U.S. patent application number 14/157944 was filed with the patent office on 2014-05-15 for vectors and methods for the efficient generation of integration/transgene-free induced pluripotent stem cells from peripheral blood cells.
This patent application is currently assigned to Loma Linda University. The applicant listed for this patent is Loma Linda University. Invention is credited to David J. Baylink, Kin-Hing William Lau, Xiaobing Zhang.
Application Number | 20140134143 14/157944 |
Document ID | / |
Family ID | 49624306 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134143 |
Kind Code |
A1 |
Baylink; David J. ; et
al. |
May 15, 2014 |
VECTORS AND METHODS FOR THE EFFICIENT GENERATION OF
INTEGRATION/TRANSGENE-FREE INDUCED PLURIPOTENT STEM CELLS FROM
PERIPHERAL BLOOD CELLS
Abstract
A vector for generating induced pluripotent stem cells from
human target cells comprising a) a vector backbone, b) exactly two,
three or four transcription and reprogramming factor genes, each
gene separated by a 2a self-cleavage peptide sequence, c) a spleen
focus-forming virus promoter, and d) a post-transcriptional
regulatory element Wpre, with or without an anti-apoptotic factor
gene. A method for generating integration-free induced pluripotent
stem cells, the method comprising: a) providing target cells, b)
providing one or more than one vector according to the present
invention, c) transducing or transfecting the target cells with the
one or more than one vector, and d) culturing the transduced or
transfected cells in a cell culture, thereby generating
integration-free induced pluripotent stem cells.
Inventors: |
Baylink; David J.;
(Redlands, US) ; Lau; Kin-Hing William; (Redlands,
CA) ; Zhang; Xiaobing; (Loma Linda, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Loma Linda University |
Loma Linda |
CA |
US |
|
|
Assignee: |
Loma Linda University
Loma Linda
CA
|
Family ID: |
49624306 |
Appl. No.: |
14/157944 |
Filed: |
January 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2013/042115 |
May 21, 2013 |
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14157944 |
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61817135 |
Apr 29, 2013 |
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61650318 |
May 22, 2012 |
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Current U.S.
Class: |
424/93.21 ;
435/320.1; 435/325; 435/455 |
Current CPC
Class: |
C07K 14/4702 20130101;
C12N 2501/48 20130101; C12N 2501/602 20130101; C12N 2830/48
20130101; C12N 2510/00 20130101; C07K 14/4747 20130101; C12N
2501/603 20130101; C07K 2319/92 20130101; C12N 5/0696 20130101;
C12N 2820/007 20130101; C12N 2501/604 20130101; C12N 15/85
20130101; C12N 2830/60 20130101 |
Class at
Publication: |
424/93.21 ;
435/320.1; 435/455; 435/325 |
International
Class: |
C12N 15/85 20060101
C12N015/85 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with United States Government
support under Basic Award W81XWH-11-1-0607 from the United States
Department of Defense, United States Army Medical Research
Acquisition Activity (USAMRAA) Grant W81XWH-08-1-0697 from the
United States Army Medical Research and Materiel Command (USAMRMC).
The United States Government has certain rights in this invention.
Claims
1. An episomal vector for generating induced pluripotent stem cells
from human target cells, the vector comprising: a) an
oriP/EBNA1-based plasmid backbone; b) exactly two transcription and
reprogramming factor genes, oct4 and sox2, separated by a 2a
self-cleavage peptide sequence; c) a spleen focus-forming virus
promoter; d) a post-transcriptional regulatory element Wpre; and e)
anti-apoptotic factor gene selected from the group consisting of
bcl-xl and bcl2.
2. An episomal vector for generating induced pluripotent stem cells
from human target cells, the vector comprising: a) an
oriP/EBNA1-based plasmid backbone; b) exactly three transcription
and reprogramming factor genes, oct4, sox2 and klf4, each separated
by a 2a self-cleavage peptide sequence; c) a spleen focus-forming
virus promoter; d) a post-transcriptional regulatory element Wpre;
and e) anti-apoptotic factor gene selected from the group
consisting of bcl-xl and bcl2.
3. An episomal vector for generating induced pluripotent stem cells
from human target cells, the vector comprising: a) an
oriP/EBNA1-based plasmid backbone; b) exactly four transcription
and reprogramming factor genes, oct4, sox2, klf4 and myc, each
separated by a 2a self-cleavage peptide sequence; c) a spleen
focus-forming virus promoter; d) a post-transcriptional regulatory
element Wpre; and e) anti-apoptotic factor gene selected from the
group consisting of bcl-xl and bcl2.
4. A vector for generating induced pluripotent stem cells from
human target cells, the vector comprising: a) a vector backbone; b)
exactly two, three or four transcription and reprogramming factor
genes, each gene separated by a 2a self-cleavage peptide sequence;
c) a spleen focus-forming virus promoter; and d) a
post-transcriptional regulatory element Wpre.
5. The vector of claim 4, where the vector backbone is an
oriP/EBNA1-based episomal vector.
6. The vector of claim 4, where the vector backbone is an
oriP/EBNA1-based plasmid backbone.
7. The vector of claim 4, where the vector is an episomal
vector.
8. The vector of claim 4, where the vector is selected from the
group consisting of a plasmid, a non-plasmid, a non-integrating
plasmid, a non-integrating vector, a viral vector, a
non-integrating viral vector, a self-inactivating vector and a
lentivirus vector.
9. The vector of claim 4, where the transcription and reprogramming
factor genes are selected from the group consisting of one or more
than one Yamanaka factor gene and one or more than one Thomson/Yu
factor gene, and a combination of the preceding.
10. The vector of claim 4, where one or more than one of the
transcription and reprogramming factor genes are selected from the
group consisting of klf4, lin28, myc, nanog, oct4, sox1, sox2,
sox3, sox15 and sox18.
11. The vector of claim 4, where a plurality of the transcription
and reprogramming factor genes are selected from the group
consisting of klf4, lin28, myc, nanog, oct4, sox1, sox2, sox3,
sox15 and sox18.
12. The vector of claim 4, where all of the transcription and
reprogramming factor genes are selected from the group consisting
of klf4, lin28, myc, nanog, oct4, sox1, sox2, sox3, sox15 and
sox18.
13. The vector of claim 4, where all of the transcription and
reprogramming factor genes are selected from the group consisting
of oct4, sox2, klf4 and myc.
14. The vector of claim 4, where the transcription and
reprogramming factor genes are exactly two transcription and
reprogramming factor genes, oct4 and sox2.
15. The vector of claim 4, where the transcription and
reprogramming factor genes are exactly three transcription and
reprogramming factor genes, oct4, sox2 and klf4.
16. The vector of claim 4, where the transcription and
reprogramming factor genes are exactly four transcription and
reprogramming factor genes, oct4, sox2, klf4 and myc.
17. The vector of claim 4, where the 2a self-cleavage peptide
sequence is selected from the group consisting of equine rhinitis A
virus, foot-and-mouth disease virus, porcine teschovirus-1 and
Thosea asigna virus.
18. The vector of claim 4, further comprising one or more than one
gene coding for an inhibitor, siRNA, or shRNA construct of a
pro-apoptotic factor.
19. The vector of claim 4, further comprising one or more than one
gene coding for an inhibitor, siRNA, or shRNA construct of a
pro-apoptotic factor, where the pro-apoptotic factor is a BAX
subfamily pro-apoptotic factor selected from the group consisting
of BAK, BAX and BOK.
20. The vector of claim 4, further comprising one or more than one
gene coding for an inhibitor, siRNA, or shRNA construct of a
pro-apoptotic factor, where the pro-apoptotic factor is a BH3
subfamily pro-apoptotic factor selected from the group consisting
of BAD, BID, BIK, BIML, BLK, BNIP3 and HRK.
21. The vector of claim 4, further comprising one or more than one
anti-apoptotic factor gene encoding one or more than one
anti-apoptotic factor.
22. The vector of claim 4, further comprising one or more than one
anti-apoptotic factor gene encoding one or more than one
anti-apoptotic factor, where the anti-apoptotic factor is a BCL-2
family anti-apoptotic factor.
23. The vector of claim 4, further comprising one or more than one
anti-apoptotic factor gene encoding one or more than one
anti-apoptotic factor, where the anti-apoptotic factor is a BCL-2
family anti-apoptotic factor selected from the group consisting of
A1, BCL2, BCL-W, BCL-XL and MCL1.
24. The vector of claim 4, further comprising one or more than one
anti-apoptotic factor gene encoding one or more than one
anti-apoptotic factor, where the anti-apoptotic factor is BCL2 or
BCL-XL.
25. A method for generating integration-free induced pluripotent
stem cells, the method comprising: a) providing target cells; b)
providing one or more than one vector of claim 4; c) transducing or
transfecting the target cells with the one or more than one vector;
and d) culturing the transduced or transfected cells in a cell
culture, thereby generating integration-free induced pluripotent
stem cells.
26. The method of claim 25, where the one or more than one vector
provided is one vector.
27. The method of claim 25, where the one or more than one vector
provided is a plurality of vectors.
28. The method of claim 25, where the one or more than one vector
provided is two vectors.
29. The method of claim 25, where the one or more than one vector
provided is three vectors.
30. The method of claim 25, where the one or more than one vector
is a first vector and a second vector, and transducing or
transfecting the target cells comprises transducing or transfecting
the target cells with a first amount of the first vector and a
second amount of a second vector, where the first amount is equal
to the second amount.
31. The method of claim 25, where the one or more than one vector
is a first vector and a second vector, and transducing or
transfecting the target cells comprises transducing or transfecting
the target cells with a first amount of the first vector and a
second amount of a second vector, where the first amount is half of
the second amount.
32. The method of claim 25, where the one or more than one vector
is three vectors.
33. The method of claim 25, where the one or more than one vector
is four vectors.
34. The method of claim 25, where the one or more than one vector
is five vectors.
35. The method of claim 25, where the one or more than one vector
is an episomal vector comprising a strong spleen focus-forming
virus promoter, a post-transcriptional regulatory element Wpre, and
exactly two transcription and reprogramming factor genes, oct4 and
sox2, and the method further comprises transducing or transfecting
the target cells with an additional episomal vector comprising a
strong spleen focus-forming virus promoter, a post-transcriptional
regulatory element Wpre, an anti-apoptotic factor gene bcl-xl, and
exactly one transcription and reprogramming factor gene, klf4.
36. The method of claim 25, where the one or more than one vector
is an episomal vector comprising a strong spleen focus-forming
virus promoter, a post-transcriptional regulatory element Wpre, and
exactly two transcription and reprogramming factor genes, oct4 and
sox2, and the method further comprises transducing or transfecting
the target cells with a first additional episomal vector comprising
a strong spleen focus-forming virus promoter, a
post-transcriptional regulatory element Wpre, and exactly one
transcription and reprogramming factor gene, klf4, and with a
second additional episomal vector comprising a strong spleen
focus-forming virus promoter, a post-transcriptional regulatory
element Wpre, and an anti-apoptotic factor gene bcl-xl, but without
any transcription and reprogramming factor gene.
37. The method of claim 25, where the target cells are
hematopoietic stem cells.
38. The method of claim 25, where the target cells are peripheral
blood mononuclear cells.
39. The method of claim 25, where the target cells are peripheral
blood myeloid cells.
40. The method of claim 25, where the target cells are peripheral
blood cells that have been enriched for one or more than one cell
type selected from the group consisting of CD33+ cells, CD34+ cells
and CD133+ cells.
41. The method of claim 25, where the target cells are peripheral
blood mononuclear cells that have been enriched for CD33+
cells.
42. The method of claim 25, where the target cells are peripheral
blood cells that have been depleted of cells that express T cell
marker CD3 or B cell maker CD19.
43. The method of claim 25, further comprising harvesting the
target cells from a body fluid or tissue.
44. The method of claim 43, where the body fluid or tissue is
selected from the group consisting of bone marrow and cord
blood.
45. The method of claim 43, where the body fluid or tissue is
peripheral blood.
46. The method of claim 25, further comprising providing cord
blood, and purifying the cord blood to obtain the target cells.
47. The method of claim 46, where the cord blood is obtained from a
cord blood bank.
48. The method of claim 25, further comprising enhancing or
purifying the target cells for cells that express a CD33
marker.
49. The method of claim 25, further comprising enhancing or
purifying the target cells for cells that express a CD34 marker or
a CD133 marker.
50. The method of claim 25, further comprising depleting the target
cells of cells that express a T cell marker CD3 or a B cell maker
CD19.
51. The method of claim 25, further comprising enhancing or
purifying the target cells for cells that express a CD33 marker,
and depleting the target cells of cells that express a T cell
marker CD3 or a B cell maker CD19.
52. The method of claim 25, further comprising purifying
integration-free induced pluripotent stem cells from the cell
culture after generating the integration-free induced pluripotent
stem cells.
53. The method of claim 25, further comprising culturing the target
cells in a cell culture for a duration of between three days and
six days before transducing or transfecting the target cells.
54. The method of claim 25, further comprising culturing the target
cells in a cell culture for a duration of four days before
transducing or transfecting the target cells.
55. Integration-free induced pluripotent stem cells generated by
the method of claim 25.
56. Integration-free induced pluripotent stem cells of claim 55
that express one or more than one marker for a mature cell type
selected from the group consisting of cardiomyocytes, hepatocytes
and mesenchymal stem cells.
57. Integration-free induced pluripotent stem cell colonies formed
by the integration-free induced pluripotent stem cells generated by
the method of claim 25.
58. Integration-free induced pluripotent stem cell colonies of
claim 57 that express one or more than one marker for a mature cell
type selected from the group consisting of cardiomyocytes,
hepatocytes and mesenchymal stem cells.
59. A method of treating a patient having a condition or disease,
the method comprising: a) identifying a patient with a condition or
disease suitable for treatment by the present method; and b)
administering integration-free induced pluripotent stem cells
according to the present invention or generated by a method
according to claim 25.
60. The method of claim 59, where the patient is a human.
61. The method of claim 59, where the condition or disease is
selected from the group consisting of an autoimmune disease,
cancer, cardiovascular disease, a connective tissue disease, an
injury, and a neurodegenerative disease.
62. The method of claim 59, where identifying the patient comprises
diagnosing the patient with one or more than one condition or
disease suitable for treatment by the method.
63. The method of claim 62, where diagnosing the patient comprises
performing one or more than one of action selected from the group
consisting of performing a physical examination, performing a
non-invasive imaging examination, and identifying one or more than
one marker for a condition or disease in the blood or other body
fluid of the patient.
64. The method of claim 59, where identifying the patient comprises
consulting patient records to determine if the patient has a
condition or disease suitable for treatment by the method.
65. A method for generating integration-free induced pluripotent
stem cells, the method comprising: a) providing target cells; b)
providing one or more than one vector of claim 1; c) transducing or
transfecting the target cells with the one or more than one vector;
and d) culturing the transduced or transfected cells in a cell
culture, thereby generating integration-free induced pluripotent
stem cells.
66. A method for generating integration-free induced pluripotent
stem cells, the method comprising: a) providing target cells; b)
providing one or more than one vector of claim 2; c) transducing or
transfecting the target cells with the one or more than one vector;
and d) culturing the transduced or transfected cells in a cell
culture, thereby generating integration-free induced pluripotent
stem cells.
67. A method for generating integration-free induced pluripotent
stem cells, the method comprising: a) providing target cells; b)
providing one or more than one vector of claim 3; c) transducing or
transfecting the target cells with the one or more than one vector;
and d) culturing the transduced or transfected cells in a cell
culture, thereby generating integration-free induced pluripotent
stem cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of International
Patent Application No. PCT/US2013/042115 titled "Vectors and
Methods for the Efficient Generation of Integration/Transgene-Free
Induced Pluripotent Stem Cells from Peripheral Blood Cells," filed
May 21, 2013, which claims the benefit of U.S. Provisional Patent
Application No. 61/817,135 titled "Efficient Generation of
Integration-Free iPS Cells from Human Adult Peripheral Blood Using
BCL-XL Together With Yamanaka Factors," filed Apr. 29, 2013; and
U.S. Provisional Patent Application No. 61/650,318 titled
"Substance and Method for Generating Induced Pluripotent Stem
Cells," filed May 22, 2012, the contents of which are incorporated
in this disclosure by reference in their entirety.
BACKGROUND
[0003] Induced pluripotent stem cells (iPSCs) that have been
generated from somatic cells have a large variety of current and
potential uses in regenerative medicine. Among these uses are
generating patient-specific cells, tissues and organs for
replacement therapy, and for modeling diseases for research.
[0004] Induced pluripotent stem cells have been generated from
somatic cells such as fibroblasts derived from a skin biopsy by the
overexpression of Yamanaka factors (KLF4, MYC, OCT4 and SOX2) or
Thomson/Yu factors (LIN28, NANOG, OCT4 and SOX2).
Disadvantageously, however, several weeks are required to prepare
cells from a skin biopsy for use in generating induced pluripotent
stem cells. Further, induced pluripotent stem cells have also been
generated from hematopoietic stem cells (progenitor cells) (HSCs)
such as CD34+ cells, CD 133+ cells, or from unenriched cells such
as mononuclear cells (MNCs) that are harvested from bone marrow,
cord blood or peripheral blood, and advantageously do not require
substantial time to prepare the cells for use in generating induced
pluripotent stem cells. Disadvantageously, however, isolating
hematopoietic stem cells or CD34+ cells from mobilized peripheral
blood and bone marrow is invasive, time-consuming and has potential
risks for the donor. Further, generating induced pluripotent stem
cells from cord blood cells has only been accomplished only at an
efficiency that is too low for widespread clinical use.
[0005] Additionally, in some clinical applications,
integration/transgene-free induced pluripotent stem cells are
preferably used to ameliorate potential adverse effects due to
retroviral or lentiviral integration, or due to the interference by
residual expression of reprogramming factors during differentiation
of induced pluripotent stem cells into progenies. Several methods
have been used to produce integration/transgene-free induced
pluripotent stem cells, including the use of adenoviruses,
artificial chromosome vectors, the Cre/loxP system or excisable
polycistronic lentiviral vectors, minicircle DNA, piggyBac
transposon, plasmids, protein transduction, the Sendai virus and
synthetic modified mRNA. Disadvantageously, however, these methods
are associated with very low efficiency of
integration/transgene-free induced pluripotent stem cells
generation, require repetitive induction or selection, or require
virus production. For example, techniques using excisable
polycistronic lentiviral vectors and transposons require a separate
step to remove the transgenes once reprogramming has been achieved,
while using synthetic modified mRNA to produce
integration/transgene-free induced pluripotent stem cells requires
the daily addition of mRNA by lipofection, and transfection by
lipofection is difficult to achieve with some cell types including
blood CD34+ cells.
[0006] Further, integration/transgene-free induced pluripotent stem
cells have been generated from somatic cells using the Epstein-Barr
virus (EBV) latent gene-based episomal vector (EBNA)-based episomal
vector) that advantageously requires only one transfection of
vector DNA by nucleofection for efficient reprogramming, and that
is lost in 5% or more of the cells after each cell division,
leading to depletion of the vector from cells after long-term
passage. Additionally, integration/transgene-free induced
pluripotent stem cells have been generated from somatic cells using
the pCEP4 vector (that contains the gene coding for the Epstein
Barr nuclear antigen (EBNA1) and OriP sequence). Disadvantageously,
however, the use of the Epstein-Barr virus (EBV) latent gene-based
episomal vector and pCEP4 vector also requires five to seven
additional reprogramming factor genes, including strong oncogenes
like Myc (c-Myc) (a regulator gene that codes for a transcription
factor) or simian virus 40 large T antigen (SV40LT) that might
raise safety concerns for general clinical use of the induced
pluripotent stem cells generated by using these factors.
[0007] The most cost effective approach for generating
integration/transgene-free induced pluripotent stem cells from
somatic cells is using EV, a plasmid comprising two elements from
Epstein-Bar virus (oriP and EBNA1), because there is no need for
packaging of viral vectors and one infection is sufficient for
successful reprogramming instead of multiple daily infection or the
multiple additions of other factors. Binding of the EBNA1 protein
to the virus replicon region oriP maintains a relatively long-term
episomal presence of the EV plasmids in mammalian cells. These
unique features of EV makes it an ideal vector for generating
integration/transgene-free induced pluripotent stem cells. EV
yields expression of reprogramming factors at sufficiently high
levels for several cell divisions, thus allowing for successful
reprogramming after only one infection, while the gradual depletion
of plasmids during each cell division leads to the generation of
integration/transgene-free induced pluripotent stem cells after
approximately 2 months of culture.
[0008] Among the various cell types used for reprogramming,
fibroblasts from skin biopsy or other sources were initially used
in many studies for the generation of iPSCs; however, mononuclear
cells (MNCs) from peripheral blood (PB) have been widely accepted
as a more convenient and almost unlimited resource for cell
reprogramming Peripheral blood mononuclear cells are a mixed
population, containing lymphoid cells, including T cells and B
cells, and non-lymphoid cells, including myeloid cells, as well as
between 0.01% and 0.1% CD34.sup.+ hematopoietic stem/progenitor
cells (HSCs). In earlier studies, mature T or B cells were
efficiently converted to induced pluripotent stem cells with Sendai
virus or EV plasmids. However, induced pluripotent stem cells
generated from T cells and B cells contain T cell receptor (TRC) or
immunoglobulin (IG) gene rearrangements, restricting their broad
applications in regenerative medicine. Therefore, attempts to
generate integration/transgene-free induced pluripotent stem cells
from non-lymphoid cells have been made, however, these attempts
generated only between one and five integration-free induced
pluripotent stem cells colonies from 1 ml of peripheral blood which
is too low for therapeutic use. More recent approaches using
factors including EBNA1 and shRNA against TP53 (also known as p53)
generate up to ten induced pluripotent stem cells colonies from 1
ml of peripheral blood in non-T cell culture conditions; however,
expression of EBNA1 and TP53 shRNA synergistically inhibits the
genome guardian p53, which raises concerns about the genomic
integrity of induced pluripotent stem cells generated using this
approach.
[0009] Therefore, there is a need for a vector and method for
generating integration-free induced pluripotent stem cells from
somatic cells that are not subject to these disadvantages, where
the vector and method generate sufficient numbers of
integration/transgene-free induced pluripotent stem cells from
somatic cells for therapeutic use in a cost-effective manner that
does not require the use of excessive number of factors such as
TP53 shRNA.
SUMMARY
[0010] According to one embodiment of the present invention, there
is provided an episomal vector for generating induced pluripotent
stem cells from human target cells, the vector comprising: a) an
oriP/EBNA1-based plasmid backbone; b) exactly two transcription and
reprogramming factor genes, oct4 and sox2, separated by a 2a
self-cleavage peptide sequence; c) a spleen focus-forming virus
promoter; d) a post-transcriptional regulatory element Wpre; and e)
anti-apoptotic factor gene selected from the group consisting of
bcl-xl and bcl2.
[0011] According to another embodiment of the present invention,
there is provided an episomal vector for generating induced
pluripotent stem cells from human target cells, the vector
comprising: a) an oriP/EBNA1-based plasmid backbone; b) exactly
three transcription and reprogramming factor genes, oct4, sox2 and
klf4, each separated by a 2a self-cleavage peptide sequence; c) a
spleen focus-forming virus promoter; d) a post-transcriptional
regulatory element Wpre; and e) anti-apoptotic factor gene selected
from the group consisting of bcl-xl and bcl2.
[0012] According to another embodiment of the present invention,
there is provided an episomal vector for generating induced
pluripotent stem cells from human target cells, the vector
comprising: a) an oriP/EBNA1-based plasmid backbone; b) exactly
four transcription and reprogramming factor genes, oct4, sox2, klf4
and myc, each separated by a 2a self-cleavage peptide sequence; c)
a spleen focus-forming virus promoter; d) a post-transcriptional
regulatory element Wpre; and e) anti-apoptotic factor gene selected
from the group consisting of bcl-xl and bcl2.
[0013] According to another embodiment of the present invention,
there is provided a vector for generating induced pluripotent stem
cells from human target cells, the vector comprising: a) a vector
backbone; b) exactly two, three or four transcription and
reprogramming factor genes, each gene separated by a 2a
self-cleavage peptide sequence; c) a spleen focus-forming virus
promoter; and d) a post-transcriptional regulatory element Wpre. In
one embodiment, the vector backbone is an oriP/EBNA1-based episomal
vector. In another embodiment, the vector backbone is an
oriP/EBNA1-based plasmid backbone. In another embodiment, the
vector is an episomal vector. In another embodiment, the vector is
selected from the group consisting of a plasmid, a non-plasmid, a
non-integrating plasmid, a non-integrating vector, a viral vector,
a non-integrating viral vector, a self-inactivating vector and a
lentivirus vector. In one embodiment, the transcription and
reprogramming factor genes are selected from the group consisting
of one or more than one Yamanaka factor gene and one or more than
one Thomson/Yu factor gene, and a combination of the preceding. In
another embodiment, one or more than one of the transcription and
reprogramming factor genes are selected from the group consisting
of klf4, lin28, myc, nanog, oct4, sox1, sox2, sox3, sox15 and
sox18. In another embodiment, a plurality of the transcription and
reprogramming factor genes are selected from the group consisting
of klf4, lin28, myc, nanog, oct4, sox1, sox2, sox3, sox15 and
sox18. In another embodiment, all of the transcription and
reprogramming factor genes are selected from the group consisting
of klf4, lin28, myc, nanog, oct4, sox1, sox2, sox3, sox15 and
sox18. In another embodiment, all of the transcription and
reprogramming factor genes are selected from the group consisting
of oct4, sox2, klf4 and myc. In another embodiment, the
transcription and reprogramming factor genes are exactly two
transcription and reprogramming factor genes, oct4 and sox2. In
another embodiment, the transcription and reprogramming factor
genes are exactly three transcription and reprogramming factor
genes, oct4, sox2 and klf4. In another embodiment, the
transcription and reprogramming factor genes are exactly four
transcription and reprogramming factor genes, oct4, sox2, klf4 and
myc. In one embodiment, the 2a self-cleavage peptide sequence is
selected from the group consisting of equine rhinitis A virus,
foot-and-mouth disease virus, porcine teschovirus-1 and Thosea
asigna virus. In another embodiment, the vector further comprises
one or more than one gene coding for an inhibitor, siRNA, or shRNA
construct of a pro-apoptotic factor. In another embodiment, the
vector further comprises one or more than one gene coding for an
inhibitor, siRNA, or shRNA construct of a pro-apoptotic factor,
where the pro-apoptotic factor is a BAX subfamily pro-apoptotic
factor selected from the group consisting of BAK, BAX and BOK. In
another embodiment, the vector further comprises one or more than
one gene coding for an inhibitor, siRNA, or shRNA construct of a
pro-apoptotic factor, where the pro-apoptotic factor is a BH3
subfamily pro-apoptotic factor selected from the group consisting
of BAD, BID, BIK, BIML, BLK, BNIP3 and HRK. In another embodiment,
the vector further comprises one or more than one anti-apoptotic
factor gene encoding one or more than one anti-apoptotic factor. In
another embodiment, the vector further comprises one or more than
one anti-apoptotic factor gene encoding one or more than one
anti-apoptotic factor, where the anti-apoptotic factor is a BCL-2
family anti-apoptotic factor. In another embodiment, the vector
further comprises one or more than one anti-apoptotic factor gene
encoding one or more than one anti-apoptotic factor, where the
anti-apoptotic factor is a BCL-2 family anti-apoptotic factor
selected from the group consisting of A1, BCL2, BCL-W, BCL-XL and
MCL1. In another embodiment, the vector further comprises one or
more than one anti-apoptotic factor gene encoding one or more than
one anti-apoptotic factor, where the anti-apoptotic factor is BCL2
or BCL-XL.
[0014] According to another embodiment of the present invention,
there is provided a method for generating integration-free induced
pluripotent stem cells. The method comprises: a) providing target
cells; b) providing one or more than one vector to the present
invention; c) transducing or transfecting the target cells with the
one or more than one vector; and d) culturing the transduced or
transfected cells in a cell culture, thereby generating
integration-free induced pluripotent stem cells. In one embodiment,
the one or more than one vector provided is one vector. In another
embodiment, the one or more than one vector provided is a plurality
of vectors. In another embodiment, the one or more than one vector
provided is two vectors. In another embodiment, the one or more
than one vector provided is three vectors. In another embodiment,
the one or more than one vector is a first vector and a second
vector, and transducing or transfecting the target cells comprising
transducing or transfecting the target cells with a first amount of
the first vector and a second amount of a second vector, where the
first amount is equal to the second amount. In another embodiment,
the one or more than one vector is a first vector and a second
vector, and transducing or transfecting the target cells comprising
transducing or transfecting the target cells with a first amount of
the first vector and a second amount of a second vector, where the
first amount is half of the second amount. In another embodiment,
the one or more than one vector is three vectors. In another
embodiment, the one or more than one vector is four vectors. In
another embodiment, the one or more than one vector is five
vectors. In another embodiment, the one or more than one vector is
an episomal vector comprising a strong spleen focus-forming virus
promoter, a post-transcriptional regulatory element Wpre, and
exactly two transcription and reprogramming factor genes, oct4 and
sox2, and the method further comprises transducing or transfecting
the target cells with an additional episomal vector comprising a
strong spleen focus-forming virus promoter, a post-transcriptional
regulatory element Wpre, an anti-apoptotic factor gene bcl-xl, and
exactly one transcription and reprogramming factor gene, klf4. In
another embodiment, the one or more than one vector is an episomal
vector comprising a strong spleen focus-forming virus promoter, a
post-transcriptional regulatory element Wpre, and exactly two
transcription and reprogramming factor genes, oct4 and sox2, and
the method further comprises transducing or transfecting the target
cells with a first additional episomal vector comprising a strong
spleen focus-forming virus promoter, a post-transcriptional
regulatory element Wpre, and exactly one transcription and
reprogramming factor gene, klf4, and with a second additional
episomal vector comprising a strong spleen focus-forming virus
promoter, a post-transcriptional regulatory element Wpre, and an
anti-apoptotic factor gene bcl-xl, but without any transcription
and reprogramming factor gene. In another embodiment, the target
cells are hematopoietic stem cells. In another embodiment, the
target cells are peripheral blood mononuclear cells. In another
embodiment, the target cells are peripheral blood myeloid cells. In
another embodiment, the target cells are peripheral blood cells
that have been enriched for one or more than one cell type selected
from the group consisting of CD33+ cells, CD34+ cells and CD133+
cells. In another embodiment, the target cells are peripheral blood
mononuclear cells that have been enriched for CD33+ cells. In
another embodiment, the target cells are peripheral blood cells
that have been depleted of cells that express T cell marker CD3 or
B cell maker CD 19. In another embodiment, the method further
comprises harvesting the target cells from a body fluid or tissue.
In one embodiment, the body fluid or tissue is selected from the
group consisting of bone marrow and cord blood. In another
embodiment, the body fluid or tissue is peripheral blood. In
another embodiment, the method further comprises providing cord
blood, and purifying the cord blood to obtain the target cells. In
one embodiment, the cord blood is obtained from a cord blood bank.
In another embodiment, the method further comprises enhancing or
purifying the target cells for cells that express a CD33 marker. In
another embodiment, the method further comprises enhancing or
purifying the target cells for cells that express a CD34 marker or
a CD133 marker. In another embodiment, the method further comprises
depleting the target cells of cells that express a T cell marker
CD3 or a B cell maker CD 19. In another embodiment, the method
further comprises enhancing or purifying the target cells for cells
that express a CD33 marker, and depleting the target cells of cells
that express a T cell marker CD3 or a B cell maker CD19. In another
embodiment, the method further comprises purifying integration-free
induced pluripotent stem cells from the cell culture after
generating the integration-free induced pluripotent stem cells. In
another embodiment, the method further comprises culturing the
target cells in a cell culture for a duration of between three days
and six days before transducing or transfecting the target cells.
In another embodiment, the method further comprises culturing the
target cells in a cell culture for a duration of four days before
transducing or transfecting the target cells.
[0015] According to another embodiment of the present invention,
there is provided integration-free induced pluripotent stem cells
generated by a method according to the present invention. In one
embodiment, the integration-free induced pluripotent stem cells
express one or more than one marker for a mature cell type selected
from the group consisting of cardiomyocytes, hepatocytes and
mesenchymal stem cells.
[0016] According to another embodiment of the present invention,
there is provided integration-free induced pluripotent stem cell
colonies formed by the integration-free induced pluripotent stem
cells generated by a method according to the present invention. In
one embodiment, the integration-free induced pluripotent stem cell
colonies express one or more than one marker for a mature cell type
selected from the group consisting of cardiomyocytes, hepatocytes
and mesenchymal stem cells.
[0017] According to another embodiment of the present invention,
there is provided a method of treating a patient having a condition
or disease. The method comprises: a) identifying a patient with a
condition or disease suitable for treatment by the present method;
and b) administering integration-free induced pluripotent stem
cells according to the present invention or generated by a method
according to the present invention. In one embodiment, the patient
is a human. In another embodiment, the condition or disease is
selected from the group consisting of an autoimmune disease,
cancer, cardiovascular disease, a connective tissue disease, an
injury, and a neurodegenerative disease. In another embodiment,
identifying the patient comprises diagnosing the patient with one
or more than one condition or disease suitable for treatment by the
method. In one embodiment, diagnosing the patient comprises
performing one or more than one of action selected from the group
consisting of performing a physical examination, performing a
non-invasive imaging examination, and identifying one or more than
one marker for a condition or disease in the blood or other body
fluid of the patient. In another embodiment, identifying the
patient comprises consulting patient records to determine if the
patient has a condition or disease suitable for treatment by the
method.
DRAWINGS
[0018] These and other features, aspects and advantages of the
present invention will become better understood with regard to the
following description, appended claims, and accompanying drawings
where:
[0019] FIG. 1 is a schematic depiction of the self-inactivating
(SIN) lentiviral vector backbones for expression of OCT4 (O), SOX2
(S) and OCT4 and SOX2 (OS), where .DELTA. indicates the SIN design
with partially deleted U3 of the 3' long terminal repeat, cPPT is a
central polypurine tract, Wpre is a post-transcriptional regulatory
element, RRE is a rev-responsive element, .psi. is a packaging
signal, and SFFV is the spleen focus-forming virus U3 promoter;
[0020] FIG. 2 is schematic depiction of the self-inactivating (SIN)
lentiviral vector backbones for expression of GFP, where .DELTA.
indicates the SIN design with partially deleted U3 of the 3' long
terminal repeat, cPPT is a central polypurine tract, Wpre is a
post-transcriptional regulatory element, RRE is a rev-responsive
element, .psi. is a packaging signal, SFFV is the spleen
focus-forming virus U3 promoter, EF1 is the Elongation factor-1
alpha promoter and PGK is the phosphoglycerokinase promoter;
[0021] FIG. 3 is a graph of the measured GFP intensity for
expression of the GFP driven by the PGK promoter (left), the EF-1
alpha promoter (center) and the SFFV promoter (right) in cord blood
cells;
[0022] FIG. 4 is a graph of the measured GFP intensity for
expression of the fusion gene OCT4GFP driven by the PGK promoter
(left), the EF1 promoter (center) or the SFFV promoter (right) in
cord blood cells;
[0023] FIG. 5 is a graph of the number of induced pluripotent stem
cell colonies generated from 1.times.10.sup.4 cord blood CD34+
cells that were transduced only with the vector mediating
overexpression of both OCT4 and SOX2 driven by the EF1 promoter
(left), transduced with both the vector mediating overexpression of
both OCT4 and SOX2 driven by the EF1 promoter and the vector
mediating overexpression of MYC driven the SFFV promoter (center),
and transduced only with the vector mediating overexpression of MYC
driven the SFFV promoter (right);
[0024] FIG. 6 is a schematic depiction of an episomal mammalian
expression vector backbone (bottom) for (from upper to lower,
respectively) co-expression of OCT4 and SOX2 (OS) without Wpre
(pCEP-OS (w/o W)), co-expression of OCT4 and SOX2 (OS) with Wpre
(pCEP-OS), expression of KLF4 (K) (pCEP-K), and expression of MYC
(MK) (pCEP-MK); where 2a is a self-cleavage site derived from
equine rhinitis A virus, Wpre is a post-transcriptional regulatory
element, SV40PolyA is a polyadenylation signal from SV40 virus,
OriP is an EBV origin of replication, and EBNA1 is Epstein-Barr
nuclear antigen 1;
[0025] FIG. 7 are graphs of the relative expression of OCT4 (left)
and SOX2 (right) for cells transfected with pCEP-OS (w/o Wpre)
(left bar) and pCEP-OS (with Wpre) (right bar);
[0026] FIG. 8 is a graph of the number of induced pluripotent stem
cells generated from 1.times.10.sup.5 cord blood CD34+ cells
transfected with the pCEP-OS episomal vector (OS) (left-most bar),
with the pCEP-OS episomal vector and the pCEP-K episomal vector
(OS+K) (center bar), or with the pCEP-OS episomal vector and the
pCEP-MK episomal vector (OS+MK) (right-most bar);
[0027] FIG. 9 is graph of the number of induced pluripotent stem
colonies generated from 1.times.10.sup.4 cord blood CD34+ cells
when reprogrammed by using balanced expression of OCT4 and SOX2
(OS) (left-most bar), OS+BCL2 (center left bar), OS+BCL-XL (center
right bar), and OS+MCL (right-most bar);
[0028] FIG. 10 is a graph of the number of induced pluripotent stem
colonies generated from 1.times.10.sup.5 peripheral blood
mononuclear cells when reprogrammed by using balanced expression of
OCT4 and SOX2 (OS) (left-most), OS+BCL2 (center left), OS+BCL-XL
(center right), and OS+MCL (right-most);
[0029] FIGS. 11A-11C are, respectively, a schematic depiction of an
episomal mammalian expression vector backbone (bottom) for (from
upper to lower, respectively) co-expression of OCT4 and SOX2
(pCEP-OS), expression of KLF4 (pCEP-K), expression of BCL-XL
(pCEP-B), co-expression of BCL-XL and KLF4 (pCEP-BK), co-expression
of OCT4, SOX2, BCL-XL and KLF4 (pCEP-OSBK), and co-expression of
MYC and KLF4 (pCEP-MK), where 2a is a self-cleavage site derived
from equine rhinitis A virus, SFFV is a spleen focus-forming virus
promoter, Wpre is a post-transcriptional regulatory element,
SV40PolyA is a polyadenylation signal from SV40 virus, OriP is an
EBV origin of replication, and EBNA1 is Epstein-Barr nuclear
antigen 1 (FIG. 11A); photographs of alkaline phosphatase staining
of induced pluripotent stem cell colonies at four weeks after
nucleofection of adult peripheral blood mononuclear cells with
episomal vectors expressing OCT4 and SOX2 (OS) (left-most); OCT4,
SOX2 (OS) and BCL-XL (OS+B) (center left); OCT4, SOX2, MYC and KLF4
(OS+MK) (center right), and OCT4, SOX2, MYC, KLF4 and BCL-XL
(OS+MK+B) (right most) (FIG. 11B); and a graph of the number of
induced pluripotent stem colonies generated from 1 ml of adult
peripheral blood mononuclear cells nucleofected with the episomal
vectors expressing reprogramming factors OCT4 and SOX2 (OS) without
BCL-XL/with BCL-XL (left-most two bars); and OCT4, SOX2 and KLF4
(OS+K) without BCL-XL/with BCL-XL (center two bars); and OCT4,
SOX2, MYC and KLF4 (OS+MK) without BCL-XL/with BCL-XL (right-most
two bars) (FIG. 11C);
[0030] FIGS. 12A-12B are, respectively, photographs of alkaline
phosphatase staining of induced pluripotent stem cell colonies at
four weeks after nucleofection of fractionated adult peripheral
blood mononuclear cells with episomal vectors expressing
reprogramming factors OCT4, SOX2, MYC, KLF4 and BCL-XL (OS+MK+B),
where the fractionated adult peripheral blood mononuclear cells
expressed the myeloid lineage marker CD33 (CD33+, left-most), did
not express the myeloid lineage marker CD33 (CD33-, center left),
expressed the T cell marker CD3 or the B cell marker CD19
(CD3+/CD19+, center right), and did not express the T cell marker
CD3 or the B cell marker CD19 (CD3-/CD19-, right-most) (FIG. 12A);
and a graph of the number of induced pluripotent stem colonies
generated from 1 ml of adult whole peripheral blood mononuclear
cells nucleofected with the episomal vectors expressing OCT4, SOX2,
MYC, KLF4 and BCL-XL (left bar), and generated from 1 ml of adult
peripheral blood mononuclear cells that were T cell/B cell
lymphocyte depleted (CD3-/CD19-) nucleofected with the episomal
vectors expressing OCT4, SOX2, MYC, KLF4 and BCL-XL (right bar)
(FIG. 12B);
[0031] FIG. 13 is a graph of the number of induced pluripotent stem
cell colonies generated from 1 ml of adult peripheral blood
mononuclear cells that were depleted of cells that expressed the T
cell marker CD3 or the B cell marker CD19 (CD3-/CD19-), and were
then nucleofected with the episomal vectors expressing OCT4, SOX2,
MYC, KLF4 and BCL-XL versus the number of days in culture before
nucleofection; and
[0032] FIGS. 14A-14G are, respectively, a photograph of a
representative induced pluripotent stem cell colony (FIG. 14A); a
photograph of a representative karyogram of an induced pluripotent
stem cell clone (FIG. 14B); representative images captured using a
Zeiss LSM 710 confocal microscope with a 20.times. objective of
induced pluripotent stem cells immunostained to show expression of
pluripotency markers OCT4 (left), SOX2 (center), and NANOG and
SSEA4 (right) by representative induced pluripotent stem cell
colonies (FIG. 14C); representative images captured using an
Olympus microscope with a 20.times. objective of cell layer
derivatives in hematoxylin and eosin (H & E) staining formed by
teratomas in immunodeficient mice produced by representative
induced pluripotent stem cell colonies, where the teratoma cell
layers included all three embryonic germ layers, cartilage
(mesoderm, left), glands (endoderm, center) and neurotubules
(ectoderm, right) (FIG. 14D); photographs of representative induced
pluripotent stem cell colonies showing differentiation into
mesenchymal stem cells (left-most), stained with Oil Red O stains
to show the oil droplets of adipocytes (center left), stained with
Alizarin Red to show bone nodules formed by osteoblasts (center
right), and stained with Alcian Blue to show acid
mucopolysaccharides synthesized and secreted by chondrocytes
(right-most) (FIG. 14E); photographs of representative induced
pluripotent stem cell colonies showing differentiation into
hepatocytes (left-most), and stained with monoclonal antibody
against alpha-fetoprotein (AFP) to show expression of
alpha-fetoprotein (center left), stained with goat anti-albumin to
show expression of albumin (ALB), and stained with goat anti-alpha
1-antitrypsin to show expression of alpha 1-antitrypsin
(.alpha.1-AT) (FIG. 14F); and a photograph of representative
induced pluripotent stem cell colonies showing expression of
Troponin I marker showing differentiation into cardiomyocytes where
the cell nuclei are counterstained with
4',6-diamidino-2-phenylindole (FIG. 14G).
DESCRIPTION
[0033] According to one embodiment of the present invention, there
is provided a vector for generating integration/transgene-free
induced pluripotent stem cells from target cells, where the target
cells are hematopoietic stem cells or somatic cells. In one
embodiment, the vector is a viral vector. In another embodiment,
the vector is an episomal vector. The vector comprises a plurality
of transcription and reprogramming factor genes. In one embodiment,
the vector comprises between two, three or four transcription and
reprogramming factor genes. In one embodiment, the vector further
comprises one or more than one anti-apoptotic factor. In one
embodiment, the vector further comprises a promoter. In one
embodiment, the vector further comprises a post-transcriptional
regulatory element. According to another embodiment of the present
invention, there is provided a method for generating
integration/transgene-free induced pluripotent stem cells. The
method comprises providing one or more than one vector according to
the present invention and transducing or transfecting target cells
with the one or more than one vector. In a preferred embodiment,
the target cells are hematopoietic stem cells. In another preferred
embodiment, the target cells are peripheral blood cells that have
been enriched for one or more than one cell type selected from the
group consisting of CD33+ cells, CD34+ cells and CD133+ cells, or
depleted of cells that express T cell marker CD3 or B cell maker CD
19. According to another embodiment of the present invention, there
are provided integration/transgene-free induced pluripotent stem
cells generated by the method. According to another embodiment of
the present invention, there is provided a method of treating a
patient having a condition or disease. The method comprises
administering integration/transgene-free induced pluripotent stem
cells according to the present invention or
integration/transgene-free induced pluripotent stem cells generated
by a method according to the present invention.
[0034] Among the various aspects of the present invention are: 1)
selecting a vector based on an oriP/EBNA1-based plasmid backbone
episomal vector (EV), 2) incorporating exactly two, exactly three
or exactly four transcription and reprogramming factor genes in the
vector rather than the five or more transcription and reprogramming
factor genes currently being used, and in particular incorporating
the combination of oct4 gene and sox2 gene alone, or the
combination of oct4 gene, sox2 gene and klf4 gene alone, with or
without a myc gene, but without other transcription and
reprogramming factor genes, 3) incorporating an anti-apoptotic
factor gene, such as a gene expressing BCL-XL or BCL2 into the
vector, 4) incorporating a strong spleen focus-forming virus (SFFV)
promoter in the vector, 5) incorporating a post-transcriptional
regulatory element such as Wpre, 6) selecting peripheral blood
cells that have been enriched for one or more than one cell type
selected from the group consisting of CD33+ cells, CD34+ cells and
CD133+ cells, or depleted of cells that express T cell marker CD3
or B cell maker CD 19 as the target cells for reprogramming; or
depleting the target cells of cells that express T cell marker CD3
or B cell maker CD19 as the target cells for reprogramming, and 7)
culturing the target cells before transduction or transfection in a
cell culture for a duration of between three days and six days, and
preferably about four days, which is optimal for generation of
integration/transgene-free induced pluripotent stem cells. Using
these techniques, integration/transgene-free induced pluripotent
stem cells can be generated from adult peripheral blood in
quantities of between twenty and thirty integration-free induced
pluripotent stem cells/colonies from 1 ml peripheral blood, an
efficiency that is substantially higher (between ten to one
thousand times higher) than previously reported. Further, the
integration/transgene-free induced pluripotent stem cells generated
according to the present invention were shown to differentiate into
cardiomyocytes, hepatocytes and mesenchymal stem cells, among other
cell types, all of which appeared to be morphologically,
phenotypically and functionally normal. The
integration/transgene-free induced pluripotent stem cells according
to the present invention and generated by a method according to the
present invention have potential applications in allogeneic cell
therapy for regenerative medicine, disease modeling, and induced
pluripotent stem cell banking, among other uses. The vectors,
methods and cells will now be disclosed in detail.
[0035] As used in this disclosure, except where the context
requires otherwise, the term "comprise" and variations of the term,
such as "comprising," "comprises" and "comprised" are not intended
to exclude other additives, components, integers or steps.
[0036] As used in this disclosure, except where the context
requires otherwise, the method steps disclosed are not intended to
be limiting nor are they intended to indicate that each step is
essential to the method or that each step must occur in the order
disclosed.
[0037] As used in this disclosure, except where the context
requires otherwise, "integration-free induced pluripotent stem
cells" is synonymous with "integration/transgene-free induced
pluripotent stem cells" and is understood to mean that after eight
passages, the average copy number of residual vector is less than
0.01 copies per genome.
[0038] As presented in this disclosure, except where otherwise
specified, data are presented as mean.+-.standard error of the mean
(SEM), two-tailed Student t test was performed, and P values of
<0.05 were considered statistically significant.
[0039] According to one embodiment of the present invention, there
is provided a vector for generating induced pluripotent stem cells
from target cells. In one embodiment, the vector is a plasmid. In a
preferred embodiment, the vector is a non-integrating plasmid. In
one embodiment, the vector is a non-plasmid. In one embodiment, the
vector is a non-integrating vector. In one embodiment, the vector
is a viral vector. In one embodiment, the vector is a
non-integrating viral vector. In one embodiment, the vector is a
self-inactivating (SIN) vector. In one embodiment, the vector is a
lentivirus. In one embodiment, the vector is an episomal vector. In
a preferred embodiment, the vector is an oriP/EBNA1-based episomal
vector. In one embodiment, the vector is an oriP/EBNA1-based
plasmid backbone episomal vector (EV).
[0040] The vector comprises a plurality of transcription and
reprogramming factor genes. In one embodiment, the plurality of
transcription and reprogramming factor genes is exactly two
transcription and reprogramming factor genes. In another
embodiment, the plurality of transcription and reprogramming factor
genes is exactly three transcription and reprogramming factor
genes. In another embodiment, the plurality of transcription and
reprogramming factor genes is exactly four transcription and
reprogramming factor genes. In another embodiment, the plurality of
transcription and reprogramming factor genes is exactly five
transcription and reprogramming factor genes.
[0041] In one embodiment, the transcription and reprogramming
factor genes are selected from the group consisting of one or more
than one Yamanaka factor gene and one or more than one Thomson/Yu
factor gene, and a combination of the preceding. In one embodiment,
the transcription and reprogramming factor genes are selected from
the group consisting of a) octamer-binding transcription factor 4
gene (Octamer-4 gene; oct4 gene, encoding Octomer-4; OCT4) (also
known as pou5f1, encoding POU5F1), b) (sex determining region
Y)-box 1 gene (sox1 gene) (encoding SOX1), (sex determining region
Y)-box 2 gene (sox2 gene) (encoding SOX2), c) (sex determining
region Y)-box 3 gene (sox3 gene) (encoding SOX3), d) (sex
determining region Y)-box 15 gene (sox15 gene) (encoding SOX15), e)
(sex determining region Y)-box 18 gene (sox18 gene) (encoding
SOX18), O Krueppel-like factor 4 gene (klf4 gene) (encoding
Krueppel-like factor 4 protein; KLF4 protein), g) myelocytomatosis
gene (myc gene; MYC) (encoding Myc protein), h) nanog (encoding
NANOG protein) and i) 1 in28 (encoding Lin-28 homolog A
protein).
[0042] In one embodiment, the transcription and reprogramming
factor genes are exactly two genes, oct4 gene and sox2 gene without
other transcription and reprogramming factor genes. In another
embodiment, the transcription and reprogramming factor genes are
exactly three genes, oct4 gene, sox2 gene and klf4 gene without
other transcription and reprogramming factor genes. In another
embodiment, the transcription and reprogramming factor genes are
exactly four genes, oct4 gene, sox2 gene, klf4 gene and myc gene
without other transcription and reprogramming factor genes.
[0043] In one embodiment, the vector further comprises one or more
than one gene coding for an inhibitor, siRNA, or shRNA construct of
a pro-apoptotic factor. In a preferred embodiment, the
pro-apoptotic factor is a BAX subfamily pro-apoptotic factor. In a
particularly preferred embodiment, the pro-apoptotic factor is a
BAX subfamily pro-apoptotic factor selected from the group
consisting of BAK, BAX and BOK. In another preferred embodiment,
the pro-apoptotic factor is a BH3 subfamily pro-apoptotic factor.
In a particularly preferred embodiment, the pro-apoptotic factor a
BH3 subfamily pro-apoptotic factor selected from the group
consisting of BAD, BID, BIK, BIML, BLK, BNIP3 and HRK.
[0044] In a preferred embodiment, the vector further comprises one
or more than one anti-apoptotic factor gene encoding one or more
than one anti-apoptotic factor. In one embodiment, the one or more
than one anti-apoptotic factor is a BCL-2 family anti-apoptotic
factor. In another preferred embodiment, the one or more than one
anti-apoptotic factor is selected from the group consisting of A1,
BCL2, BCL-W, BCL-XL and MCL1. In a particularly preferred
embodiment, the anti-apoptotic factor is BCL-XL (B-cell
lymphoma-extra large) or BCL2.
[0045] In one embodiment, at least two of the plurality of
transcription and reprogramming factor genes are linked with a
cleavage sequence. In a preferred embodiment, the cleavage sequence
is a 2a self-cleavage peptide sequence. In a particularly preferred
embodiment, the 2a self-cleavage peptide sequence is selected from
the group consisting of equine rhinitis A virus (E2A),
foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and
Thosea asigna virus (T2A).
[0046] In one embodiment, the vector further comprises a promoter
suitable for promoting transcription of at least one of the
plurality of transcription and reprogramming factor genes.
[0047] In one embodiment, the promoter is selected from the group
consisting of CAG promoter, CMV promoter, EF1a promoter and
ubiquitin promoter. In a preferred embodiment, the promoter is
strong spleen focus forming virus (SFFV) promoter (strong spleen
focus forming virus (SFFV) long terminal repeat (LTR) promoter;
spleen focus-forming virus U3 promoter).
[0048] In one embodiment, the vector further comprises a
post-transcriptional regulatory element. In a preferred embodiment,
the post-transcriptional regulatory element is Wpre. In a preferred
embodiment, the post-transcriptional regulatory element is Wpre at
the 3' end of the transgene and in front of a PolyA signal.
[0049] In one embodiment, the vector is an episomal vector
comprising a strong spleen focus-forming virus promoter, a
post-transcriptional regulatory element Wpre, and exactly two
transcription and reprogramming factor genes, oct4 and sox2. In one
embodiment, vector is an episomal vector comprising a strong spleen
focus-forming virus promoter, a post-transcriptional regulatory
element Wpre, an anti-apoptotic factor gene bcl-xl, and exactly two
transcription and reprogramming factor genes, oct4 and sox2. In
another embodiment, the vector is an episomal vector comprising a
strong spleen focus-forming virus promoter, a post-transcriptional
regulatory element Wpre, and exactly three transcription and
reprogramming factor genes, oct4, sox2 and klf4. In another
embodiment, the vector is an episomal vector comprising a strong
spleen focus-forming virus promoter, a post-transcriptional
regulatory element Wpre, an anti-apoptotic factor gene bcl-xl, and
exactly three transcription and reprogramming factor genes, oct4,
sox2 and klf4. In another embodiment, the vector is an episomal
vector comprising a strong spleen focus-forming virus promoter, a
post-transcriptional regulatory element Wpre, and exactly four
transcription and reprogramming factor genes, oct4, sox2, klf4 and
myc. In another embodiment, the vector is an episomal vector
comprising a strong spleen focus-forming virus promoter, a
post-transcriptional regulatory element Wpre, an anti-apoptotic
factor gene bcl-xl, and exactly four transcription and
reprogramming factor genes, oct4, sox2, klf4 and myc.
[0050] According to another embodiment of the present invention,
there is provided a method for generating integration-free induced
pluripotent stem cells. The method comprises providing target
cells, providing one or more than one vector according to the
present invention, and transducing or transfecting the target cells
with the one or more than one vector. In one embodiment, the one or
more than one vector is one vector. In another embodiment, the one
or more than one vector is a plurality of vectors. In another
embodiment, the one or more than one is two vectors. In another
embodiment, one or more than one vector is three vectors.
[0051] In one embodiment, one of the one or more than one vector is
an episomal vector comprising a strong spleen focus-forming virus
promoter, a post-transcriptional regulatory element Wpre, and
exactly two transcription and reprogramming factor genes, oct4 and
sox2. In one embodiment, the vector is an episomal vector
comprising a strong spleen focus-forming virus promoter, a
post-transcriptional regulatory element Wpre, an anti-apoptotic
factor gene bcl2 or bcl-xl, and exactly two transcription and
reprogramming factor genes, oct4 and sox2. In another embodiment,
one of the one or more than one vector is an episomal vector
comprising a strong spleen focus-forming virus promoter, a
post-transcriptional regulatory element Wpre, and exactly three
transcription and reprogramming factor genes, oct4, sox2 and klf4.
In another embodiment, one of the one or more than one vector is an
episomal vector comprising a strong spleen focus-forming virus
promoter, a post-transcriptional regulatory element Wpre, an
anti-apoptotic factor gene bcl2 or bcl-xl, and exactly three
transcription and reprogramming factor genes, oct4, sox2 and klf4.
In another embodiment, one of the one or more than one vector is an
episomal vector comprising a strong spleen focus-forming virus
promoter, a post-transcriptional regulatory element Wpre, and
exactly four transcription and reprogramming factor genes, oct4,
sox2, klf4 and myc. In another embodiment, one of the one or more
than one vector is an episomal vector comprising a strong spleen
focus-forming virus promoter, a post-transcriptional regulatory
element Wpre, an anti-apoptotic factor gene bcl2 or bcl-xl, and
exactly four transcription and reprogramming factor genes, oct4,
sox2, klf4 and myc.
[0052] In one embodiment, the one or more than one vector is a
first vector and a second vector. By way of example only, in one
embodiment, the first vector is an episomal vector comprising a
strong spleen focus-forming virus promoter, a post-transcriptional
regulatory element Wpre, and exactly two transcription and
reprogramming factor genes, oct4 and sox2, and the second vector is
an episomal vector comprising a strong spleen focus-forming virus
promoter, a post-transcriptional regulatory element Wpre, and
exactly two transcription and reprogramming factor genes, klf4 and
myc. The first vector and the second vector can, however, be any
suitable vectors for generating the integration-free induced
pluripotent stem cells, as will be understood by those with skill
in the art with respect to this disclosure. Similarly, where the
one or more than one vector is three vectors, four vectors or five
vectors, each of the vectors can be any suitable vector for
generating the integration-free induced pluripotent stem cells, as
will be understood by those with skill in the art with respect to
this disclosure.
[0053] In a preferred embodiment, the one or more than one vector
is a first vector and a second vector, and transducing or
transfecting the target cells comprises transducing or transfecting
the target cells with a first amount of the first vector and a
second amount of a second vector, where the first amount is equal
to the second amount. In another preferred embodiment, the one or
more than one vector is a first vector and a second vector, and
transducing or transfecting the target cells comprises transducing
or transfecting the target cells with a first amount of the first
vector and a second amount of a second vector, where the first
amount is half of the second amount. The first amount of the first
vector and the second amount of a second vector can, however, be in
any suitable ratio for generating the integration-free induced
pluripotent stem cells, as will be understood by those with skill
in the art with respect to this disclosure.
[0054] In one embodiment, the one or more than one vector is an
episomal vector comprising a strong spleen focus-forming virus
promoter, a post-transcriptional regulatory element Wpre, and
exactly two transcription and reprogramming factor genes, oct4 and
sox2, and the method further comprises transducing or transfecting
the target cells with an additional episomal vector comprising a
strong spleen focus-forming virus promoter, a post-transcriptional
regulatory element Wpre, an anti-apoptotic factor gene bcl-xl, and
exactly one transcription and reprogramming factor gene, klf4. In
one embodiment, the one or more than one vector is an episomal
vector comprising a strong spleen focus-forming virus promoter, a
post-transcriptional regulatory element Wpre, and exactly two
transcription and reprogramming factor genes, oct4 and sox2, and
the method further comprises transducing or transfecting the target
cells with a first additional episomal vector comprising a strong
spleen focus-forming virus promoter, a post-transcriptional
regulatory element Wpre, and exactly one transcription and
reprogramming factor gene, klf4, and with a second additional
episomal vector comprising a strong spleen focus-forming virus
promoter, a post-transcriptional regulatory element Wpre, and an
anti-apoptotic factor gene bcl-xl, but without any transcription
and reprogramming factor gene. Other combinations of vectors
according to the present invention and additional vectors are
suitable, as will be understood by those with skill in the art with
respect to this disclosure.
[0055] In one embodiment, the target cells are hematopoietic stem
cells. In another embodiment, the target cells are peripheral blood
mononuclear cells. In another embodiment, the target cells are
peripheral blood myeloid cells. In another embodiment, the target
cells are peripheral blood cells that have been enriched for one or
more than one cell type selected from the group consisting of CD33+
cells, CD34+ cells and CD133+ cells. In another embodiment, the
target cells are peripheral blood mononuclear cells that have been
enriched for CD33+ cells. In a preferred embodiment, the target
cells are peripheral blood cells that have been depleted of cells
that express T cell marker CD3 or B cell maker CD19.
[0056] In one embodiment, the method further comprises harvesting
the target cells from a body fluid or tissue. In one embodiment,
the body fluid or tissue is selected from the group consisting of
bone marrow, cord blood and peripheral blood. In a preferred
embodiment, the body fluid or tissue is peripheral blood. In one
embodiment, the method further comprises providing cord blood, and
further comprises purifying the cord blood to obtain the target
cells. In one embodiment, the cord blood is obtained from a cord
blood bank. In another embodiment, the method further comprises
enhancing or purifying the target cells for cells that express a
CD33 marker. In another embodiment, the method further comprises
enhancing or purifying the target cells for cells that express a
CD34 marker or a CD133 marker. In another embodiment, the method
further comprises depleting the target cells of cells that express
a T cell marker CD3 or a B cell maker CD 19. In another embodiment,
the method further comprises enhancing or purifying the target
cells for cells that express a CD33 marker, and depleting the
target cells of cells that express a T cell marker CD3 or a B cell
maker CD19.
[0057] Next, the method further comprises culturing the transduced
or transfected cells in a cell culture, thereby generating
integration-free induced pluripotent stem cells. In one embodiment,
the method further comprises purifying integration-free induced
pluripotent stem cells from the cell culture after generating the
integration-free induced pluripotent stem cells.
[0058] In another embodiment, the method further comprises
culturing the target cells in a cell culture for a duration of
between three days and six days before transducing or transfecting
the target cells. In another embodiment, the method further
comprises culturing the target cells in a cell culture for a
duration of four days before transducing or transfecting the target
cells.
[0059] In a preferred embodiment, the method comprises
incorporating sodium butyrate into the cell culture. In a preferred
embodiment, the sodium butyrate is incorporated into the cell
culture at a concentration of between 0.1 and 1.0 mM. In a
particularly preferred embodiment, the sodium butyrate is
incorporated into the cell culture at a concentration of 0.25 mM.
In one embodiment, the transduced or transfected cells are cultured
in hematopoietic stem cell culture condition such as Iscove's
modified Dulbecco's medium (IMDM)/10% FBS supplemented with the
cytokines FlT3-ligand (FL), granulocyte colony-stimulating factor
(G-CSF), stem cell factor (SCF) and thrombopoietin (TPO) each at
100 ng/ml, and Interleukin 3 (IL-3) at 10 ng/ml (ProSpec-Tany
Technogene Ltd., East Brunswick, N.J., US). After two days
pre-stimulation, 1.times.10.sup.4 cells per well are seeded into
non-tissue culture, treated twenty-four well plates that were
pre-coated with RetroNectin (CH-296; Takara Bio, Inc., Shiga, JP)
for lentiviral transduction for four to five hours. A second
transduction is conducted twenty-four hours later. One day after
transduction, the cells are harvested and transferred to six-well
plates, which are pre-seeded with a mitomycin C-inactivated CF-1
mouse embryonic fibroblast (MEF) feeder layer (Applied Stemcell,
Inc., Menlo Park, Calif., US). Cells are maintained in the
hematopoietic stem cell culture condition for two more days before
being replaced with induced pluripotent stem cell media, such as
for example Knockout DMEM/F12 medium supplemented with 20% Knockout
Serum Replacement (KSR), 1 mM GlutaMAX, 2 mM nonessential amino
acids, 1.times. penicillin/streptomycin (all from Invitrogen, Grand
Island, N.Y., US), 0.1 mM .beta.-mercaptoethanol (Sigma-Aldrich
Corp., St. Louis, Mo., US), 20 ng/ml FGF2 (ProSpec). In a preferred
embodiment, sodium butyrate is added at 0.25 mM from day two to
twelve, and cells are cultured under hypoxia by placing culture
plates in a Hypoxia Chamber (Stemcell Technologies, inc.,
Vancouver, BC, CA) that is flushed with mixed air composed of 92%
N2/3% O2/5% CO2. Starting from day ten, mouse embryonic
fibroblast-conditioned medium is used.
[0060] According to another embodiment of the present invention,
there are provided integration-free induced pluripotent stem cells
generated by the method. In one embodiment, the integration-free
induced pluripotent stem cells express one or more than one marker
for a mature cell type selected from the group consisting of
cardiomyocytes, hepatocytes and mesenchymal stem cells. According
to another embodiment of the present invention, there are provided
integration-free induced pluripotent stem cell colonies formed by
the integration-free induced pluripotent stem cells generated by
the method. In one embodiment, the integration-free induced
pluripotent stem cell colonies express one or more than one marker
for a mature cell type selected from the group consisting of
cardiomyocytes, hepatocytes and mesenchymal stem cells.
[0061] According to another embodiment of the present invention,
there is provided a method of treating a patient having a condition
or disease. The method comprises identifying a patient with a
condition or disease suitable for treatment by the present method,
and administering integration-free induced pluripotent stem cells
according to the present invention or generated by a method
according to the present invention. In a preferred embodiment, the
patient is a human. In one embodiment, the condition or disease is
selected from the group consisting of an autoimmune disease,
cancer, cardiovascular disease, a connective tissue disease, an
injury, and a neurodegenerative disease. In one embodiment,
identifying the patient comprises diagnosing the patient with one
or more than one condition or disease suitable for treatment by the
present method. In one embodiment, diagnosing the patient comprises
performing one or more than one of action selected from the group
consisting of performing a physical examination, performing a
non-invasive imaging examination (such as for example computerized
tomography, magnetic resonance imaging and ultrasound), and
identifying one or more than one marker for a condition or disease
in the blood or other body fluid of the patient. In another
embodiment, identifying the patient comprises consulting patient
records to determine if the patient has a condition or disease
suitable for treatment by the present method.
Example 1
Determination of Whether Co-Expression of Both OCT4 and SOX2 in a
Single Vector Driven by a Strong Promoter Generated More Induced
Pluripotent Stem Cells than Simultaneous Expression of Both OCT4
and SOX2 by Separate Vectors
[0062] A determination was made as follows, whether co-expression
of both OCT4 and SOX2 in a single vector driven by a strong
promoter generated more induced pluripotent stem cells than
simultaneous expression of both OCT4 and SOX2 by separate vectors
from cord blood CD 133+ cells. It has been previously shown that
overexpression of OCT4 together with SOX2 using two separate
retroviral vectors (O+S) can generate induced pluripotent stem
cells from cord blood CD133+ cells. However, the two retroviral
vector combination yielded a generation efficiency of between
0.002% and 0.005% which is too low for practical clinical use. To
determine if this low efficiency was due to inadequate retroviral
vector-mediated overexpression of OCT4 and SOX2, lentiviral vectors
mediating overexpression of OCT4 alone (O), overexpression of SOX2
alone (S), and overexpression of both OCT4 and SOX2 in a single
vector with a self-cleavage peptide sequence 2a between the OCT4
gene and the SOX2 gene (OS) were produced, where expression in each
of the three vectors was driven by spleen focus-forming virus
(SFFV) promoter (a strong promoter in primary hematopoietic cells
and hematopoietic cell lines). Referring now to FIG. 1, there is
shown a schematic depiction of the self-inactivating (SIN)
lentiviral vector backbones for expression of OCT4 (O), SOX2 (S)
and OCT4 and SOX2 (OS), where .DELTA. indicates the SIN design with
partially deleted U3 of the 3' long terminal repeat, cPPT is a
central polypurine tract, Wpre is a post-transcriptional regulatory
element, RRE is a rev-responsive element, .psi. is a packaging
signal, and SFFV is the spleen focus-forming virus U3 promoter.
[0063] CD34+ cells were purified from cord blood with a
CD34+MicroBead Kit (Miltenyi Biotec, Auburn, Calif., US). The
purified CD34+ cells were transduced with either the combination of
the lentiviral vector mediating overexpression of OCT4 and the
lentiviral vector mediating overexpression of SOX2 (0+S), or were
transduced with the single lentiviral vector mediating
overexpression of both OCT4 and SOX2 (OS). The transduced cells
were cultured on mouse embryonic fibroblasts (MEFs).
[0064] Four to five days after seeding the transduced cord blood
CD34+ cells onto the mouse embryonic fibroblasts, the O+S cells had
formed dozens of small colonies in each well; however,
morphologically induced pluripotent stem cells did not appear until
approximately twelve days after seeding. The cells that appeared in
the first four to five days were analyzed by flow cytometry and
many of these cells expressed mesenchymal markers. The O+S cells
produced between 300 and 600 total colonies in each well from
10,000 transfected cord blood CD34+ cells eight to ten days after
transduction. However, the majority of colonies were
morphologically not induced pluripotent stem cells and alkaline
phosphatase (ALP) staining showed that only about 20% of the
colonies stained like induced pluripotent stem cells.
[0065] By contrast, the cells transduced with the single lentiviral
vector mediating overexpression of both OCT4 and SOX2 (OS cells)
did not produce any colonies at all in the first week after
transduction, but produced the first morphologically induced
pluripotent stem cells-like colonies eight to ten days after
transduction. The OS cells produced between 200 and 250 total
colonies in each well, with about 80% of the colonies being
morphologically induced pluripotent stem cells. Alkaline
phosphatase (ALP) staining showed that about 80% of the colonies
stained like induced pluripotent stem cells. Fluorescence-activated
cell sorting (FACS) analysis was also performed on both groups of
cultures. About 9% of the cells generated from the O+S cells
expressed the induced pluripotent stem cells marker TRA-1-60, while
about 40% of the cells generated from the OS cells expressed the
induced pluripotent stem cells marker TRA-1-60. Therefore,
simultaneous expression of both OCT4 and SOX2 by separate vectors
driven by a strong promoter is sufficient to generate induced
pluripotent stem cells from cord blood cells, while the
co-expression of both OCT4 and SOX2 in a single vector driven by
the same promoter generated more induced pluripotent stem cells
than the separate simultaneous expression, and additionally
inhibited the growth of other non-induced pluripotent stem
cells.
Example 2
Determination of Whether KLF4 Increased Efficiency of Generation of
Induced Pluripotent Stem Cells by Co-Expression of Both OCT4 and
SOX2 in a Single Vector Driven by a Strong Promoter
[0066] Next, a determination was made as follows, whether adding
another transcription and reprogramming factor known to generate
induced pluripotent stem cells from somatic cells increased
efficiency of generation of induced pluripotent stem cells by
co-expression of both OCT4 and SOX2 in a single vector driven by a
strong promoter. The test transcription and reprogramming factor
used was Krueppel-like factor 4 (KLF4, a protein encoded by klf4).
A single lentiviral vector mediating simultaneous overexpression of
OCT4, SOX2 and KLF4 was produced, and used to transduce CD34+ cells
as in Example 1. Expression of KLF4 by the transfected cells was
confirmed. Approximately 2% of the CD34+ cells transfected
converted into induced pluripotent stem cells, about the same
amount as using the lentivirus simultaneously expressing both OCT4
and SOX2 only without the KLF4 in Example 1. Approximately 40% of
the cells in the culture expressed the induced pluripotent stem
cells marker TRA-1-60, slightly higher than using the lentivirus
simultaneously expressing both OCT4 and SOX2 only without the KLF4
in Example 1; however, the difference did not rise to the level of
statistical significance. Therefore, addition of another
transcription and reprogramming factor known to generate induced
pluripotent stem cells from target cells did not significantly
increase the efficiency of generation of induced pluripotent stem
cells by co-expression of both OCT4 and SOX2 in a single vector
driven by a strong promoter.
Example 3
Determination of Whether Sffv Promoter was More Effective in
Driving Transgene Expression in Cord Blood CD34+Cells than Other
Promoters
[0067] The efficiency of generation of induced pluripotent stem
cells from cord blood cells from the single vector co-expressing
both OCT4 and SOX2 (OS) driven by the spleen focus-forming virus
(SFFV) promoter as shown in Example 1 was approximately 1000-fold
greater than the efficiency of generation of induced pluripotent
stem cells from cord blood CD34+ cells previously reported.
Therefore, a determination was next made whether use of the SFFV
promoter was partially responsible for the increased efficiency by
determining whether the SFFV promoter was more effective in driving
transgene expression in cord blood CD34+ cells than other
promoters. First, lentiviral vectors were cloned in which green
fluorescent protein (GFP) expression was driven by either the
phosphoglycerokinase (PGK) promoter, the human elongation factor-1
alpha (EF1 alpha) promoter or the spleen focus-forming virus (SFFV)
promoter to determine the relative strength of these promoters in
CD34+ cells. Referring now to FIG. 2, there is shown a schematic
depiction of the self-inactivating (SIN) lentiviral vector
backbones for expression of GFP, where .DELTA. ndicates the SIN
design with partially deleted U3 of the 3' long terminal repeat,
cPPT is a central polypurine tract, Wpre is a post-transcriptional
regulatory element, RRE is a rev-responsive element, .psi. is a
packaging signal, SFFV is the spleen focus-forming virus U3
promoter, EF1 is the Elongation factor-1 alpha promoter and PGK is
the phosphoglycerokinase promoter. Cord blood CD+34 cells were
transduced with the vectors. Fluorescence-activated cell sorting
analysis was performed on the transduced cells. Referring now to
FIG. 3, there is shown a graph of the measured GFP intensity for
expression of the GFP driven by the PGK promoter (left), the EF-1
alpha promoter (center) and the SFFV promoter (right) in cord blood
cells. As can be seen, GFP expression in the cord blood CD34+ cells
driven by the PGK promoter was about 20% of the expression driven
by the SFFV promoter, and GFP expression in the cord blood CD34+
cells driven by the EF1 promoter was about 35% of the expression
driven by the SFFV promoter showing that the SFFV promoter was more
than twice as efficient at driving transgene expression than the
PGK promoter and the EF1 promoter. Next, a fusion gene of GFP and
OCT4 was produced. The fusion gene OCT4GFP was cloned into
lentiviral vectors where expression was driven by either the PGK
promoter, the EF1 promoter or the SFFV promoter. Cord blood CD+34
cells were transduced with the vectors. Fluorescence-activated cell
sorting analysis was performed. GFP expression as measured by
fluorescence intensity was assumed to reflect the co-expression
level of OCT4. Referring now to FIG. 4, there is shown a graph of
the measured GFP intensity for expression of the fusion gene
OCT4GFP driven by the PGK promoter (left), the EF1 promoter
(center) or the SFFV promoter (right) in cord blood cells. As can
be seen, OCT4GFP expression in the cord blood CD34+ cells driven by
the PGK promoter was about 35% of the expression driven by the SFFV
promoter expression, and OCT4GFP expression in the cord blood CD34+
cells driven by the EF 1 promoter was about 50% of the expression
driven by the SFFV promoter showing that the SFFV promoter was
significantly more efficient at driving transgene expression than
the PGK promoter and the EF1 promoter. Therefore, the SFFV promoter
was at least twice as effective in driving transgene expression in
cord blood CD34+ cells as the PGK promoter and the EF1
promoter.
Example 4
Determination of Whether Transgene Expression Level Affected
Generation of Induced Pluripotent Stem Cells
[0068] Then, a determination was made as follows, whether the
increased level of transgene expression from the SFFV promoter
demonstrated in Example 3 had an effect on the generation of
induced pluripotent stem cells from cord blood CD34+ cells.
1.times.10.sup.4 cord blood CD34+ cells were transduced with a
single lentiviral vector mediating overexpression of both OCT4 and
SOX2 (OS transgene) driven by either the phosphoglycerokinase (PGK)
promoter, the human elongation factor-1 alpha (EF1 alpha) promoter
or the spleen focus-forming virus (SFFV). In six independent
experiments, no induced pluripotent stem cells were generated from
the transduced cells where the OS transgene was driven by either
the PGK promoter or by the EF1 promoter, while approximately 200
colonies were generated from 10,000 CD34+ cells using the SFFV
promoter. Given that expression of OCT4 was decreased by about 50%
when driven by the EF1 promoter as compared to the SFFV promoter
(Example 3), these experiments indicate that a 50% decrease in OS
expression leads to failure to generate induced pluripotent stem
cells from the transduced cord blood CD34+ cells. Further, a
synthetic OS transgene (synOS) was synthesized that was codon
optimized (DNA 2.0, Menlo Park, Calif., US) and expressed in a
lentiviral vector driven by the SFFV promoter. Analysis of protein
expression indicated that the level of the protein encoded by synOS
was about 20% lower than the level of the protein encoded by the
wild type OS, while the number of induced pluripotent stem cell
colonies generated by the cord blood CD34+ cells transduced with
the synOS vector was about 25% of the number of induced pluripotent
stem cell colonies generated by the cord blood CD34+ cells
transduced with the vector comprising the wild-type OS. Combined
with the data above, these experiments show that a 20% drop in
protein level from expression of transgene resulted in generation
of only 25% efficiency of induced pluripotent stem cells generation
while a 50% drop in expression of transgene resulted in failure to
generate any induced pluripotent stem cell colonies. Therefore, the
level of OS transgene expression is critical to generation of
induced pluripotent stem cells from cord blood CD34+ cells.
Example 5
Determination of Whether Additional Transcription and Reprogramming
Factors Increased the Efficiency of Generation of Induced
Pluripotent Stem Cells Using Co-Expression of OCT4 and SOX2 in a
Viral-Based Vector
[0069] Next, a determination was made as follows, whether adding
transcription and reprogramming factor genes beside oct4 and sox2
to the viral-based vector affected the transgene expression level
needed to generate induced pluripotent stem cells. The two
additional transcription and reprogramming factors tested were myc
and klf4, both of which have previously been shown to generate
induced pluripotent stem cells from hematopoietic stem cells in
various vectors. Lentiviral vectors were produced where one vector
mediated overexpression of both OCT4 and SOX2 driven by the EF1
promoter, and one vector mediated overexpression of MYC driven by
the SFFV promoter. Referring now to FIG. 5, there is shown a graph
of the number of induced pluripotent stem cell colonies generated
from 1.times.10.sup.4 cord blood CD34+ cells that were transduced
only with the vector mediating overexpression of both OCT4 and SOX2
driven by the EF1 promoter (left), transduced with both the vector
mediating overexpression of both OCT4 and SOX2 driven by the EF1
promoter and the vector mediating overexpression of MYC driven by
the SFFV promoter (center), and transduced only with the vector
mediating overexpression of MYC driven by the SFFV promoter
(right). As can be seen, no induced pluripotent stem cells were
generated from the cells transduced only with the vector mediating
overexpression of both OCT4 and SOX2 driven by the EF1 promoter
(left), or transduced only with the vector mediating overexpression
of MYC driven by the SFFV promoter (right). However, induced
pluripotent stem cells were generated from 0.1% of the cells that
were transduced with both the vector mediating overexpression of
both OCT4 and SOX2 driven by the EF1 promoter and with the vector
mediating overexpression of MYC driven by the SFFV promoter
(center). Analysis of alkaline phosphatase (ALP) staining and
fluorescence-activated cell sorting (FACS) of the generated induced
pluripotent stem cells did not show any obvious differences in the
expression of pluripotency markers when compared with induced
pluripotent stem cells generated from cells transduced with a
vector mediating overexpression of both OCT4 and SOX2 driven by the
SFFV promoter, as above. Further, cord blood CD34+ cells were
transduced with a vector mediating overexpression of both MYC and
KLF4 driven by the EF1 promoter, but no induced pluripotent stem
cells were generated from the cells. Therefore, these experiments
show that high-level expression of OCT4 and SOX2 (driven by the
SFFV promoter, Examples 1 and 4) without other transcription and
reprogramming factors is sufficient to generate induced pluripotent
stem cells from cord blood CD34+ cells using a viral-based vector,
while lower-level expression of OCT4 and SOX2 (driven by the EF1
promoter (Example 5) requires additional transcription and
reprogramming factors to generate induced pluripotent stem cells
from cord blood CD34+ cells using a viral-based vector.
Example 6
Determination of Whether Co-Expression of OCT4 and SOX2 in a
Nonviral Vector Generates Induced Pluripotent Stem Cells
[0070] Then, a determination was made as follows, whether
co-expression of OCT4 and SOX2 in a nonviral vector generates
induced pluripotent stem cells from cord blood CD34+ cells, such as
for example, co-expression of OCT4 and SOX2 in an episomal vector.
Referring now to FIG. 6, there is shown a schematic depiction of an
episomal mammalian expression vector backbone (bottom) for (from
upper to lower, respectively) co-expression of OCT4 and SOX2 (OS)
without Wpre (pCEP-OS (w/o W)), co-expression of OCT4 and SOX2 (OS)
with Wpre (pCEP-OS), expression of KLF4 (K) (pCEP-K), and
expression of MYC (MK) (pCEP-MK); where 2a is a self-cleavage site
derived from equine rhinitis A virus, Wpre is a
post-transcriptional regulatory element, SV40PolyA is a
polyadenylation signal from SV40 virus, OriP is an EBV origin of
replication, and EBNA1 is Epstein-Barr nuclear antigen 1 which
plays essential roles in replication and persistence of episomal
DNA in infected cells. First, the OCT4 and SOX2 (OS) transgene
driven by the SFFV promoter was shuttle cloned from the lentiviral
vector (Examples 1 and 4) into the EBNA1/OriP-based episomal
mammalian expression vector (Invitrogen), where the hygromycin
resistance gene element and CMV promoter were removed from the
pCEP4 vector by digestion with endonucleases NruI and BamHI, and
inserts were cut from the counterparts of lentiviral vectors. Then,
1.times.10.sup.5 cord blood CD34+ cells were cultured in IMDM/10%
FBS supplemented with the cytokines FlT3-ligand (FL), stem cell
factor (SCF) and thrombopoietin (TPO) at 100 ng/ml. Three days
later, cells were harvested for nucleofection with a total of 12 ug
episomal vector. Nucleofection was performed with Amaxa
Nucleofector II using program U-008 Immediately after
nucleofection, the cells were cultured in a CH-296 pretreated well
plate to facilitate the cord blood cell recovery. The next day,
half of the cells were transferred to each well of MEF-coated
six-well plates. The cells were cultured the same way as for
reprogramming with lentiviral vector, above. The total number of
induced pluripotent stem cell colonies was counted on day sixteen
post-transfection after ALP staining At day fourteen to seventeen,
colonies were picked for further culture or harvested for FACS
analysis. After three days of culture, the total cell number
increased by about five-fold and all the cells were harvested for
nucleofection with the pCEP-OS (w/o W) vector. No induced
pluripotent stem cells were generated in three independent
attempts. Next, the post-transcriptional regulatory element Wpre
was cloned into the pCEP-OS (w/o W) vector producing the pCEP-OS
episomal vector to enhance transgene expression levels to determine
whether the failure to generate induced pluripotent stem cells by
the pCEP-OS (w/o W) vector was due to low OS transgene expression
levels mediated by the pCEP-OS (w/o W) vector. HE 293T cells were
transfected with same amount of the pCEP-OS episomal vector.
Referring now to FIG. 7, there are shown, respectively, graphs of
the relative expression of OCT4 (left) and SOX2 (right) for cells
transfected with pCEP-OS (w/o Wpre) (left bar) and pCEP-OS (with
Wpre) (right bar). As can be seen, the inclusion of Wpre in the
episomal vector led to a 50% increase in OCT4 expression (right
graph) and a 55% increase in SOX2 expression (left graph),
P<0.05. Then, 1.times.10.sup.5 freshly thawed cord blood CD34+
cells were transfected with the pCEP-OS episomal vector and
generated about twenty induced pluripotent stem cell colonies from
the progeny. Therefore, co-expression of OCT4 and SOX2 driven by
SFFV alone in a nonviral vector such as an episomal vector
generated induced pluripotent stem cells from cord blood CD34+
cells.
Example 7
Determination of Whether Additional Transcription and Reprogramming
Factors Increased the Efficiency of Generation of Induced
Pluripotent Stem Cells Using Co-Expression of OCT4 and SOX2 in a
Nonviral Vector
[0071] Next, a determination was made as follows, whether
additional transcription and reprogramming factors increased the
efficiency of generation of induced pluripotent stem cells using
co-expression of OCT4 and SOX2 in a nonviral vector. The test
transcription and reprogramming factors used was KLF4
(Krueppel-like factor 4 encoded by klf4) (K) and MYC (encoded by
myc; c-myc) (MK). 1.times.10.sup.5 cord blood CD34+ cells were
transfected with the pCEP-OS episomal vector (OS), with the pCEP-OS
episomal vector and the pCEP-K episomal vector (OS+K), or with the
pCEP-OS episomal vector and the pCEP-MK episomal vector (OS+MK).
Referring now to FIG. 8, there is shown a graph of the number of
induced pluripotent stem cells generated from 1.times.10.sup.5 cord
blood CD34+ cells transfected with the pCEP-OS episomal vector (OS)
(left-most bar), with the pCEP-OS episomal vector and the pCEP-K
episomal vector (OS+K) (center bar), or with the pCEP-OS episomal
vector and the pCEP-MK episomal vector (OS+MK) (right-most bar). As
can be seen, the cells transfected with (OS+K) generated eight
times the number of induced pluripotent stem cells as the cells
transfected with OS only, and the cells transfected with (OS+MK)
generated twenty-four times the number of induced pluripotent stem
cells as the cells transfected with OS only (OS vs. OS+K:
P<0.05; OS+K vs. OS+MK: P<0.05). Further, the appearance of
the first induced pluripotent-like stem cell colonies was observed
at nine to ten days, six to seven days and four to five days after
transfection with OS, OS+K, and OS+MK, respectively. Additionally,
1.times.10.sup.5 cord blood CD34+ cells transfected with the
pCEP-OS+MK episomal vector generated approximately six hundred
induced pluripotent stem cells compared with about eighty colonies
from the same amount of cord blood CD34+ cells even with 5 factors
(OSMK+LIN28) using the EF1 promoter. Further, the induced
pluripotent stem cells generated from the cord blood CD34+ cells
transfected with the nonviral vectors OS, OS+K, and OS+MK were
tested with immunostaining and fluorescence-activated cell sorting
(FACS) analysis twenty days after nucleofection to determine
differences in the expression of pluripotency markers. 20-30% of
the cells expressed the induced pluripotent stem cells markers
NANOG and TRA-1-60 in all the cells groups, however, while the
cells transfected with OS+MK had significantly less Tra-1-60
positive induced pluripotent stem cells (22%) than cells
transfected with OS (30%) or with OS+K (31%). Therefore, additional
transcription and reprogramming factors increased the efficiency of
generation of induced pluripotent stem cells using co-expression of
OCT4 and SOX2 in a nonviral vector; however, additional
transcription and reprogramming factors do not necessarily increase
the expression of pluripotency markers in the generated cells.
Example 8
Determination of Whether Co-Expression of OCT4 and SOX2 in a
Nonviral Vector Generates Functional Transgene-Free Induced
Pluripotent Stem Cells
[0072] Then, a determination was made as follows, whether
co-expression of OCT4 and SOX2 in a nonviral vector generates
functional transgene-free induced pluripotent stem cells. Ten
induced pluripotent stem cell colonies were randomly picked from
the colonies generated from cord blood CD34+ cells as above
(Example 6), and were passaged for more than three months.
Real-time analysis conducted using two pairs of primers
demonstrated that no copies of the vector for one primer were
detected in any cell, and approximately 0.5 copies of the vector
for the other primer were detected per cell. After eight passages,
the average copy number of residual vector decreased to 0.001-0.007
copies per genome using either primer and in two of ten clones, the
vector was undetectable. After twelve passages, vector was
undetectable using either primer in the majority of the clones.
Further, several of the clones were randomly picked and
characterized. Immunostaining showed that all clones expressed
typical human induced pluripotent stem cells transcription factors
OCT4, SOX2, NANOG, and surface markers SSEA-3, SSEA-4 and Tra-1-60.
Karyotype analysis indicated that all clones possessed a normal
human karyotype. Sulphite sequencing showed that both the OCT4 and
NANOG promoters were demethylated in 3 randomly picked induced
pluripotent stem cells. Induced pluripotent stem cells formed
teratomas consisting of derivatives of all three embryonic germ
layers when injected into immunodeficient NOD scid IL2 receptor
gamma chain knockout (NSG) mice, demonstrating the pluripotency of
the induced pluripotent stem cells. Therefore, co-expression of
OCT4 and SOX2 in a nonviral vector generates transgene-free induced
pluripotent stem cells that appear to be morphologically,
phenotypically and functionally identical to pluripotent stem
cells.
Example 9
Determination of Whether Expression of BCL-XL in a Viral Vector
Increases the Efficiency of OCT4/SOX2-Mediated Reprogramming of
Cord Blood CD34+Cells into Induced Pluripotent Stem Cells
[0073] Next, a determination was made as follows, whether
simultaneous expression of an anti-apoptotic factor selected from
the group consisting of BCL2, BCL-XL (an isoform of Bch X(L) of
BCL2L1) and MCL1, along with balanced expression of OCT4 and SOX2
(OS) in a lentiviral vector increases reprogramming efficiency of
cord blood CD34+ cells into induced pluripotent stem cells (iPSCs).
The BCL2, BCL-XL, or MCL1 genes were each cloned into a lentiviral
vector under the control of the spleen focus-forming virus (SFFV)
promoter. Cord blood CD34+ cells were cultured for two days before
lentiviral transduction. Cord blood iPSC colonies were enumerated
at two weeks after transduction of reprogramming factors. Referring
now to FIG. 9, there is shown a graph of the number of induced
pluripotent stem colonies generated from 1.times.10.sup.4 cord
blood CD34+ cells when reprogrammed by using balanced expression of
OCT4 and SOX2 (OS) (left-most bar), OS+BCL2 (center left bar),
OS+BCL-XL (center right bar), and OS+MCL (right-most bar). Data
shown are presented as mean.+-.SEM (n=4). * indicates P<0.05. As
can be seen, between 1 and 2% of cord blood CD34+ cells were
reprogrammed to iPSCs using balanced expression of OCT4 and SOX2
(OS) (left-most bar) only. Inclusion of BCL2 or BCL-XL increased
reprogramming efficiency by approximately three-fold (P<0.05),
while the inclusion of MCL1 had no apparent enhancing effect on
OS-mediated reprogramming efficiency. While the inclusion of BCL-XL
demonstrated increased reprogramming efficiency over the inclusion
of BCL2, as shown, but the difference in efficiency was not
statistically significantly. Therefore, inclusion of either BCL2 or
BCL-XL significantly increases efficiency of OCT4/SOX2-mediated
reprogramming of cord blood CD34+ cells.
Example 10
Determination of Whether BCL-XL Increases the Efficiency of
OCT4/SOX2-Mediated Reprogramming of Adult Peripheral Blood
Mononuclear Cells
[0074] Then, a determination was made as follows, whether
simultaneous expression of an anti-apoptotic factor selected from
the group consisting of BCL2, BCL-XL and MCL1, along with balanced
expression of OCT4 and SOX2 (OS) in a lentiviral vector increases
reprogramming efficiency of adult peripheral blood mononuclear
cells (PB MNCs) into induced pluripotent stem cells (iPSCs). The
BCL2, BCL-XL, or MCL1 gene was each cloned into a lentiviral vector
under the control of the spleen focus-forming virus (SFFV)
promoter. Adult peripheral blood mononuclear cells were isolated
from several male and female donors aged 22 to 43 years old by
Ficoll-Hypaque density gradient centrifugation or were purchased
from AllCells (Emeryville, Calif., US), and cultured for four to
six days. To generate adult peripheral blood iPSCs, the human
peripheral blood mononuclear cells were cultured in hematopoietic
stem cell (HSC) culture conditions. Iscove's modified Dulbecco's
medium (IMDM)/10% fetal bovine serum (FBS), supplemented with TPO,
SCF, FL, and G-CSF (purchased from ProSpec, East Brunswick, N.J.,
US; and StemRegeninl (SRL Cellagen Technology, San Diego, Calif.,
US), each at 10 ng/ml, IL-3 at 10 ng/ml. After six to eight days of
culture, 1.times.10.sup.5 cells per culture well were seeded into
non-tissue culture-treated 24-well plates that were pre-coated with
fibronectin fragment RetroNectin or CH-296 (Takara Bio, Inc.,
Shiga, Japan). The cells were then transduced with a lentiviral
vector co-expressing OCT4 and SOX2, along with or without a
lentiviral vector expressing BCL2, BCL-XL, or MCL1, with a
multiplicity of infection (MOI) of four. One day after viral
transduction, cells were harvested and transferred to 6-well
culture plates, which were pre-seeded with inactivated rat
embryonic fibroblasts (REF) feeder cells (Applied Biological
Materials (ABM), Richmond, BC, Canada). The cells were maintained
in the HSC culture condition for two additional days before being
gradually replaced with iPSC medium, which comprised Knockout
DMEM/F12 medium (Invitrogen, Carlsbad, Calif., US) supplemented
with 20% Knockout Serum Replacement (KSR) (Invitrogen), 1 mM
GlutaMAX (Invitrogen), 2 mM nonessential amino acid (ABM), 1.times.
penicillin/streptomycin (ABM), 0.1 mM .beta.-mercaptoethanol
(Sigma), 20 ng/ml FGF2 (ABM), and 50 .mu.g/ml ascorbic acid. The
culture medium was changed to fresh medium every two days. To
increase reprogramming efficiency, an inhibitor of histone
deacetylase sodium butyrate was added at 0.25 mM every two days
from day two to day ten, and the cells were cultured under hypoxia
throughout the reprogramming procedure by placing cells in culture
plates in a hypoxia chamber (Stemcell Technologies, Inc.,
Vancouver, BC, Canada) that was flushed with mixed air composed of
92% N2/3% O2/5% CO.sub.2. Starting from day ten, only
REF-conditioned medium was used in the culture. The peripheral
blood mononuclear cell iPSC colonies were enumerated at three weeks
after transduction of reprogramming factors.
[0075] Referring now to FIG. 10, there is shown a graph of the
number of induced pluripotent stem colonies generated from
1.times.10.sup.5 peripheral blood mononuclear cells when
reprogrammed by using balanced expression of OCT4 and SOX2 (OS)
(left-most), OS+BCL2 (center left), OS+BCL-XL (center right), and
OS+MCL (right-most). Data shown are presented as mean.+-.SEM (n=4).
* indicates P<0.05. As can be seen, while OS alone could also
induce adult peripheral blood mononuclear cells into pluripotency,
the efficiency was 100-fold lower than the efficiency of
reprogramming of cord blood CD34+ cells. Inclusion of BCL2 or
BCL-XL increased reprogramming efficiency by approximately
three-fold (P<0.05), while the inclusion of MCL1 had no apparent
enhancing effect on OS-mediated reprogramming efficiency. While the
inclusion of BCL-XL demonstrated increased reprogramming efficiency
over the inclusion of BCL2, as shown, the difference in efficiency
was not statistically significantly. As can be seen, the relative
efficiency of the three anti-apoptotic factors on OS-mediated
reprogramming of peripheral blood mononuclear cells (FIG. 10) were
identical to that of cord blood CD34+ cells (FIG. 9). Therefore,
inclusion of either BCL2 or BCL-XL significantly increases
efficiency of OCT4/SOX2-mediated reprogramming of adult peripheral
blood mononuclear cells.
Example 11
Determination of Whether Co-Expression of OCT4 and SOX4 in an
Episomal Vector Generates Induced Pluripotent Stem Cells from Adult
Peripheral Blood Mononuclear Cells, and Whether Co-Expression of
Bcl-XL in the Episomal Vector Increases the Efficiency of
OCT4/SOX2-Mediated Reprogramming of Adult Peripheral Blood
Mononuclear Cells into Induced Pluripotent Stem Cells
[0076] Next, a determination was made as follows, whether balanced
expression of OCT4 and SOX2 in a single episomal (nonviral) vector,
generates induced pluripotent stem cells from adult peripheral
blood mononuclear cells. Referring now to FIG. 11, there are shown,
respectively, a schematic depiction of an episomal mammalian
expression vector backbone (bottom) for (from upper to lower,
respectively) co-expression of OCT4 and SOX2 (pCEP-OS), expression
of KLF4 (pCEP-K), expression of BCL-XL (pCEP-B), co-expression of
BCL-XL and KLF4 (pCEP-BK), co-expression of OCT4, SOX2, BCL-XL and
KLF4 (pCEP-OSBK), and co-expression of MYC and KLF4 (pCEP-MK),
where 2a is a self-cleavage site derived from equine rhinitis A
virus, SFFV is a spleen focus-forming virus promoter, Wpre is a
post-transcriptional regulatory element, SV40PolyA is a
polyadenylation signal from SV40 virus, OriP is an EBV origin of
replication, and EBNA1 is Epstein-Barr nuclear antigen 1 (FIG.
11A); photographs of alkaline phosphatase staining (a measure of
pluripotency) of induced pluripotent stem cell colonies at four
weeks after nucleofection of adult peripheral blood mononuclear
cells with episomal vectors expressing reprogramming factors OCT4
and SOX2 (OS) (left-most); OCT4, SOX2 and BCL-XL (OS+B) (center
left); OCT4, SOX2, MYC and KLF4 (OS+MK) (center right), and OCT4,
SOX2, MYC, KLF4 and BCL-XL (OS+MK+B) (right most) (FIG. 11B); a
graph of the number of induced pluripotent stem colonies generated
from 1 ml of adult peripheral blood mononuclear cells nucleofected
with the episomal vectors expressing reprogramming factors OCT4 and
SOX2 (OS) without BCL-XL/with BCL-XL (left-most two bars); and
OCT4, SOX2 and KLF4 (OS+K) without BCL-XL/with BCL-XL (center two
bars); and OCT4, SOX2, MYC and KLF4 (OS+MK) without BCL-XL/with
BCL-XL (right-most two bars) (data are presented as mean.+-.SEM
(n=6), where * indicates P<0.05) (FIG. 11C). As indicated in
Example 6, episomal vector constructs comprising only the two
transcription and reprogramming factor genes OCT4 and SOX2
successfully generated integration-free induced pluripotent stem
cells from cord blood CD34+ cells. To test the effect of the
various transcription and reprogramming factor genes on generation
of integration-free induced pluripotent stem cells from adult
peripheral blood mononuclear cells (PB MNCs), adult peripheral
blood mononuclear cells were cultured for four to eight days in
conditions that favored expansion of hematopoietic stem cells and
myeloid cells before nucleofection, and then nucleofected with the
various vectors shown in FIG. 11A. After nucleofection of cultured
peripheral blood mononuclear cells with the various episomal
vectors, 1.times.10.sup.6 cells were transferred to 6-well plates,
pre-coated with feeder cells, for three to four weeks of culture.
Referring now to FIG. 11B and FIG. 11C, in contrast to cord blood
CD34+ cells (Example 6), episomal vectors expressing OCT4 and SOX2
alone (OS) essentially failed to reprogram peripheral blood
mononuclear cells (FIG. 11B (left-most) and FIG. 11C (left)) in any
significant amount, while inclusion of BCL-XL along with OCT4 and
SOX2 in the episomal vector successfully reprogrammed the
peripheral blood mononuclear cells (FIG. 11B (center left) and FIG.
11C (left)). Further as can be seen, the addition of KLF4, or KLF4
and MYC without BCL-XL did reprogram some peripheral blood
mononuclear cells into induced pluripotent stem cells (FIG. 11C
(center and right)) in small amounts. Further as can be seen,
inclusion of BCL-XL in the episomal vectors OS+K and OS+MK also
reprogrammed the peripheral blood mononuclear cells into induced
pluripotent stem cells (FIG. 11B (right-most) and FIG. 11C (center
and right)), where the inclusion of BCL-XL increased reprogramming
efficiency by up to 10-fold over to 10 iPSC colonies per ml of
peripheral blood mononuclear cells. The inclusion of MYC, however,
along with OCT4, SOX2, KLF4 and BCL-XL did not further increase the
efficiency of generation of induced pluripotent stem cells from
peripheral blood mononuclear cells beyond that of using OCT4, SOX2,
KLF4 and BCL-XL alone (FIG. 12C (right)).
Example 12
Determination of Optimal Cell Population for Generation of Induced
Pluripotent Stem Cells from Adult Peripheral Blood Mononuclear
Cells
[0077] Then, a determination was made as follows, of the optimal
cell population for generation of induced pluripotent stem cells
from adult peripheral blood cells. Referring now to FIG. 12, there
are shown, respectively, photographs of alkaline phosphatase
staining of induced pluripotent stem cell colonies at four weeks
after nucleofection of fractionated adult peripheral blood
mononuclear cells with episomal vectors expressing reprogramming
factors OCT4, SOX2, MYC, KLF4 and BCL-XL (OS+MK+B), where the
fractionated adult peripheral blood mononuclear cells expressed the
myeloid lineage marker CD33 (CD33+, left-most), did not express the
myeloid lineage marker CD33 (CD33-, center left), expressed the T
cell marker CD3 or the B cell marker CD19 (CD3+/CD19+, center
right), and did not express the T cell marker CD3 or the B cell
marker CD19 (CD3-/CD19-, right-most) (FIG. 12A); and a graph of the
number of induced pluripotent stem colonies generated from 1 ml of
adult whole peripheral blood mononuclear cells nucleofected with
the episomal vectors expressing OCT4, SOX2, MYC, KLF4 and BCL-XL
(left bar), and generated from 1 ml of adult peripheral blood
mononuclear cells that were T cell/B cell lymphocyte depleted
(CD3-/CD19-) nucleofected with the episomal vectors expressing
OCT4, SOX2, MYC, KLF4 and BCL-XL (right bar), (data are presented
as mean.+-.SEM (n=4), where * indicates P<0.05) (FIG. 12B). As
can be seen, nucleofection of fractionated adult peripheral blood
mononuclear cells with episomal vectors expressing reprogramming
factors OCT4, SOX2, MYC, KLF4 and BCL-XL (OS+MK+B) generated
induced pluripotent stem cells from cells expressing the myeloid
lineage marker CD33 (CD33+, FIG. 12A, left-most), but not from
cells that did not express the myeloid lineage marker CD33 (CD33-,
FIG. 12A, center left). Further, nucleofection of fractionated
adult peripheral blood mononuclear cells with episomal vectors
expressing reprogramming factors OCT4, SOX2, MYC, KLF4 and BCL-XL
(OS+MK+B) did not generate induced pluripotent stem cells from
cells that expressed the T cell marker CD3 or the B cell marker
CD19 (CD3+/CD19+, FIG. 12A, center right), but did generate induced
pluripotent stem cells from cells that did not express the T cell
marker CD3 or the B cell marker CD19 (CD3-/CD19-, FIG. 12A,
right-most). Quantifying these results indicated that depleting the
adult whole peripheral blood mononuclear cells of cells that
expressed the T cell marker CD3 or the B cell marker CD19
(CD3-/CD19-) increased the generation of induced pluripotent stem
cells by about ten-fold (on a per cell basis, where only about 30%
of the peripheral blood mononuclear cells were left after
purification to delete cells that expressed the T cell marker CD3
or the B cell marker CD19 (CD3-/CD19-)) (FIG. 12B). Therefore,
integration-free induced pluripotent stem cells can be generated
from non-lymphoid cells, and in particular, from T cell and B
cell-depleted non-lymphoid cells.
Example 13
Determination of Optimal Culture Duration Before Nucleofection for
the Generation of Induced Pluripotent Stem Cells from Adult
Peripheral Blood Mononuclear Cells
[0078] Next, a determination was made as follows, of the optimal
culture duration before nucleofection for the generation of induced
pluripotent stem cells from adult peripheral blood mononuclear
cells. Based on the results from Example 11, peripheral blood
mononuclear cells depleted of cells that expressed the T cell
marker CD3 or the B cell marker CD19 (CD3-/CD19- cells) were used
to determine the optimal culture duration before nucleofection for
reprogramming The numbers of induced pluripotent stem cell colonies
were counted at three to four weeks after nucleofection. Referring
now to FIG. 13, there is shown a graph of the number of induced
pluripotent stem cell colonies generated from 1 ml of adult
peripheral blood mononuclear cells that were depleted of cells that
expressed the T cell marker CD3 or the B cell marker CD19
(CD3-/CD19-), and were then nucleofected with the episomal vectors
expressing OCT4, SOX2, MYC, KLF4 and BCL-XL versus the number of
days in culture before nucleofection (data are presented as
mean.+-.SEM (n=6), where * indicates P<0.05). As can be seen,
when the days cultured before nucleofection was two days or eight
days, only a few induced pluripotent stem cell colonies were
obtained, whereas culturing the cells for four days before
nucleofection resulted in optimal generation of induced pluripotent
stem cells of more than twenty iPSC colonies from 1 ml of cells.
This optimal culture duration is consistent with analysis showing
that more progenitors (CD34+ cells) were expanded after four to six
days of culture, while culturing for a longer duration led to
differentiation of myeloid progenitors (data not shown). Therefore,
the optimal culture duration before nucleofection for the
generation of induced pluripotent stem cells from adult peripheral
blood mononuclear cells depleted of cells that expressed the T cell
marker CD3 or the B cell marker CD19 (CD3-/CD19-) is about four
days.
Example 14
Determination of Morphology, Phenotype and Function of Induced
Pluripotent Stem Cells Generated from Adult Peripheral Blood With
Integration-Free Yamanaka Factors
[0079] Then, a determination was made as follows, of the
morphology, phenotype and function of integration-free induced
pluripotent stem cells generated from adult peripheral blood
mononuclear cells depleted of cells that expressed the T cell
marker CD3 or the B cell marker CD 19 (CD3-/CD 19- cells) using
integration-free Yamanaka factors in accordance with Example 12 and
Example 13. The generated integration-free induced pluripotent stem
cells were robustly proliferated under human induced pluripotent
stem cells culture conditions for more than twenty passages.
Referring now to FIG. 14, there are shown, respectively, a
photograph of a representative induced pluripotent stem cell colony
(FIG. 14A); a photograph of a representative karyogram of an
induced pluripotent stem cell clone (FIG. 14B); representative
images captured using a Zeiss LSM 710 confocal microscope with a
20.times. objective of induced pluripotent stem cells immunostained
to show expression of pluripotency markers OCT4 (left), SOX2
(center), and NANOG and SSEA4 (right) by representative induced
pluripotent stem cell colonies (FIG. 14C); representative images
captured using an Olympus microscope with a 20.times. objective of
cell layer derivatives in hematoxylin and eosin (H & E)
staining formed by teratomas in immunodeficient mice produced by
representative induced pluripotent stem cell colonies, where the
teratoma cell layers included all three embryonic germ layers,
cartilage (mesoderm, left), glands (endoderm, center) and
neurotubules (ectoderm, right) (FIG. 14D); photographs of
representative induced pluripotent stem cell colonies showing
differentiation into mesenchymal stem cells (left-most), stained
with Oil Red O stains to show the oil droplets of adipocytes
(center left), stained with Alizarin Red to show bone nodules
formed by osteoblasts (center right), and stained with Alcian Blue
to show acid mucopolysaccharides synthesized and secreted by
chondrocytes (right-most) (FIG. 14E); photographs of representative
induced pluripotent stem cell colonies showing differentiation into
hepatocytes (left-most), and stained with monoclonal antibody
against alpha-fetoprotein (AFP) to show expression of
alpha-fetoprotein (center left), stained with goat anti-albumin to
show expression of albumin (ALB), and stained with goat anti-alpha
1-antitrypsin to show expression of alpha 1-antitrypsin
(.alpha.1-AT) (FIG. 14F); and a photograph of representative
induced pluripotent stem cell colonies showing expression of
Troponin I marker showing differentiation into cardiomyocytes where
the cell nuclei are counterstained with
4',6-diamidino-2-phenylindole (FIG. 14G).
[0080] As can be seen, the integration-free induced pluripotent
stem cells showed typical morphology for human pluripotent stem
cells (FIG. 14A). Further, karyotyping and Giemsa banding
(GTG-banding) chromosome analysis were carried out on
representative induced pluripotent stem cell clones following
standard DNA spectral karyotyping procedures using HiSKY Complete
Cytogenetic System (Applied Spectral Imaging, Inc., Vista, Calif.,
US). For each clone, 10 metaphases were analyzed and karyotyped. As
can be seen in the representative karyogram, FIG. 14B, all
integration-free induced pluripotent stem cell clones were normal.
Additionally, the integration-free induced pluripotent stem cells
were cultured in chamber slides for four to five days. The cells
were then treated with fixation buffer and permeabilization buffer
(eBioscience, Inc., San Diego, Calif., US) for thirty minutes
before being stained overnight at 4.degree. C. with PE or FITC
conjugated antibodies anti-OCT4 (eBioscience), anti-SOX2 (BD
Pharmingen; San Diego, Calif., US), anti-NANOG (BD Pharmingen), and
anti-SSEA-4 (eBioscience). Confocal imaging was performed using the
Zeiss LSM 710 NLO laser scanning confocal microscope with a
20.times. objective. High resolution monochrome images were
captured using a Zeiss HRm CCD camera. As can be seen in FIG. 14C,
the integration-free induced pluripotent stem cells expressed the
pluripotency markers OCT4, SOX2, NANOG and SSEA4. Further,
immunodeficient NSG mice (purchased from the Jackson Laboratory,
Sacramento, Calif., US) were injected subcutaneously with
1.times.10.sup.6 integration-free induced pluripotent stem cells
according to the present invention that were suspended in 200 ul
DMEM/F12 diluted (1:1) Matrigel solution (BD) and the cells formed
teratomas. Two months after injection, the teratomas were
dissected, fixed in 10% formalin, stained with hematoxylin and
eosin, and examined by a board certified pathologist. The teratomas
and were found to contain derivatives of all three embryonic germ
layers as represented by cartilage (mesoderm, left), glands
(endoderm, center) and neurotubules (ectoderm, right) (FIG. 14D)
indicating that these integration-free induced pluripotent stem
cells automatically differentiated into tissues and cells of
mesoderm, endoderm and ectoderm after implantation in animals
cells.
[0081] Induced pluripotent stem cells according to the present
invention were then investigated to determine if the
integration-free induced pluripotent stem cells could differentiate
into cells of different lineages in culture. First,
integration-free induced pluripotent stem cells were cultured with
Mesenchymal Stem Cell (MSC) Medium Kit (ABM) for four to five days.
The cells were then treated with Accutase (Innovative Cell
Technologies, Inc., San Diego, Calif., US) and further cultured in
fibronectin (BD)-pre-coated non-tissue culture treated well plates
and readily differentiated into mesenchymal stem cells (MSCs) as
can be seen in FIG. 14E (left-most), where more than 90% of the
differentiated cells expressed typical markers of mesenchymal stem
cells including CD73, CD105 and CD 166. After three weeks of
culture in differentiation medium, the mesenchymal stem cells were
stained with Oil Red O, Alizarin Red, and Alcian Blue, showing
differentiation into adipocytes, osteoblasts and chondrocytes (FIG.
14E, center left, center right and right-most, respectively). These
data suggest that the mesenchymal stem cells differentiated from
integration-free induced pluripotent stem cells are morphologically
and functionally indistinguishable to bone marrow-derived
mesenchymal stem cells. Second, integration-free induced
pluripotent stem cells were cultured under conditions that produced
hepatocytes. Initially, induction of the integration-free induced
pluripotent stem cells definitive endoderm (DE) was initiated in
RPMI 1640 medium containing 100 ng/ml Activin A (R&D Systems,
Minneapolis, Minn., US) and 2 mM L-glutamine for two days. B27
(Invitrogen) and 0.5 mM sodium butyrate were then supplemented for
additional seven to nine days. The definitive endoderm cells were
treated with Accutase for a short period of time and rapidly
transferred to collagen I-coated plates and cultured in RPMI 1640
medium supplemented with FGF-4, HGF, BMP2, BMP4 (R&D Systems),
dexamethasone, and DMSO. Fourteen days after differentiation
induction, the cells were maintained in hepatocyte culture medium
supplemented with FGF-4, HGF, Oncostatin M (R&D Systems),
dexamethasone and DMSO for an additional two to three weeks. At
eighteen days after differentiation, cells were stained with
monoclonal antibody against AFP (Dako North America, Inc.,
Carpinteria, Calif., US), goat anti-albumin (Bethyl Laboratories,
Inc., Montgomery, Tex., US), and goat anti-alpha 1-antitrypsin
(Bethyl), following standard protocols. Successful differentiation
into hepatocytes was evidenced by a series of morphological changes
resulting in a polygonal shape and round single or double nuclei
with many cytoplasmic vesicles characteristic of mature
hepatocytes, as can bee seen in FIG. 14F. Immunohistochemical
analysis showed that about 90% of the differentiated cells
expressed liver-specific genes at eighteen days after culture,
including alpha fetoprotein (AFP), albumin (ALB), and alpha
1-antitrypsin (.alpha.1-AT) (FIG. 14F, center left, center right
and right-most, respectively), further indicating differentiation
into mature hepatocytes. Third, integration-free induced
pluripotent stem cells were cultured under conditions that produced
cardiomyocytes. Small clusters of integration-free induced
pluripotent stem cells were cultured in differentiation medium
consisting of StemPro-34 (Invitrogen), supplemented with 2 mM
GlutaMAX, 50 .mu.g/ml ascorbic acid, and 4.times.10.sup.-4 M
monothioglycerol (MTG) (Sigma). Cytokine Activin A (R &D
Systems) was used at 50 ng/ml for one day and 10 ng/ml BMP4
(R&D Systems) was added for four days. After twelve days of
culture, dozens of beating colonies of cardiomyocytes were observed
in each well of 6-well plates after two weeks of culture
Immunostaining of these cells with 4',6-diamidino-2-phenylindole
(DAPI) showed the majority of cells expressed the Troponin I marker
(R&D Systems), confirming differentiation into mature
cardiomyocytes (FIG. 14G). Fourth, integration-free induced
pluripotent stem cells were also differentiated into neuron cells
after one month of induction culture (not shown). Finally, qPCR
analysis of induced pluripotent stem cells after 10 passages showed
that the average copy number of residual episomal vectors decreased
to less than 0.01 copy per cell or was undetectable in six out of
six induced pluripotent stem cells clones, suggesting that after
long-term culture, the episomal vectors were depleted from almost
all cells.
[0082] Therefore, integration-free induced pluripotent stem cells
produced according to the present invention are morphologically,
phenotypically and functionally identical to pluripotent stem
cells, and can be induced to differentiate into fully functional
mesenchymal stem cells, hepatocytes, cardiomyocytes and
neurons.
[0083] Although the present invention has been discussed in
considerable detail with reference to certain preferred
embodiments, other embodiments are possible. Therefore, the scope
of the appended claims should not be limited to the description of
preferred embodiments contained in this disclosure. All references
cited herein are incorporated by reference in their entirety.
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