selected from the group consisting of H and C 1-4 alkyl;
the subscript n is 0 or 1; and
the subscript m is 0 or 1.
24 . The method of claim 1 , wherein the compound of Formula (I) has the structure of Formula IIIa1
or a pharmaceutically acceptable salt, hydrate, or solvate thereof; wherein,
R 1 is —NH—C(O)—R 1a ;
R 1a is selected from the group consisting of C 2-6 alkyl and C 2-6 haloalkyl;
R 2 is —OH; and
the subscript n is 0 or 1.
25 . The method of claim 24 , wherein n is 0 and R 1a is C 2-4 alkyl.
26 . The method of claim 24 , wherein n is 0 and R 1a is C 2-4 haloalkyl.
27 . The method of claim 24 , wherein the compound of Formula IIIa is selected from the group consisting of
28 . The method of claim 24 , wherein the compound of Formula IIIa is
or a pharmaceutically acceptable salt thereof.
29 . The method of claim 24 , wherein the compound of Formula IIIa1 is
or a pharmaceutically acceptable salt thereof.
30 . The method of claim 24 , wherein the compound of Formula IIIa1 is
or a pharmaceutically acceptable salt thereof.
31 . The method of claim 4 , wherein the compound of Formula (I) has the structure of Formula IIIa1
or a pharmaceutically acceptable salt, hydrate, or solvate thereof; wherein,
R 1 is —NH—C(O)—R 1a ;
R 1a is selected from the group consisting of C 2-6 alkyl and C 2-6 haloalkyl;
R 2 is —OH; and
the subscript n is 0 or 1.
32 . The method of claim 31 , wherein the compound of Formula IIIa1 is
or a pharmaceutically acceptable salt thereof.
33 . The method of claim 31 , wherein the compound of Formula IIIa1 is
or a pharmaceutically acceptable salt thereof.
34 . The method of claim 31 , wherein the compound of Formula IIIa1 is
or a pharmaceutically acceptable salt thereof.
35 . A medium for expanding hematopoietic stem cells in culture comprising:
(a) (i) a base medium or (ii) a feed medium; and
(b) a compound selected from the group consisting of
36 . A method for expanding hematopoietic stem cells in culture, the method comprising contacting a source of CD34+ cells in culture with the medium of claim 35 , thereby expanding hematopoietic stem cells in the culture.
37 . A system for expanding hematopoietic stem cells in culture, the system comprising (a) a source of CD34+ cells in culture; and (b) the medium of claim 35 .
38 . A kit comprising:
(a) the medium of claim 35 ; and
(b) thrombopoietin (TPO), stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (FLT3L), and interleukin 6 (IL-6).
39 . A method for producing a cell culture media for culturing hematopoietic stem cells (HSC), the method comprising: combining (a) the medium of claim 35 ; and (b) thrombopoietin (TPO), stem cell factor (SCE), fms-related tyrosine kinase 3 ligand (PLT3L), and interleukin 6 (IL-6).