U.S. patent application number 17/827338 was filed with the patent office on 2022-09-29 for induction of arterial-type of hemogenic endothelium (ahe) and enhancement of t cell production from pscs through overexpression of ets factors or modulating mapk/erk signalling pathways.
The applicant listed for this patent is Wisconsin Alumni Research Foundation. Invention is credited to Akhilesh Kumar, Mi Ae Park, Igor I. Slukvin.
Application Number | 20220306988 17/827338 |
Document ID | / |
Family ID | 1000006406214 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220306988 |
Kind Code |
A1 |
Slukvin; Igor I. ; et
al. |
September 29, 2022 |
INDUCTION OF ARTERIAL-TYPE OF HEMOGENIC ENDOTHELIUM (AHE) AND
ENHANCEMENT OF T CELL PRODUCTION FROM PSCS THROUGH OVEREXPRESSION
OF ETS FACTORS OR MODULATING MAPK/ERK SIGNALLING PATHWAYS
Abstract
The present invention is a method of creating a population of
hemogenic endothelial cells with arterial specification. In one
embodiment, the method uses ETS transgene induction at the
mesodermal stage of differentiation. In another embodiment, the
method activates ERK signaling at the mesodermal stage of
differentiation.
Inventors: |
Slukvin; Igor I.; (Verona,
WI) ; Park; Mi Ae; (Madison, WI) ; Kumar;
Akhilesh; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wisconsin Alumni Research Foundation |
Madison |
WI |
US |
|
|
Family ID: |
1000006406214 |
Appl. No.: |
17/827338 |
Filed: |
May 27, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15816914 |
Nov 17, 2017 |
11345895 |
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17827338 |
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62424144 |
Nov 18, 2016 |
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62572066 |
Oct 13, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70503 20130101;
C12N 2501/42 20130101; A61K 35/17 20130101; C12N 2506/45 20130101;
C12N 2501/60 20130101; C12N 5/0647 20130101; C12N 2501/727
20130101; C12N 5/069 20130101; C07K 14/4702 20130101; C12N 5/0636
20130101; C12N 2510/00 20130101; C12N 2501/602 20130101; A61K
2035/124 20130101; C12N 2506/02 20130101; C12N 2506/28
20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; A61K 35/17 20060101 A61K035/17; C07K 14/705 20060101
C07K014/705; C12N 5/0789 20060101 C12N005/0789; C12N 5/0783
20060101 C12N005/0783; C07K 14/47 20060101 C07K014/47 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
HL116221, HL099773 and OD011106 awarded by the National Institutes
of Health. The government has certain rights in the invention.
Claims
1. A method of enhancing arterial specification of hemogenic
endothelium, the method comprising: (a) introducing an ETS
transcription factor transgene into a mesoderm cell population; and
(b) culturing the mesoderm cells under conditions sufficient to
express the ETS transcription factor transgene within the mesoderm
population and differentiate the mesoderm cells to arterial
hemogenic endothelium (AHE) cells.
2. The method of claim 1, wherein step (a) comprises introducing a
vector comprising the ETS transcription factor transgene into the
mesoderm cell population.
3. The method of claim 2, wherein the vector comprises an inducible
promoter operably linked to the ETS transcription factor
transgene.
4. The method of claim 1, wherein the mesoderm cell population is
differentiated from human pluripotent stem cells (hPSCs).
5. The method of claim 1, wherein the mesoderm cells population
expresses the ETS transcription factor for at least 2 days to
differentiate to AHE cells.
6. A method of enhancing arterial specification of hemogenic
endothelium in differentiating hPSC, comprising the steps of (a)
introducing an ETS transcription factor transgene into a hPSC
population, (b) culturing the hPSC cells under conditions to
differentiate the hPSC into mesoderm cells at two days of
differentiation, and (b) inducing expression of the transgene at
day two of differentiation, such that arterial hemogenic
endothelium cells (AHE) are obtained by day four of
differentiation.
7. The method of claim 6, wherein the expression is under inducible
control.
8. The method of claim 6 wherein the ETS transgene is ETS1.
9. The method of claim 6 wherein the ETS transgene is selected from
the group of ETV2, ETS2 and ERG.
10. The method of claim 6, wherein the cells are further
differentiated into lympho-myeloid and erythroid cell lines.
11. The method of claim 6, wherein a population of hemogenic
endothelium cells that are CD144+CD43-CD73-DLL4+CXCR4.sup.+/- HE
and express high level of one or more arterial markers selected
from the group consisting of EFNB2, NOTCH1, NOTCH 4 and SOX17 is
obtained.
12. A cell population produced by the method of claim 1.
13. A method of creating a cell population, comprising the steps of
(a) obtaining a cell population of AHE cells, (b) further
differentiating the AHE cells into an at least 90% pure population
of cells, wherein the cell type of the cell population is selected
from the group consisting of T-cells, B-cells, definitive
(adult-type) red blood cells, myeloid progenitors and mature
myelomonocytic cells.
14. A method of enhancing arterial specification of hemogenic
endothelium in differentiating hPSC, comprising the steps of (a)
culturing human mesoderm cells in defined cell culture medium
comprising an effective amount of a factor capable of activating
ERK signaling to differentiate the mesoderm cells into arterial
hemogenic endothelium cells (AHE); and (b) obtaining the arterial
hemogenic endothelium cells.
15. The method of claim 14, wherein the factor activates ERK
signaling through the VEGF receptor pathway.
16. The method of claim 14, wherein the factor capable of
activating ERK signaling is a PI3K inhibitor.
17. The method of claim 11, wherein the PI3K inhibitor is
LY294002.
18. The method of claim 14, wherein step (a) is performed for about
3 days.
19. The method of claim 14, wherein the human mesoderm cells are
obtained from a method comprising culturing human pluripotent stem
cells in a chemically defined culture medium for about 2 to about 3
days, whereby a cell population comprising human KDR.sup.+ mesoderm
cells is obtained.
20. The method of claim 14, wherein the AHE cells are
CD144+CD43-CD73-DLL4.sup.+CXCR4.sup.+/-.
21. The method of claim 20, wherein the AHE cells obtained further
express one or more arterial markers selected from the group
consisting of EFNB2, NOTCH1, NOTCH 4 and SOX17.
22. The method of claim 14, wherein the hPSCs are embryonic stem
cells (ESCs) or induced pluripotent stem cells (iPSCs).
23. A cell population produced by the method of claim 14.
24. A method of creating a cell population, comprising the steps of
(a) obtaining a cell population of AHE DLL4.sup.+ cells, (b)
further differentiating the AHE DLL4.sup.+ cells into an at least
90% pure population of cells, wherein the cell type of the cell
population is selected from the group consisting of T-cells,
B-cells, definitive (adult-type) red blood cells, myeloid
progenitors and mature myelomonocytic cells.
25. The method of claim 24, wherein the cell population is T cells
and wherein step (b) comprises co-culturing the AHE DLL4.sup.+
cells with stromal cells expressing NOTCH ligand DLL4 or DLL1 in T
cell differentiating medium for an effective amount of time to
differentiate the AHE cells into T-cells.
26. The method of claim 24, wherein the derived T-cells are
engineered to express an exogenous chimeric antigen receptor
(CAR).
27. The method of claim 26, wherein the CAR comprises a CD19
chimeric antigen receptor.
28. A method of killing tumor cells, the method comprising
contacting the tumor cells with T cells derived from the method of
claim 26 in an effective amount to kill the tumor cells.
29. The method of claim 28, wherein the tumor cells are selected
from the group consisting of lymphoma, leukemia, and myeloma
cells.
30. A method of enhancing arterial specification of hemogenic
endothelium in mesoderm cells, the method comprising culturing the
mesoderm cells in defined medium comprising an effective amount of
a factor capable of activating NOTCH signaling to differentiate the
mesoderm cells into arterial hemogenic endothelium (AHE) cells.
31. The method of claim 30, wherein the factor capable of
activating NOTCH signaling is selected from the group consisting of
DLL4, DLL1-Fc, DLL1-expressing feeder cells, plates coated with
DLL4-Fc, and plates coated with DLL1-Fc.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 15/816,914 which will grant as U.S. Pat. No.
11,345,895 which claims priority to U.S. Provisional Application
Nos. 62/424,144 and 62/572,066 filed on Nov. 18, 2016 and Oct. 13,
2017, respectively, the contents of which are incorporated by
reference in their entireties.
REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0003] This application includes an electronically submitted
Sequence Listing in .txt format. The .txt file contains a sequence
listing entitled
"2022-05-27_960296-04314_SEQ_Listing_P170133US04.txt" and was
created on May 27, 2022 and is 4,096 bytes in size. The Sequence
Listing contained in this .txt file is part of the specification
and is hereby incorporated herein in its entirety.
BACKGROUND OF THE INVENTION
[0004] De novo production of hematopoietic stem cells (HSCs) from
in vitro expandable human cells, such as pluripotent stem cells
(hPSCs), represents a promising approach for stem cell-based
therapies and modeling of hematologic diseases. However, generation
of HSCs from hPSCs remains a significant challenge.sup.63-65. Since
HSCs are specified from hemogenic endothelium (HE) with definitive
hematopoietic program, understanding molecular mechanisms
regulating the establishment of HE with broad lymphoid and myeloid
potentials is essential to advance the HSC manufacturing
technology.
[0005] During development, blood cells and HSCs arise from
hemogenic endothelium (HE). In contrast to the first wave of
primitive hematopoiesis lacking of lymphoid and granulocytic
potential, definitive hematopoiesis produces the entire spectrum of
adult-type erythro-myeloid progenitors (EMP), lymphoid cells, and
cells capable of limited engraftment (second wave), and HSCs with
capacity of long-term repopulation of adult recipient (third
wave).sup.6-8. While some definitive hematopoietic cells such as
EMPs can be produced from HE in venous vessels and
capillaries.sup.9-11, production of lymphoid cells and HSCs is
mostly restricted to arterial vasculature.sup.12-16. The lack of
venous contribution to HSCs when considered along with the shared
requirements for Notch, VEGF, and Hedgehog signaling in both
arterial fate acquisition and HSC formation.sup.17-21, suggests
that arterial specification is an essential prerequisite for
establishing of definitive hematopoiesis with lymphoid potential.
Although previous studies demonstrated arterial commitment within
nonHE fraction of hPSC-derived endothelium.sup.22, little is known
about the effect of arterial programming on HE.
SUMMARY OF THE INVENTION
[0006] The present disclosure provides methods for promoting
arterial hemogenic endothelium cell differentiation for human
pluripotent stem cell populations in vitro. In one aspect, the
method provides a method of promoting AHE differentiation by
overexpression of ETS family transcription factor (e.g. ETS1)
during hPSCs differentiation at the mesoderm cell population stage,
which enhances AHE formation. In one aspect, the overexpression of
ETS family transcription factor, ETS1, was associated with
promotion of HE formation with DLL4.sup.+CXCR4.sup.+/- arterial
phenotype and TB lymphoid and definitive erythroid potentials.
[0007] In another aspect, arterialization of HE and enhancement of
definitive hematopoiesis can be achieved through modulating of
MAPK/ERK pathways, specifically by contacting the cells with a PI3K
inhibitor. Methods of activating ERK pathway by inhibiting PI3K
results in the enhanced production of DLL4.sup.+CXCR4.sup.+/-
arterial type HE.
[0008] In another aspect, the disclosure provides a method of
enhancing arterial specification in mesoderm cell population, the
method comprising: (a) introducing an ETS transcription factor
transgene into the mesoderm cell population; and (b) culturing the
mesoderm cells under conditions sufficient to express the ETS
transcription factor transgene within the mesoderm population and
differentiating the mesoderm cells to arterial hemogenic
endothelium (AHE) cells.
[0009] In another aspect, the disclosure provides a method of
enhancing arterial specification in differentiating hPSC,
comprising the steps of (a) introducing an ETS transcription factor
transgene into a hPSC population, (b) culturing the hPSC cells
under conditions to differentiate the hPSC cells into mesoderm
cells at two days of differentiation, and (b) inducing expression
of the transgene at day two of differentiation, such that arterial
hemogenic endothelium cells (AHE) are obtained by day four of
differentiation.
[0010] In yet another aspect, the disclosure provides a method of
creating a cell population, comprising the steps of (a) obtaining a
cell population of AHE cells, (b) further differentiating the AHE
cells into an at least 90% pure population of cells, wherein the
cell type of the cell population is selected from the group
consisting of T-cells, B-cells, definitive (adult-type) red blood
cells, myeloid progenitors and mature myelomonocytic cells.
[0011] In yet another aspect, the method provides a method of
enhancing arterial specification in differentiating hPSC,
comprising the steps of (a) culturing human mesoderm cells in
defined cell culture medium comprising an effective amount of a
factor capable of activating ERK signaling to differentiate the
mesoderm cells into arterial hemogenic endothelium cells (AHE); and
(b) obtaining the arterial hemogenic endothelium cells. In some
aspects, the factor capable of activating ERK signaling is a PI3K
inhibitor.
[0012] In another aspect, the disclosure provides a method of
creating a cell population, comprising the steps of (a) obtaining a
cell population of AHE DLL4.sup.+ cells, (b) further
differentiating the AHE DLL4.sup.+ cells into an at least 90% pure
population of cells, wherein the cell type of the cell population
is selected from the group consisting of T-cells, B-cells,
definitive (adult-type) red blood cells, myeloid progenitors and
mature myelomonocytic cells.
[0013] The foregoing and other aspects and advantages of the
invention will appear from the following description. In the
description, reference is made to the accompanying drawings which
form a part hereof, and in which there is shown by way of
illustration a preferred embodiment of the invention. Such
embodiment does not necessarily represent the full scope of the
invention, however, and reference is made therefore to the claims
and herein for interpreting the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The patent or patent application file contains at least one
drawing in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0015] FIGS. 1A-1E demonstrate that ETS1 induction enhances
arterial specification of hPSCs. (a) Experimental scheme. iETS1
hESC were differentiated in defined conditions with or without Dox
for 4 days and evaluated for expression of arterial markers in
CD144.sup.+ endothelial cells. (b) The effect of DOX treatment on
generation of CD144.sup.+ endothelial cells. (c) Flow cytometric
analysis of arterial markers expression by hESC-derived endothelial
cells following DOX treatment (1, 1.5 or 2 .mu.g/ml) for 2-4 days.
Representative experiment of three independent experiments is
shown. (d) Heat map of arterial and venous genes expression in day
4 KDR.sup.+CD144.sup.+ endothelial cells obtained with or without
ETS1 induction as determined by RNAseq analysis. Gene expression is
estimated in tpm values. (e) RT-qPCR analysis confirms upregulation
of arterial genes following DOX treatment. Bar graphs in (b) and
(e) are mean.+-.s.d. of at least three independent experiments;
*p<0.05; **p<0.01;***p<0.001
[0016] FIGS. 2A-2E show the stage-specific effect of ETS1 on
hematopoietic development. (a) Experimental scheme. (b) Flow
cytometric analysis of the hematopoietic progenitors obtained from
iETS1 hESCs treated with DOX at indicated time points.
Representative experiment of three independent experiments is
shown. (c) The percentage of hematopoietic and endothelial cells in
day 8 of differentiation cultures following DOX treatment at
indicated time points. (d) Pie charts display the composition of
CD43.sup.+ subsets. (e) Hematopoietic colony-forming potential of
iETS1 hESCs treated with the DOX at indicated time points. Bar
graphs in (c) and (e) are mean.+-.s.d. for two independent
experiments performed in duplicates; *p<0.05;**p<0.01;
***p<0.001 compared to No DOX treatment.
[0017] FIGS. 3A-3F demonstrate that ETS1 induction suppresses
primitive and promotes definitive hematopoiesis. (a) Experimental
scheme. (b) Effect of ETS1 induction on hemangioblast (HB)-CFCs.
(c) Flow cytometric analysis shows the increase of
CD144.sup.+CD73.sup.-CD235a/43.sup.- HE population at day 5 of
differentiation of iETS1-hESC cultures treated with DOX for 2-5
days. (d) CD43.sup.+ cells generated in DOX-treated and No-DOX
cultures possess T cell potential. Representative experiment of 3
independent experiments shows the expression of T cell markers on
CD45-gated cells in T cell differentiation cultures. (e) Bar graph
shows the total number of T cells generated from 10.sup.4
CD43.sup.+ cells obtained from iETS1-hESCs in DOX-treated and
untreated conditions. (f) Ratio of .beta./.epsilon. and
.beta./.gamma. globin chain and BCL11a gene expression as measured
by RT-qPCR in red blood cell cultures generated from CD43.sup.+
cells obtained from DOX-treated and untreated iETS1-hESC cultures.
Bar graphs in (b), (c), (e) and (f) are mean.+-.s.d. of at least
three independent experiments. *p<0.05; **p<0.01;
***p<0.001 compared to No DOX treatment.
[0018] FIGS. 4A-4I demonstrate that ETS1 induction promotes
hematopoiesis through upregulation of NOTCH signaling. (a)
Schematic diagram of experiment with chimeric hESC cultures.
Mixture of iETS1 (tdTomato.sup.-) and tdTomato H1 hESCs were
cultured with or without Dox (2 .mu.g/ml) for 2-6 days during
hematopoietic differentiation in chemically defined culture
conditions. (b) Flow cytometric analysis of hematopoietic
development on day 8 of differentiation following gating
tdTomato.sup.+ or tdTomato.sup.- (iETS1) cells in DOX-treated and
untreated cultures. (c) Hematopoietic colony-forming potential of
tdTomato.sup.+ or tdTomato.sup.- cells. (d) Flow cytometric
analysis of DLL4 expression in day 4 KDR.sup.+CD144.sup.+
population following gating tdTomato.sup.+ or tdTomato.sup.-(iETS1)
cells in DOX-treated and untreated cultures. (e) Schematic diagram
of experiment to assess the role of NOTCH signaling in
ETS1-mediated effect on hematopoiesis. The effect of DAPT treatment
on blood production (f) and CFC potential (g) in cultures treated
with DOX. The effect of DLL4 neutralizing antibodies on blood
production (h) and CFC potential (i) in cultures treated with DOX.
Bar graphs in (b), (c), (g) and (i) are mean.+-.s.d. of three
independent experiments; *p<0.05, **p<0.01;
***p<0.001.
[0019] FIGS. 5A-5K demonstrate that ETS1 promotes specification of
arterial type HE. (a) and (b) DOX treatment enhances specification
of DLL4.sup.+CXCR4.sup.+/- arterial type HE in dose-dependent
manner. (c) DOX-treatment enhances production of
CD34.sup.+CD43.sup.+ hematopoietic progenitors expressing
CXCR4.sup.+ in a dose-dependent manner. (d) Schematic
representation of the experimental strategy to assess hematopoietic
potential of DLL4.sup.+ and DLL4.sup.- HE. (e) RT-qPCR analysis of
arterial genes in DLL4.sup.+ and DLL4.sup.- HE. Hematopoietic (f)
and CFC (g) potential of DLL4.sup.+ and DLL4.sup.- HE. (h) Ratio of
.beta./.epsilon. and .beta./.gamma. globin chain and BCL11a gene
expression as measured by RT-qPCR in red blood cell cultures
generated from DLL4.sup.+ and DLL4.sup.- HE. (i) B cell potential
of DLL4.sup.+ and DLL4.sup.- HE. (j) T cell potential of DLL4.sup.+
and DLL4.sup.- HE. Flow cytometry plot depicts percentage of
CD4.sup.+CD8.sup.+ T cells. Bar graph shows the total number of T
cells generated from 10.sup.4 CD43.sup.+ cells obtained from
DLL4.sup.+ and DLL4.sup.- HE. (k) Limiting dilution assay to
determine frequency of T cell progenitors in DLL4.sup.+ and
DLL4.sup.- HE cultures. Bars in (b), (c), (e)-(j) are mean s.d. of
at least three independent experiments; *p<0.05, **p<0.01;
***p<0.001.
[0020] FIGS. 6A-6H show modulation of MAPK/ERK signaling enhances
arterial specification and definitive hematopoiesis from hESCs. (a)
Experimental scheme. Effect of LY294002 and U0126 on arterial HE
specification (b) and (c), hematopoietic (d)-(e), CFC (f) and T
cell development (g) and (h) from hESCs. Bars in (c), (e), (f) and
(h) are mean.+-.s.d. of at least three independent experiments;
*p<0.05, **p<0.01;***p<0.001.
[0021] FIGS. 7A-7B demonstrate that gene expression profiling
reveals activation of NOTCH and SOXF- mediated transcriptional
programs in DLL4.sup.+ arterial HE. (a) Transcriptional regulatory
network reconstructed based on analysis of differentially expressed
genes in DLL4.sup.+ and DLL4.sup.- RE cells as described in
materials and methods. Size of the nodes represents relative
abundance of mRNA of the respective gene, computed as log 2 (fold
change) in DLL4.sup.+ and DLL4.sup.- HE cells. Both up- and
down-regulation effects are mapped onto the node size. The color
density represents enrichment (red) or depletion (blue) of known
targets of that transcription factor (regulon members) among the
differentially expressed genes. Network visualization was performed
using Cytoscape ver. 3.4.0.sup.62. (b) Schematic of arterial HE
induction from hPSCs by overexpression of ETS1 or modulation of
MAPK/ERK signaling pathways.
[0022] FIGS. 8A-8D show generation of conditional H1 hESC cell line
(iETS1-hESC). (a) Schematic diagram of PiggyBac system used to
generate iETS1. (b) Expression of Venus reported in
undifferentiated and day 5 differentiated iETS1 cells.
Dose-dependent effect of DOX on ETS1 expression in undifferentiated
iETS1-hESCs as determined by RT-qPCR (c) and Western Blot (d).
[0023] FIGS. 9A-9C Effect of ETS1 overexpression on development of
primitive posterior mesoderm. (a) Schematic diagram depicts the
major stages of hematopoietic development from hESCs. A+P+PM is
APLNR+PDGFR.alpha.+ primitive posterior mesoderm; HB-CFC is
hemangioblast CFCs; KDR.sup.hiP.sup.lo/- HVMPs is
KDR.sup.highPDGFR.alpha..sup.low/- hematovascular mesodermal
progenitors; HE, hemogenic endothelium; MHPs, multipotent
hematopoietic progenitors; EMkPs, erythromegakaryocytic
progenitors. (b) Representative contour plots show the DOX effect
on A+P+PM. (c) Percentage of A+P+PM cells in DOX-treated and
untreated cultures. Bars are mean s.d. of three independent
experiments; ***p<0.001.
[0024] FIG. 10 shows evaluation of ETS1 effect using chimeric (wild
type tdTomato+ and iETS1 tdTomato-) H1 hESCs. Representative
contour plots show flow cytometric analysis of hematopoietic
development on day 8 of differentiation following gating
tdTomato.sup.+ or tdTomato-(iETS1) cells in DOX-treated and
untreated cultures.
[0025] FIGS. 11A-11E show hematopoietic potential of CXCR4+ and
CXCR4- DLL4+ arterial HE. (a) Schematic diagram of experiments. (b)
and (c) flow cytometric analysis of hematopoiesis from CXCR4+ and
CXCR4-DLL4+HE. (d) CFC potential of CD43+ cells generated from
CXCR4+ and CXCR4-DLL4+HE. (e) Limiting dilution assay to determine
the frequency of T cell progenitors from CXCR4+ and CXCR4-DLL4+HE.
Bars in (c) and (d) are mean.+-.s.d. of three independent
experiments; *p<0.05 and **p<0.01.
[0026] FIG. 12 lists the antibodies used for FACS.
[0027] FIG. 13 lists the primers used for RT-qPCR.
[0028] FIG. 14 is a schematic representation of generation of T
Lymphoid cells from hematopoietic progenitor subsets using hESC/OP9
coculture system. Scheme shows the emerging progenitor subsets
according to different co culture days. KDR.sup.+ mesodermal
hematovascular progenitors at coculture day4, CD144.sup.+ hemogenic
endothelial progenitors at coculture day5, and CD43.sup.+
hematopoietic progenitors at coculture day 8.5 were isolated and
used for T cell differentiation. T cell differentiation was
accomplished on Op9-DLL4 using the respective subsets.
[0029] FIGS. 15A-15F demonstrate T cell differentiation from
hESC-derived hematopoietic progenitor subsets. (A) KDR.sup.+
mesodermal hematovascular progenitors were generated from hESC/OP9
coculture at day4, KDR.sup.brightCD31.sup.-/+ subsets were sorted
for lymphoid differentiation. (B) Percentage of T cell phenotypes
from day 4 cell subsets, as detected by flow cytometry. (C)
CD144.sup.+ hemogenic endothelial progenitors were generated from
hESC/OP9 coculture at day5, different endothelial subsets were
sorted for lymphoid differentiation. (D) Percentage of T cell
phenotypes from day 5 cell subsets, as detected by flow cytometry.
(E) CD43.sup.+ hematopoietic progenitors were generated from
hESC/OP9 coculture at day 8.5, subsets were sorted for lymphoid
differentiation. (F) Percentage of T cell phenotypes from day 8.5
cell subsets, as detected by flow cytometry. All gates represent
target cell population sorted by MACS and FACS.
[0030] FIGS. 16A-16C show the characterization of T cell from
different progenitor subsets. (A) Proliferative potential of T cell
generated from various subsets. Expansion and proliferation
potential of KDR.sup.brightCD31.sup.- subsets is higher in
comparison to other subsets. (B) Limiting dilution assay to
determine the frequency of T cell progenitors from different
subsets. (C) Comparison of hematopoietic colony-forming potential
of subsets differentiated on OP9 and OP9-DLL4.
[0031] FIG. 17 depict the analysis of TCR gene rearrangement in
HVMP derived T cells. PCR analysis of TCR gene rearrangement in
TCR.beta. and TCR.gamma. locus of HVMP derived T cells and specimen
control. M, 50 bp DNA ladder. T cells, genomic DNA from Hlderived T
cells. H1, genomic DNA from hESC (negative control). PB, genomic
DNA from peripheral blood (positive control) and undifferentiated
H1 ESCs.
[0032] FIGS. 18A-18C show the results of the T cell Functional
assay. (A) HVMP derived T cell were stimulated with PMA and
ionomycin for 24 hours before analysis of activation markers CD25
and CD69 and intracellular protein IFN-.gamma. and perforin. (B)
Flow cytometry analysis of T cells transduced with CD19 CAR. (C)
Cytotoxicity assay of HVMP derived CAR-T cells. CAR-T cells
(effector) and Raji (target cells) were combined in ratio 1:1, 2:1,
4:1 and 10:1. Target cells (Raji) were labeled with PKH67 (green
fluorescent cell linker) to distinguish from cell mixture.
DETAILED DESCRIPTION OF THE INVENTION
In General
[0033] The present disclosure demonstrates methods that allow for
the promoting of arterial patterning in hPSC cultures that can aid
in in vitro approaches to instruct definitive hematopoiesis with
lymphoid and HSC potentials from hPSCs. Arterial program from hPSCs
can be enhanced by overexpression of ETS family transcription
factor, ETS1 which was associated with promotion of HE formation
with DLL4.sup.+CXCR4.sup.+/- arterial phenotype and TB lymphoid and
definitive erythroid potentials, as described in the Examples.
Further, arterialization of HE and enhancement of definitive
hematopoiesis can be achieved through modulating of MAPK/ERK
pathways, further demonstrated in the examples. Methods of
activating ERK pathway by inhibiting PI3K results in the enhanced
production of DLL4.sup.+CXCR4.sup.+/- arterial type HE. Together,
the Examples demonstrate different approaches in providing
arterialization of HE and enhanced definitive hematopoiesis.
[0034] In the Examples, how arterial programming affects
specification of definitive HE from hPSCs was investigated. During
vascular development, arterial fate is specified following
induction of DLL4 expression.sup.23 initiated by signaling through
arterial-specific enhancer located within the third intron of DLL4
that is controlled by ETS factors.sup.24-25.
[0035] The inventors found that arterial program from hPSCs could
be enhanced by overexpression of ETS family transcription factor,
ETS1. The boost in arterial programming by ETS1 was associated with
promotion of definitive HE formation with lymphoid and definitive
erythroid potential. The observed increase in arterial programming
by ETS1 was associated with promotion of HE formation with
DLL4.sup.+CXCR4.sup.+/- arterial phenotype and TB lymphoid and
definitive erythroid potentials. The ETS1 effect was associated
with upregulation of SOXF factors and DLL4 in endothelial cells.
Inhibition of NOTCH signaling with DAPT or DLL4 neutralizing
antibodies abrogated the effect of ETS1 overexpression in
hematopoiesis, thereby indicating that enhancement of arterial
patterning is mediated through upregulation of NOTCH signaling.
Together these findings suggest that promotion of arterial
patterning in hPSC cultures could aid in vitro approaches to
instruct definitive hematopoiesis and HSC fate from hPSCs.
[0036] In one embodiment, the disclosure provides a population of
hPSCs or mesoderm cells (e.g. KDR+ mesoderm cells) comprising an
exogenous vector comprising the ETS transcription factor. This
population of cells is capable of differentiation into arterial
hemogenic endothelial cells upon expression of the exogenous
vector. In some embodiments, the exogenous vector includes an
inducible promoter that allows for inducing of the ETS
transcription factor within the cells. Suitable exogenous vectors,
including viral vectors, are discussed more below.
[0037] We expect that the result with ETS1 can be replicated with
other ETS transcriptional factors. In one embodiment of the
invention, the ETS factor is ETS1. In another embodiment of the
invention, the ETS factor is selected from the group consisting of
ETV2, ERG and ETS2. (See Y. Sato, Cell Structure and Function 26:
19-24 (2001), incorporated by reference in its entirety). We note
that the ETS transcription factor FLI1 was not able to induce AHE
from hPSCs (see Example 1).
[0038] In another embodiment, the present disclosure provides a
method of enhancing arterial specification of hemogenic endothelium
in mesoderm cells, the method comprising culturing the mesoderm
cells in defined medium comprising an effective amount of a factor
capable of activating NOTCH signaling to differentiate the mesoderm
cells into arterial hemogenic endothelium (AHE) cells. As
demonstrated in the Examples, the activation of NOTCH signaling
allows for the arterial specification of hemogenic endothelium in
mesoderm cells.
[0039] One goal of the present invention is to produce a population
of arterial type hemogenic endothelial cells (AHEs). Arterial type
cells (AHE) of the present invention are CD144+CD73- DLL4+ HE that
express high level of EFNB2 and NOTCH1 arterial markers. These
cells have broad lympho-myeloid and definitive erythroid
potentials.
[0040] As described in the Examples, arterial type cells are
referred to as having a CD144+CD43- CD73-DLL4+ phenotype.
Applicants note that the vast majority of CD43+ cells are DLL4- by
default. In other words, selection of arterial type
CD144+CD73-DLL4+ phenotype does not typically require CD43
exclusion.
[0041] In one embodiment, the present invention is a population of
arterial hemogenic endothelium cells (AHE) that are
CD144+CD73-DLL4+HE that express high level of EFNB2 and NOTCH1
arterial markers. These cells have broad lympho-myeloid and
definitive erythroid potentials. As described above, definitive
hematopoiesis produces the entire spectrum of adult-type
erythro-myeloid progenitors (EMP), lymphoid cells, and cells
capable of limited engraftment (second wave), and HSCs with
capacity of long-term repopulation of an adult recipient.
[0042] In some embodiments, the population of arterial hemogenic
endothelium cells is produced by expression of an exogenous ETS
factor (e.g. ETS1) in differentiating hPSCs (e.g. by use of an
exogenous vector or exogenous viral vector).
[0043] In another embodiment, the present invention is a method of
making AHE cells and a method of differentiating AHE cells to
obtain cells of interest.
[0044] In another embodiment, methods of enhancing arterial
specification in differentiating hPSCs by activating ERK signaling
are provided. Specifically, the method comprises culturing of human
mesodermal progenitors derived from hPSCs in chemically defined
culture medium containing an effective amount of an activator of
ERK signaling to obtain arterial hemogenic endothelium (AHE) cells.
In one embodiment, the activator for ERK signaling is an inhibitor
of phosphoinositide 3-kinase (PI3K) downstream of VEGF receptor
signaling.
Methods of the Present Invention
[0045] In one embodiment of the present invention, we disclose a
method of enhancing arterial specification in differentiating hPSC.
In general, our method involves increase of ETS factor gene
expression in hPSCs or mesoderm cells (KDR+ cells) by introducing
an ETS transgene, preferably an inducible gene, into a hESC or
mesoderm cell population. The ETS factor is selected from the group
consisting of ETS1, ETV2, ERG and ETS2. In a preferred embodiment,
the ETS factor is ETS1. The ETS factor gene may be obtained by
amplifying the gene cDNA from human PSCs differentiated into
endothelial and blood cells or cDNA clones can be obtained
commercially (e.g. Sino Biological, Origene, etc.). In preferred
embodiments, the ETS transgene is provided within a vector or
plasmid.
[0046] In one embodiment of the present invention, we disclose a
method of enhancing arterial specification in differentiating hPSC.
In general, our method involves increase of ETS gene expression in
hPSCs by introducing an ETS transgene (e.g. ETS1 transgene),
preferably an inducible gene, into a hESC population. A typical ETS
gene may be obtained by amplifying ETS cDNA from human PSCs
differentiated into endothelial and blood cells or cDNA clones can
be obtained commercially (e.g. Sino Biological, Origene, etc.). In
a preferred embodiment, the ETS transgene is an ETS1 transgene. In
another embodiment, the ETS transgene is a selected from the group
consisting ETS1, ETV2, ERG and ETS2 transgenes.
[0047] The transgene can be inserted into the cell via any suitable
method, for example by transfection or transduction.
[0048] In one embodiment, the ETS transgene comprises nucleic acid
sequence able to express the human ETS1 protein (see. GenBank
accession no. NP 001137292). In some embodiments, the ETS1
transgene further comprises a vector capable of expressing the ETS1
transgene within the cell. In some embodiments, the vector
comprises an inducible promoter before the ETS1 transgene.
[0049] In another embodiment, the ETS transgene comprises nucleic
acid sequence able to express the human ETS1 protein (see. GenBank
accession no. NP 001137292), the ETV2 protein (GenBank accession
no. NP 055024), ETS2 protein (GenBank accession no. NP 001243224)
or the ERG protein (GenBank accession no. NP 891548). The ETS
transgene may comprise a vector capable of capable of expressing
the ETS transgene within the cell. In some embodiments, the vector
comprises an inducible promoter before the ETS transgene.
[0050] In some embodiments, the PSCs or mesoderm cells are
transduced with an exogenous vector encoding for the ETS factor,
for example a recombinant vector (recombinant expression vector)
such as a plasmid or viral vector. The exogenous vector allows for
the expression of the ETS factor within the cell, in some examples,
the exogenous vector is an inducible vector allowing for the
controlled expression of the ETS factor within the cells during
different stages of differentiation. In another embodiment, the
PCSs or mesoderm cells are transduced with an exogenous modified
mRNA of the ETS factor. In yet another embodiment, the PSCs or
mesoderm cells are transduced with the ETS factor protein.
[0051] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors." The vector include exogenous genetic material
that allow for the expression of the transgene.
[0052] In some embodiments, the expression vector is a viral
vector. Suitable viral vectors are known in the art and include,
but are not limited to, for example, an adenovirus vector; an
adeno-associated virus vector; a pox virus vector, such as a
fowlpox virus vector; an alpha virus vector; a baculoviral vector;
a herpes virus vector; a retrovirus vector, such as a lentivirus
vector; a Modified Vaccinia virus Ankara vector; a Ross River virus
vector; a Sindbis virus vector; a Semliki Forest virus vector; and
a Venezuelan Equine Encephalitis virus vector. In a preferred
embodiment, the viral vector is a lentiviral vector, an adenovirus
vector or an adeno-associated virus vector.
[0053] In a preferred embodiment, expression of the ETS1 gene is at
the mesoderm stage of development (day 2 of differentiation). (See
Uenishi et al. 2014, incorporated by reference in its entirety, for
typical developmental protocol.)
[0054] In one embodiment, the ETS factor may be inserted (e.g.
transduced) into a hPSC using a vector comprising an inducible
promoter, and the ETS factor may then be induced to be expressed at
the mesoderm stage of development, e.g. day 2 of differentiation.
In another embodiment, mesoderm cells are transduced with a vector
comprising the ETS factor able to be expressed in the mesoderm cell
(e.g. that may or may not be inducible). In some embodiments, the
vector may be transient.
[0055] In one example, a typical vector would include inserting the
ETS1 gene cloned downstream of a conditional promoter such as
TREtight promoter that requires Doxycycline for activation. The
gene could be introduced along with M2rtTA transactivator using,
for example, a lentivirus system, PiggyBac transposon system or a
plasmid.
[0056] Alternatively one can increase ETS factor expression in the
cells by transfecting hPSCs on day 2 of differentiation with ETS
factor modified mRNA. For example, the cells can be transfected
with ETS1 modified mRNA.
[0057] The methods of the present invention would be suitable for
any type of hPSC, including both embryonic stem cells and inducible
pluripotent stem cells. One may wish to confirm the arterial
specification by observing increased formation of CD144+
endothelial cells and induced expression of DLL4 and CXCR4 on
endothelial cells in a dose-dependent manner. Molecular profiling
of endothelial cells isolated on day 4 of differentiation will show
marked increased expression of genes associated with arterial
specification including CXCR4, NOTCH ligand DLL4, NOTCH1, NOTCH4,
HEY1, SOXF group genes (SOX7, SOX17, SOX18), as well CD93 gene
associated with emerging HSCs in the aorta-gonada-mesonephros (AGM)
region in the embryo.
[0058] One may also wish to further differentiate the ETS1 induced
cells. In general, one would consult standard procedures for cell
differentiation to obtain cell populations of hematopoietic cells,
such as T cells, beta-hemoglobin-producing red blood cells and
multipotential myeloid progenitors, including granulocyte,
erythrocyte, megakaryocyte, macrophage (GEMM) and
granulocyte-macrophage (GM) colony-forming cells (CFCs) and mature
myelomonocytic cells. See: [0059] Uenishi, Gene, et al. "Tenascin C
promotes hematoendothelial development and T lymphoid commitment
from human pluripotent stem cells in chemically defined
conditions." Stem cell reports 3.6 (2014): 1073-1084. [0060] Choi,
Kyung-Dal, Maxim Vodyanik, and Igor I. Slukvin. "Hematopoietic
differentiation and production of mature myeloid cells from human
pluripotent stem cells." Nature protocols 6.3 (2011): 296-313.
[0061] Dias, Jessica, et al. "Generation of red blood cells from
human induced pluripotent stem cells." Stem cells and development
20.9 (2011): 1639-1647. [0062] Vodyanik, Maxim A., et al. "Human
embryonic stem cell--derived CD34+ cells: efficient production in
the coculture with OP9 stromal cells and analysis of
lymphohematopoietic potential." Blood 105.2 (2005): 617-626.
[0063] Example 1 discloses that an AHE cell fraction cultured on
DLL4-OP9 cells underwent endothelial-to-hematopoietic transition
and produced blood cells. Our evaluation of lymphoid and CFC
potential revealed that CD144+CD73-DLL4+arterial type hemogenic
endothelium population has a more potent T cell potential.
[0064] Example 1 discloses a number of embodiments of the present
invention. To evaluate effect of ETS1 on arterial programming and
hematopoiesis from hPSCs, H1 human embryonic stem cells (hESC) were
engineered carrying doxycycline (DOX)-inducible ETS1 transgene
(FIG. 8) and differentiated them into endothelial and hematopoietic
cells in chemically defined conditions.sup.26.
[0065] Methods of differentiating hPSCs to mesoderm cells (e.g.
KDR+ mesoderm cells) are known in the art. For example, the hPSCs
may be cultured in chemically defined medium or co-cultured with
OP9 cells as known in the art. For example, in one embodiment the
hPSCs are cultured in chemically defined medium comprising BMP4,
activin A, LiCl and FGF2 on coated plates (e.g. collagen IV coated
or TenC coated) wherein the hPSCs are differentiated into mesoderm
cells (e.g. cells expressing KDR+). A suitable method is described
in Uenishi et al. 2014, incorporated by reference herein.
[0066] In some embodiments, the cells are attached to a culture
plate via extracellular matrix proteins. For example, in one
embodiment, the cells are attached via collagen, fibronectin,
Matrigel.TM. or Tenascin C (TenC). In a preferred embodiment, the
cells are cultured on plates coated with TenC or Collagen IV as
described in Uenishi et al.
[0067] To determine whether ETS1 overexpression promotes arterial
specification, we treated cultures with DOX beginning at mesodermal
stage of development (day 2 of differentiation) and analyzed the
expression of the arterial markers DLL4 and CXCR4.sup.23,27 on
CD144.sup.+ endothelial cells emerging on day 4 of differentiation
(FIG. 1A).
[0068] As shown in FIGS. 1B and 1C, DOX treatment increased
formation of CD144.sup.+ endothelial cells and induced expression
of DLL4 and CXCR4 on endothelial cells in dose-dependent manners.
Molecular profiling of endothelial cells isolated on day 4 of
differentiation, revealed that ETS1 upregulation led to marked
increased expression of genes associated with arterial
specification including CXCR4, EFNB2, NOTCH ligand DLL4, NOTCH1,
NOTCH4, HEY1, SOXF group genes (SOX7, SOX17, SOX18), as well CD93
gene associated with emerging HSCs in AGM region.sup.24,27-34, but
downregulated the expression of NR2F2 and APLNR venous markers
(FIG. 1D). Based on these findings, we concluded that ETS1
upregulation enhances arterial specification from hPSCs.
[0069] Previous studies demonstrated that
VEC.sup.+CD43.sup.-CD73.sup.- HE is lacking arterial marker
CXCR4.sup.37,22 and that hemogenic potential within
VEC.sup.+CD43.sup.-CD73.sup.- could be further enriched by
excluding cells expressing the earliest arterial marker
DLL4.sup.22. However, we found that increased blood production
following ETS1 overexpression was associated with a marked increase
of DLL4.sup.+ fraction within CD144.sup.+CD73.sup.- HE population
that acquires expression of CXCR4, thereby suggesting that
enhancement of definitive hematopoietic program could be attributed
to DLL4.sup.+ HE population that acquires arterial characteristics.
To find out whether arterial type HE has hematopoietic potential we
sorted DLL4.sup.+ and DLL4'' cells and assessed their hematopoietic
potential in defined conditions on matrix in presence of
hematopoietic cytokines. DLL4.sup.+ population in contrast to
DLL4'' failed to produce blood in these conditions. However, when
we cultured DLL4.sup.+ fraction on DLL4-OP9, we found that these
cells undergo endothelial-to-hematopoietic transition and produced
blood cells. Evaluation of lymphoid and CFC potential revealed that
CD144.sup.+CD73.sup.-DLL4.sup.+ arterial type hemogenic endothelium
population has a more potent T cell potential than the DLL4-
population.
[0070] As demonstrated by the Examples, the ETS1 effect is mediated
through the upregulation of DLL4 expression and activation of NOTCH
signaling. Further Example 2 demonstrates that activation of NOTCH
signaling at the mesoderm stage can enhance arterial specification
and increase the production of T cells. Thus, in another
embodiment, the disclosure provides methods of enhancing arterial
specification of hemogenic endothelium by activating NOTCH
signaling in mesoderm cells. In one embodiment, a method of
enhancing arterial specification of hemogenic endothelium in
mesoderm cells is provided, the method comprising culturing the
mesoderm cells in defined medium comprising an effective amount of
a factor capable of activating NOTCH signaling to differentiate the
mesoderm cells into arterial hemogenic endothelium (AHE) cells. In
another embodiment, a method of enhancing arterial specification of
hemogenic endothelium is provided, the method comprising: (a)
introducing into a mesoderm cell population a transgene able to
upregulate NOTCH signaling within the mesoderm cell population and
(b) culturing the mesoderm cells under conditions sufficient to
upregulate NOTCH signaling and differentiate the mesoderm cells to
arterial hemogenic endothelium (AHE) cells.
[0071] Examples of suitable factors that activate NOTCH signaling
include, but are not limited to, for example, NOTCH ligands, feeder
cells expressing NOTCH ligands and solid surfaces with immobilized
NOTCH ligands (e.g. plates coated with NOTCH ligands). Suitable
NOTCH ligands include, for example, DLL1-Fc (which has been
described in other papers as Delta1ext-IgG), Jag1 ligand, and DLL4.
Other examples of suitable factors that activate NOTCH signaling
include an immobilized synthetic molecule that can bind to NOTCH
and sufficiently activate the NOTCH receptor and the ectopic
expression of the active, intracellular domain of NOTCH1
(Notch-ICD).
[0072] The mesoderm cells may be plated onto an NOTCH activation
agent, such as immobilized Notch ligands, to activate NOTCH
signaling (Hadland et al., 2015; Ohishi et al., 2002). Activation
of NOTCH signaling by any means is suitable. For example,
overexpression of the active form of NOTCH receptor or NOTCH
ligands. See [0073] Bigas, A., D'Altri, T., and Espinosa, L.
(2012). The Notch pathway in hematopoietic stem cells. Curr Top
Microbiol Immunol 360, 1-18. [0074] Bigas, A., and Espinosa, L.
(2012). Hematopoietic stem cells: to be or Notch to be. Blood 119,
3226-3235. [0075] Butko, E., Pouget, C., and Traver, D. (2016).
Complex regulation of HSC emergence by the Notch signaling pathway.
Dev Biol 409, 129-138. [0076] Lu, Y F., Cahan, P., Ross, S.,
Sahalie, J., Sousa, P M., Hadland, B. K., Cai, W., Serrao, E.,
Engelman, A N., Bernstein, I D., Daley, G Q. (2016) Engineered
Murine HSCs Reconstitute Multi-lineage Hematopoiesis and Adaptive
Immunity. Cell Report 17, 3178-3192 the contents of which are
incorporated by reference in their entirety.
[0077] In one example, the factor capable of activating NOTCH
signaling is selected from the group consisting of DLL4, DLL1-Fc,
DLL1-expressing feeder cells (e.g. DLL1-expressing OP9 cells),
DLL4-expressing feeder cells (e.g. DLL4-expressing OP9 cells),
plates coated with DLL4-Fc, and plates coated with DLL1-Fc.
[0078] In another embodiment, a method of enhancing arterial
specification in differentiating hPSCs is achieved by activating
ERK signaling. Specifically, the method comprises culturing of
human mesodermal progenitors derived from hPSCs in chemically
defined culture medium containing an effective amount of an
activator of ERK signaling to obtain arterial hemogenic endothelium
(AHE) cells.
[0079] In one embodiment, the activator for ERK signaling is an
inhibitor of phosphoinositide 3-kinase (PI3K) downstream of VEGF
receptor signaling. Suitable PI3K inhibitors are known in the art
and include, but are not limited to, LY294002, GS4894, and
wortmannin, among others. See Hong et al. "Artery/Vein
Specification Is Governed by Opposing Phosphatidylinositol-3 Kinase
and MAP Kinase/ERK Signaling, Current Biology, 16, 1366-1372
(2006), incorporated by reference in its entirety. In a preferred
embodiment, the PI3K inhibitor is LY294002.
[0080] Suitable effective amounts of the PI3K inhibitors include,
but are not limited to about 0.01 .mu.M-about 20 .mu.M, preferably
about 0.1 .mu.M-10 .mu.M. Suitable ranges for specific PI3K
inhibitors include, but are not limited to, for example, for
LY294002 about 0.1 .mu.M-50 .mu.M, preferably about 0.1 .mu.M-10
.mu.M, preferably about 0.5 .mu.M-5 .mu.M, including any amount or
ranges in between, including, for example, about 0.1 .mu.M-20
.mu.M, 0.1 .mu.M-10 .mu.M, 0.1 .mu.M-5 .mu.M, 0.1-3 .mu.M, 0.5
.mu.M-20 .mu.M, 0.5-10 .mu.M, 0.5 .mu.M-5 .mu.M, 0.5-4 .mu.M, 0.5-3
.mu.M; for wortmannin about 0.01 .mu.M to about preferably about
0.01 .mu.M to about 5 .mu.M, including, but not limited to, for
example, 0.05 .mu.M-10 .mu.M, 0.05 .mu.M-5 .mu.M, 0.1 .mu.M to 1.0
.mu.M, 0.1 .mu.M to 2 .mu.M, 0.5 .mu.M to about 10 .mu.M, 0.5 to
about 5 .mu.M, and any amount or range in between, for GS4894,
amount of about 0.1 .mu.M-20 .mu.M, preferably about 0.1 .mu.M-10
.mu.M, including, but not limited to, 0.1 .mu.M-5 .mu.M, 0.1-3
.mu.M, 0.5 .mu.M-20 .mu.M, 0.5-10 .mu.M, 0.5 .mu.M-5 .mu.M, 0.5-4
.mu.M, 0.5-3 .mu.M. In a preferred embodiment, the amount of the
PI3K inhibitor is about 0.1 .mu.M-5 .mu.M, suitably about 0.1
.mu.M-3 .mu.M, and preferably is LY294002.
[0081] The Example demonstrates that the indirect ERK activation
through inhibition of Phosphoinositide 3-kinase (PI3K) downstream
of VEGF receptor signaling, enhances arterial specification of
hPSCs, while inhibition of ERK branch blocks arterial
specification.
[0082] In some embodiments, the differentiation cultures were
treated with the ERK activation factor, e.g. the PI3K inhibitor
(for example LY294002), on days 3 through day 6, demonstrating
enhanced production of DLL4+arterial type HE, including the
DLL4+CXCR4+ fraction. Treatment using a MAPK inhibitor, U0126,
almost completely abrogated formation of DLL4+HE (FIGS. 6B and 6C).
A direct correlation between definitive hematopoiesis efficacy and
arterial specification was seen. When ERK pathways were activated
following HE specification, production of multipotent
CD235a/CD41a-CD45+ hematopoietic progenitors and CFC potential was
dramatically increased, while ERK inhibition abrogated production
of these types of cells (FIG. 6D-6F). In addition, T lymphoid
potential was significantly increased in cultures treated with
LY294002 and entirely abrogated in cultures treated with U0126.
Overall, treatment with a PI3K inhibitor during differentiation of
hPSCs enhanced arterial specification of HE which is essential to
establish a definitive hematopoietic program with lymphomyeloid
potentials from hPSCs.
[0083] The methods described herein induced formation of
DLL4+CXCR4+/- arterial type HE that are highly enriched in
definitive hematopoietic progenitors with T and B lymphoid
potentials. In addition, arterial program activation enhanced
production of CD34+CD43+ hematopoietic progenitors expressing HSC
homing receptor CXCR4, which is typically not present in
hematopoietic progenitors in traditional hESC differentiation
cultures. DLL4 is expressed by HE underlying intra-aortic
hematopoietic clusters in the AGM.sup.47 and recent mouse studies
have revealed significant enrichment in pre-HSCs in the DLL4+
fraction of AGM HE. The in vitro data of Example 1 correlates with
in vivo observation and suggest that induction of HE
arterialization is critical to mimic the proper specification of
definitive hematopoiesis and HSC formation from hPSCs in vitro.
[0084] This disclosure also provides methods of improving T cell
progenitor production. As described herein, PSCs that undergo
arterial programming in lymphoid development results in
significantly improved T cell progenitor production in defined
conditions by applying small molecules to enhance formation of
arterial type HE. Scalable T cell production is essential to
advance translation of iPSC-based immunotherapies into the
clinic.
[0085] In one embodiment, the specification provides a method of
creating a cell population, comprising the steps of (a) obtaining a
cell population of AHE DLL4.sup.+ cells, (b) further
differentiating the AHE DLL4.sup.+ cells into an at least 90% pure
population of T-cells. In some embodiments, step (b) comprises
co-culturing the AHE DLL4.sup.+ cells with stromal cells expressing
NOTCH ligand DLL4 or others (DLL1, DLL3, JAG1 or JAG2) (for
example, OP9-DLL4+ cells) in T cell differentiating medium for an
effective amount of time to differentiate the AHE cells into
T-cells.
[0086] Example 1 further demonstrates methods of enhancing arterial
specification in differentiating hPSC by activating ERK signaling.
The method comprises (a) culturing human mesoderm cells in defined
cell culture medium comprising a factor capable of activating ERK
signaling in a sufficient amount and for a sufficient time to
differentiate the mesoderm cells into arterial hemogenic
endothelium cells (AHE); and (b) obtaining the arterial hemogenic
endothelium cells.
[0087] Suitable methods of obtaining the arterial hemogenic
endothelium cells may be isolating the AHE via expression of cell
surface markers (e.g. CD144+CD73-DLL4+HE) as described herein.
[0088] Suitable methods of differentiating PSCs to mesoderm
progenitor cells are known in the art. In one embodiment, the human
mesoderm cells are obtained from a method comprising culturing
human PSCs in a chemically defined culture medium for about 2 to
about 4 days, whereby a cell population comprising human KDR+
mesoderm cells is obtained. Mesoderm may be obtained in any
conditions and include, but are not limited to, OP9 system or
Uenishi defined system as described in Uenishi et al. 2014, which
is incorporated by reference in its entirety (medium comprising
BMP4, Activin A, LiCl and FGF2 on coated plates under hypoxic
conditions (5% O.sub.2, 5% CO.sub.2). Thus, the method of culturing
AHE is described in FIG. 7, wherein PSCs are cultured to mesoderm
progenitor cells for 1-4 days, at which time the mesoderm
progenitors cells are further cultured in defined cell culture
medium comprising a factor capable of activating ERK signaling to
differentiate the mesoderm cells into AHE cells. The AHE cells
obtained further express one or more arterial markers selected from
the group consisting of EFNB2, NOTCH1, NOTCH 4 and SOX17.
[0089] The terms "defined culture medium," "defined cell culture
medium," "defined medium," and the like, as used herein, indicate
that the identity and quantity of each medium ingredient is known.
As used herein, the terms "chemically-defined culture conditions,"
"fully defined, growth factor free culture conditions," and
"fully-defined conditions" indicate that the identity and quantity
of each medium ingredient is known and the identity and quantity of
supportive surface is known. As used herein, the term
"albumin-free" indicates that the culture medium used contains no
added albumin in any form, including without limitation Bovine
Serum Albumin (BSA) or any form of recombinant albumin.
Standardizing culture conditions by using a chemically defined
culture medium minimizes the potential for lot-to-lot or
batch-to-batch variations in materials to which the cells are
exposed during cell culture. Accordingly, the effects of various
differentiation factors are more predictable when added to cells
and tissues cultured under chemically defined conditions. As used
herein, the term "serum-free" refers to cell culture materials that
do not contain serum or serum replacement, or that contain
essentially no serum or serum replacement. For example, an
essentially serum-free medium can contain less than about 1%, 0.9%,
0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum. "Serum
free" also refers to culture components free of serum obtained from
animal (e.g., fetal bovine) blood or animal-derived materials,
which is important to reduce or eliminate the potential for
cross-species viral or prion transmission. For avoidance of doubt,
serum-containing medium is not chemically defined.
[0090] Suitable defined medium includes, but is not limited to, E8
medium.
[0091] The AHE cells can be identified as CD144+CD43-CD73-DLL4+.
AHE cells are CXCR4.sup.-/-
[0092] In some embodiments, the AHE cells are sorted from the cell
culture.
[0093] Suitably, the PSCs can be selected from the group consisting
of embryonic stem cells (ESCs) or induced pluripotent stem cells
(iPSCs).
Compositions of the Present Invention
[0094] In another embodiment, the present invention is a cell line
created from the methods of the present invention. This hemogenic
cell line will contain an ETS transgene, such as an ETS1 transgene,
and be at least 90, 95% or 99% pure.
[0095] In further embodiments, one would wish to obtain the
following cell lines:
(1) CD144.sup.+CD43-CD73-DLL4+,
[0096] (2) T cells, (3) B cells, (4) Definitive (adult-type) red
blood cells, (5) myeloid progenitors or mature myelomonocytic cells
by use of the methods described herein. As discussed above, the
methods described herein provide an enrichment in T cell
progenitors, for example at least a 3 fold enrichment for T cell
progenitors, alternatively at least a 10 fold, alternatively at
least 25 fold, alternatively at least 50 fold, alternatively at
least 75 fold, alternatively at least 100 fold enrichment of T cell
progenitors. In some embodiments, the methods described herein
using ETS1 have produced about 50 to 100 fold enhancement of T cell
progenitors, e.g. 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100
fold. In other embodiments, the methods provided herein can be used
to obtain the following cell lines: a) CD235a/CD41a.sup.-CD45.sup.+
progenitors, granulocyte-macrophage colony-forming cells (GM-CFC),
granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM), and
erythroid cells expressing .beta.-hemoglobin and BCL11a. In some
embodiments, the methods provide a population of ARE-DLL4.sup.+
cells that have B cell potential.
[0097] As demonstrated in Example 2, the ability to enrich in T
cell progenitors allows for the ability to generate a population of
T cell progenitors and ultimately T cells. The ability to produce
large populations of T cells can be used in combination with the
ability to transduece the T cells and express an engineered
chimeric antigen receptor (CAR) within these cell populations.
These engineered T cells can further be used for treatment of
cancer as a form of cancer therapy. Example 2 demonstrates the
transduction of T cells made by the methods described herein with
exogenous CD19 CAR. These engineered CAR-expressing T cells were
further shown to be able to kill tumor cells (Raji cells, cultured
cell line of lymphoblastoid cells derived from a Burkitt
lymphoma).
[0098] In one embodiment, the CAR expressing T cells can be used to
kill tumor cells. The tumor cells are contacted with the CAR
expressing T cells in an effective amount in order to kill the
tumor cells.
[0099] In another embodiment, the CAR expressing T cells can be
used to treat a subject having cancer. The CAR expressing T cells
can be administered in an effective amount to treat the cancer. The
CAR expressing T cells can be adoptively transferred to the
patient. Suitable engineered CAR for use in treating a subject
having cancer are known in the art and include, CAR that are
specific to a tumor-associated antigen. For example, in one
embodiment, the CAR is a CD19 chimeric antigen receptor.
[0100] Design and methods of making CARs are known in the art and
include, but are not limited to the first, second, third and fourth
generation of CARs. Genetically engineered CARs are contemplated
herein. These genetically engineered receptors, CARs, comprise an
antigen-specific recognition domain that binds to specific target
antigen or cell and a transmembrane domain linking the
extracellular domain to an intracellular signaling domain. Design
and methods of making CAR are known in the art. In one embodiment,
the antigen-specific recognition domain in the extracellular domain
redirect cytotoxicity of the effector cell toward tumor cells. In
Example 2, a CAR expressing CD19, which is expressed on certain
kinds of leukemia or lymphoma, is expressed in T cells derived by
these methods and used to kill tumor cells (Riji cells, which are a
cell line derived from Burkitt Lymphoma patient).
[0101] The T cells produced by the methods herein can be engineered
to express CAR specific for tumor or cancer cells, and used in the
treatment of such cancers. Suitable cancers that can be treated
using the T cells expressing the engineered CAR receptors include,
but are not limited to, hematologic malignancies, for example,
forms of cancer that begin in the cells of blood-forming tissue,
such as the bone marrow, or in the cells of the immune system.
Examples of hematologic cancer include, but are not limited, to,
for example, acute and chronic leukemias, lymphomas, multiple
myeloma and myelodysplastic syndromes.
[0102] The term "treating" can be characterized by one or more of
the following: (a) the reducing, slowing or inhibiting the growth
of tumor cells; (b) preventing the further growth of tumor cells;
(c) reducing or preventing the metastasis of tumor cells within a
patient, and (d) reducing or ameliorating at least one symptom of
the tumor or cancer. In some embodiments, the optimum effective
amounts can be readily determined by one of ordinary skill in the
art using routine experimentation. As used herein, the terms
"effective amount" and "therapeutically effective amount" refer to
the quantity of active therapeutic agent or agents sufficient to
yield a desired therapeutic response without undue adverse side
effects such as toxicity, irritation, or allergic response. The
specific "effective amount" will, obviously, vary with such factors
as the particular condition being treated, the physical condition
of the subject, the type of animal being treated, the duration of
the treatment, the nature of concurrent therapy (if any), and the
specific formulations employed and the structure of the compounds
or its derivatives.
[0103] As used herein "subject" or "patient" refers to mammals and
non-mammals. The term "subject" does not denote a particular age or
sex. In one specific embodiment, a subject is a mammal, preferably
a human. In some embodiments, the subject suffers from a cancer,
particularly a hemotologic malignancy.
Kits
[0104] Aspects of the present disclosure that are described with
respect to methods can be utilized in the context of the
compositions or kits discussed in this disclosure. Similarly,
aspects of the present disclosure that are described with respect
to the compositions can be utilized in the context of the methods
and kits, and aspects of the present disclosure that are described
with respect to kits can be utilized in the context of the methods
and compositions.
[0105] This disclosure provides kits. The kits can be suitable for
use in the methods described herein.
[0106] In one embodiment, the disclosure provides a kit for
culturing a population of AHE cells from human pluripotent cells or
mesoderm cells, the kit comprising (1) defined medium sufficient
for differentiation of the pluripotent cells into mesoderm cells,
(2) an exogenous vector comprising a ETS transcription factor
transgene or mRNA comprising a ETS transcription factor transgene;
and (3) instructions for introducing the ETS transcription factor
into the hPSCs or mesoderm cells and methods for culturing the AHE
cells.
[0107] In another embodiment, the disclosure provides a kit for
culturing AHE cells from mesoderm cells, the kit comprising an
inhibitor of the ERK pathway. In one embodiment, the kit comprises
a PI3K inhibitor. Instructions for methods of culturing are also
provided.
[0108] The following non-limiting examples are included for
purposes of illustration only, and are not intended to limit the
scope of the range of techniques and protocols in which the
compositions and methods of the present invention may find utility,
as will be appreciated by one of skill in the art and can be
readily implemented. The present invention has been described in
terms of one or more preferred embodiments, and it should be
appreciated that many equivalents, alternatives, variations, and
modifications, aside from those expressly stated, are possible and
within the scope of the invention.
EXAMPLES
Example 1: Activation of Arterial Program Drives Development of
Definitive Hemogenic Endothelium with Lymphoid Potential
[0109] This Example shows that activation of arterial program
through ETS1 overexpression or by modulating MAPK/ERK signaling
pathways, at the mesodermal stage of development, dramatically
enhanced formation of arterial type HE expressing DLL4 and CXCR4.
Blood cells generated from arterial HE were more than 100-fold
enriched in T cell precursor frequency and possessed the capacity
to produce B lymphocytes and red blood cells exhibiting high
expression of BCL11a and .quadrature.-globin. Together, these
findings demonstrated that promotion of arterial specification in
cultures provides a novel strategy to generate lymphoid cells and
eventually HSCs from hPSCs.
[0110] De novo production of hematopoietic and lymphoid cells from
in vitro expandable human cells, such as human pluripotent stem
cells (hPSCs) can be used for transplantation and immunotherapies
of hematologic diseases and cancers. Although the feasibility of
generating engraftable hematopoietic cells and T lymphoid cells
from hPSCs has been demonstrated.sup.1-5, further translation of
these technologies from bench-to-bedside requires development of
clinically safe protocols for scalable production of therapeutic
cells in defined physiological conditions. Thus, identifying the
proper molecular pathways guiding specification of multipotential
lymphomyeloid progenitors from hPSCs is essential to advance T
lymphoid cell and HSC manufacturing technologies.
[0111] During development, blood cells and HSCs arise from
hemogenic endothelium (HE). In contrast to the first wave of
primitive hematopoiesis lacking of lymphoid and granulocytic
potential, definitive hematopoiesis produces the entire spectrum of
adult-type erythro-myeloid progenitors (EMP), lymphoid cells, cells
capable of limited engraftment (second wave), and HSCs with the
capacity for long-term repopulation of an adult recipient (third
wave) (reviewed in.sup.6-8). While some definitive hematopoietic
cells such as EMPs can be produced from HE in venous vessels and
capillaries.sup.9-11, production of lymphoid cells and HSCs is
mostly restricted to arterial vasculature.sup.12-16. The lack of
venous contribution to HSCs when considered along with the shared
requirements for Notch, VEGF, and Hedgehog signaling in both
arterial fate acquisition and HSC formation.sup.17-21 suggests that
arterial specification is an essential prerequisite for
establishing of definitive hematopoiesis with lymphoid potential.
Although previous studies demonstrated arterial commitment within
nonHE fraction of hPSC-derived endothelium.sup.22, little is known
about the effect of arterial programming on HE.
[0112] This example investigated how arterial programming affects
specification of definitive HE and hematopoietic cells from hPSCs.
During vascular development, arterial fate is specified following
induction of DLL4 expression.sup.23 initiated by signaling through
an arterial-specific enhancer located within the third intron of
DLL4 that is controlled by ETS factors.sup.24, 25. Here, the
inventors found that arterial program from hPSCs could be enhanced
by overexpression of ETS family transcription factor, ETS1. The
observed boost in arterial programming by ETS1 was associated with
promotion of HE formation with DLL4.sup.+CXCR4.sup.+/- arterial
phenotype and TB lymphoid and definitive erythroid potentials. In
addition, we demonstrated that arterialization of HE and
enhancement of definitive hematopoiesis could be achieved through
modulating of MAPK/ERK pathways. Promoting arterial patterning in
hPSC cultures can be used to aid in vitro approaches to instruct
definitive hematopoiesis with lymphoid and HSC potentials from
hPSCs.
[0113] ETS1 Induction Upregulates SOXF and NOTCH-Signaling
Associated Genes and Enhances Arterial Specification
[0114] To evaluate the effect of ETS1 on arterial programming and
hematopoiesis from hPSCs, we engineered H1 human embryonic stem
cells (hESC) carrying doxycycline (DOX)-inducible ETS1 transgene
(iETS1-hESCs; FIGS. 8A-8D) and differentiated them to endothelial
and hematopoietic cells in chemically defined conditions.sup.26. We
treated cultures with DOX beginning at mesodermal stage of
development (day 2 of differentiation) and analyzed the expression
of the arterial markers DLL4 and CXCR4.sup.23, 27 on CD144.sup.+
(VE-cadherin.sup.+) endothelial cells emerging on day 4 of
differentiation (FIG. 1A). As shown in FIGS. 1B and 1C, DOX
treatment increased formation of CD144.sup.+ endothelial cells and
induced expression of DLL4 and CXCR4 on endothelial cells in a
dose-dependent manners. Molecular profiling of endothelial cells
isolated on day 4 of differentiation, revealed that ETS1
upregulation led to a marked increase expression of genes
associated with arterial specification-associated genes including
CXCR4, NOTCH ligand DLL4, NOTCH1, NOTCH4, HEY1, SOXF group genes
(SOX7, SOX17, and SOX18), as well as CD93, a gene associated with
emerging HSCs in AGM region.sup.24, 27-34, but downregulated the
expression of NR2F2 and APLNR venous markers (FIG. 1D). The
upregulation of arterial genes was confirmed by RT-qPCR (FIG. 1E).
Based on these findings, we concluded that ETS1 upregulation
enhances arterial specification from hPSCs.
[0115] ETS1 induction at mesodermal stage enhances definitive
hematopoiesis from hESCs. To determine how ETS1 affects
hematopoiesis and whether it's effect on hematopoiesis is
associated with activation of arterial program in HE, we treated
cells with DOX in a stepwise fashion as depicted in FIG. 2A. In our
differentiation system, hPSCs undergo a stepwise progression toward
APLNR+PDGFR.alpha.+ mesoderm with hemangioblast colony forming
cells (HB-CFCs) that reflects primitive hematopoiesis,
KDR.sup.highPDGFR.alpha..sup.low/- hematovascular mesodermal
progenitors with definitive hematopoietic potential,
CD144.sup.+CD43.sup.-CD73.sup.- definitive HE and CD43.sup.+
hematopoietic progenitors which include CD235a.sup.+CD41a.sup.+
erythromegakaryocytic progenitors (E-MK) and
CD235a/41a.sup.-CD45.sup.+/- hematopoietic progenitors with
lin.sup.-CD34.sup.+CD90.sup.+CD38.sup.-CD45RA.sup.- hematopoietic
stem progenitor cells (HSPC) phenotype.sup.26, 35-37 (FIG. 9A).
Stepwise DOX treatment experiments, revealed that upregulation of
ETS1 during hematovascular mesoderm and HE specification on days
2-4 or 2-6 of differentiation produced the most profound effect on
generation of CD43.sup.+ and CD45.sup.+ hematopoietic progenitors
(FIG. 2B-2C). Importantly, ETS1 upregulation increased the
proportion of multipotential CD235a/CD41a.sup.- CD45.sup.+
progenitors and GEMM-CFCs (FIGS. 2D and 2E). Typically, colonies
from DOX+ were much larger as compared to DOX- cultures (FIG. 2E).
ETS1 induction before mesoderm establishment (days 0-2) or post-HE
stage (days 6-8) had minimal effect or inhibited blood production
(FIG. 2B-2E). Thus, we concluded that the window for the optimal
effect of ETS1 on hematopoiesis coincided with amplification of
arterial program by ETS1.
[0116] To define which type of hematopoiesis is affected by ETS1
overexpression, we evaluated the effect of DOX treatment on i)
hemangioblast (HB) CFCs that reflect the primitive wave of
hematopoiesis.sup.37,38 and ii) on T lymphocytes and
.beta.-hemoglobin-producing red blood cells that reflect definitive
hematopoiesis (FIG. 3A).sup.37, 39. When cultures were treated with
DOX on day 2 of differentiation, we observed on day 3 a significant
decrease in APLNR+PDGFR.alpha..sup.+ primitive mesodermal cells and
HB-CFCs compared to control. This effect was more profound when
cultures were treated with DOX from day 0 through day 3 to ensure
maximum ETS1 overexpression on day 3 (FIG. 3B and FIGS. 9B-9C). In
contrast, DOX treatment starting from day 2 of differentiation
increased formation of CD144.sup.+CD43.sup.-CD73.sup.- definitive
HE.sup.37 on day 5 of differentiation in a dose-dependent manner
(FIG. 3C). To determine the effect of ETS1 upregulation on T cell
potential, we collected CD43.sup.+ cells from DOX+ and DOX-
cultures and subcultured on OP9-DLL4 stromal cells. Although cells
collected from both conditions generated a similar percentage of
CD5.sup.+CD7.sup.+ and CD4.sup.+CD8.sup.+ T lymphoid cells (FIG.
3D), CD43.sup.+ cells from DOX+ cultures produced a dramatically
(.about.8 fold) greater number of T lymphoid cells per 10.sup.4 of
CD43 cells (FIG. 3E). In addition, we found that CD43 cells
collected following DOX treatment and cultured in erythroid
conditions upregulated adult .beta.-hemoglobin and BCL11a genes
associated with definitive erythropoiesis.sup.40 (FIG. 3F).
Overall, these studies suggest that ETS1 upregulation suppresses
primitive and promotes definitive hematopoiesis from hPSCs, most
likely through enhancement of definitive HE specification at the
mesodermal stage.
[0117] ETS1 Overexpression Promotes Definitive Hematopoiesis
Through NOTCH-Mediated Signaling
[0118] To determine whether ETS1 induction promotes definitive
hematopoietic program in a cell-autonomous or non-autonomous
manner, we mixed tdTomato (tdT) transgenic H1 hESC with iETS1 H1
hESCs and analyzed hematopoietic development from chimeric cultures
with and without induction of ETS1 expression (FIG. 4A).
[0119] These studies revealed that ETS1 upregulation enhanced the
production of CD43.sup.+ hematopoietic progenitors, including
CD45.sup.+ progenitors, from both, tdT.sup.+ and tdT.sup.- cells
(FIG. 4B and FIG. 10). When cells were collected and assayed for
CFC potential, the number of hematopoietic colonies in both
tdT.sup.+ and tdT.sup.- were increased following DOX treatment
(FIG. 4C). Interestingly, endothelial cells in D4 tdT.sup.-
fraction expressed greater levels of DLL4 as compared to tdT.sup.+
cells (FIG. 4D).
[0120] These results suggest that ETS1 overexpression expands
DLL4-expressing arterial endothelial cells, and promotes definitive
hematoendothelial program through upregulation of NOTCH
signaling.
[0121] To confirm the role of NOTCH activation in promoting
definitive hematopoiesis by ETS1, we evaluated hematopoiesis
following ETS1 upregulation in the presence of NOTCH signaling
inhibitor DAPT, and DLL4 neutralizing antibodies (FIG. 4E). As
shown in FIG. 4F-4I, treatment of hESC cultures with NOTCH
signaling inhibitor DAPT, or DLL4 antibodies, abrogated effect of
ETS1 upregulation on hematopoiesis, thereby confirming the
important role of DLL4 expression and NOTCH activation in
ETS1-mediated promotion of definitive hematoendothelial
program.
[0122] ETS1 Overexpression Induces HE with DLL4.sup.+CXCR4.sup.+
Arterial Phenotype.
[0123] Although previous studies found that DLL4.sup.+ endothelial
cells in hPSC cultures have reduced hematopoietic potential as
compared to DLL4.sup.- cells.sup.22, 41, we noticed that increased
definitive hematopoietic cell production following ETS1
overexpression was correlated with marked increase of DLL4.sup.+
and DLL4.sup.+CXCR4.sup.+ fraction within the
CD144.sup.+CD43.sup.-CD73.sup.- HE population (FIGS. 5A and 5B),
thereby suggesting that enhancement of the definitive hematopoietic
program could be attributed to DLL4.sup.+ HE population that
acquires arterial characteristics, as determined by analysis of
EFNB2, SOX17 and NOTCH1 arterial markers by RT-qPCR (FIG. 5E). To
determine whether arterial type HE has hematopoietic potential, we
sorted DLL4.sup.+ and DLL4.sup.- cells and assessed blood formation
from them following 5 days secondary culture on OP9-DLL4 (FIG. 5D).
Although DLL4.sup.+ produced a relatively lower number of
CD43.sup.+ cells, the proportion of multipotential
CD235a/CD41a.sup.-CD45.sup.+ progenitors was greater in DLL4.sup.+
cultures compared to DLL4.sup.- (FIG. 5F). Hematopoietic
progenitors collected from DLL4.sup.+ HE also produced a greater
number of multipotential CFC-GM and -GEMM (FIG. 5G) and generated
erythroid cells with substantially higher expression of adult
.beta.-hemoglobin and BCL11a (FIG. 5H). Importantly, the most
significant difference was observed in the lymphoid potentials of
DLL4.sup.+ and DLL4.sup.- HE (FIG. 5I-5K). As shown in FIG. 5I,
only DLL4.sup.+ cells had B cell potential. While both DLL4.sup.+
and DLL4.sup.- cells possessed T cell potential (FIG. 5J), the
limiting dilution analysis revealed more than a 100-fold enrichment
in T cell progenitors in DLL4.sup.+ fraction. Interestingly, we
have previously shown that in contrast to fetal liver HSCs,
PSC-derived hematopoietic progenitors have decreased expression of
HSC homing receptor CXCR4.sup.42. As demonstrated in FIG. 5C,
following ETS1 induction, not only HE, but CD34.sup.+CD45.sup.+
hematopoietic progenitors upregulated CXCR4 expression.
[0124] Together, these data suggest that arterial specification of
HE is associated with acquisition of definitive hematopoietic
program. To further characterize DLL4.sup.+ arterial HE, we
evaluated hematopoietic potential of CXCR4.sup.+ and CXCR4.sup.-
cells (FIG. 11A). As shown in FIGS. 11B-11E, both CXCR4.sup.+ and
CXCR4.sup.- fractions of DLL4.sup.+ HE cells generated
multipotential CFCs and T cells. However, a more than 3-fold
enrichment in the T cell progenitors was observed in blood cells
generated from CXCR4.sup.+ cells.
[0125] Promotion of Arterial Specification of HE and Definitive
Hematopoiesis by Modulation of MAPK/ERK Signaling
[0126] Arterial specification in the embryo is modulated by
multiple pathways, including MAPK/ERK signaling. It has been shown,
that indirect ERK activation through inhibition of Phosphoinositide
3-kinase (PI3K) downstream of VEGF receptor signaling, enhances
arterial specification in zebrafish, while inhibition of ERK branch
blocks arterial specification.sup.43, 44. To determine whether
modulating MAPK/ERK signaling affects arterial specification of HE
from hPSCs, we treated differentiation cultures on days 3 through
day 6 with PI3K inhibitor LY294002, or MEK1 and MEK2 inhibitor
U0126 (FIG. 6A). Indeed, we revealed that treatment with LY294002
enhanced production of DLL4.sup.+ arterial type HE, including the
DLL4.sup.+CXCR4.sup.+ fraction, while U0126 treatment almost
completely abrogated formation of DLL4.sup.+ HE (FIGS. 6B and 6C).
We also observed a direct correlation between definitive
hematopoiesis efficacy and arterial specification. When ERK
pathways were activated following HE specification, production of
multipotent CD235a/CD41a.sup.-CD45.sup.+ hematopoietic progenitors
and CFC potential was dramatically increased, while ERK inhibition
abrogated production of these types of cells (FIG. 6D-6F). In
addition, T lymphoid potential was significantly increased in
cultures treated with LY294002 and entirely abrogated in cultures
treated with U0126. Overall, these observations provide additional
evidence for our hypothesis that enhancing arterial specification
of HE is essential to establish a definitive hematopoietic program
with lymphomyeloid potentials from hPSCs.
[0127] NOTCH and SOXF-mediated transcriptional program is activated
in DLL4.sup.+ arterial HE To determine the molecular program
associated with establishing arterial HE, we performed RNAseq
analysis of DLL4.sup.+ and DLL4.sup.- HE. As a basis for the
analysis, genes that were differentially expressed in a 3-way
Bayesian model involving DLL4.sup.+ vs DLL4.sup.- wild type HE and
DLL4.sup.+ vs DLL4.sup.-iETS1 HE from DOX cultures (FIG.
12-Supplementary Table S1) were used. The transcriptional network
relevant to the observed responses was visualized as described in
Methods. Every node of the network reflects both regulon-level
signal strength related to a particular transcription factor and
the change in mRNA level of the transcript of the gene encoding
that transcription factor. The relative abundance of mRNA
expression in these networks was coded as a node size, while color
density represents enrichment (red) or depletion (blue) of known
targets of that transcription factor (regulon members) among the
differentially expressed genes. As shown in FIG. 7A, the increased
expression and regulon activity for NOTCH1, SOXF (SOX17, SOX18),
KLF5 and BCL6B genes was a distinct feature of DLL4.sup.+ arterial
HE from wild type and iETS1 hESCs in DOX cultures, although these
features were more pronounced in iETS1 DLL4.sup.+ HE. As previously
shown, proinflammatory signaling plays an important role in HSC
development.sup.45, 46. Interestingly, the regulons of NFKB1 and
IRF6 factors were activated in DLL4.sup.+ cells suggesting that
arterialization of HE is associated with activation of
proinflammatory signaling. Despite ETS1 overexpression in DOX
cultures, ETS1 regulon signal in iETS1-DLL4.sup.+ HE was poor. This
is consistent with our findings that the effect of ETS1 is
primarily mediated through upregulation of signaling from NOTCH1
and likely SOXF transcription factors, rather than from any
immediate activity of ETS1. Overall, these studies suggest that
activation of arterial program in HE is primarily driven by the
NOTCH and SOXF-driven transcriptional programs.
[0128] In present example, the inventors demonstrated that
definitive hematopoiesis from hPSCs could be promoted through
activation of arterial program in HE through a number of
mechanisms: overexpression of transcription factors ETS1, which has
the capacity to activate arterial-specific enhancer in the third
intron of DLL4 gene.sup.24,25, through modulation of MAPK/ERK
signaling by small molecules, or through upregulation of NOTCH
signaling. These approaches induced formation of
DLL4.sup.+CXCR4.sup.+/- arterial type HE that is highly enriched in
definitive hematopoietic progenitors with T and B lymphoid
potentials. In addition, arterial program activation enhanced
production of CD34.sup.+CD43.sup.+ hematopoietic progenitors
expressing HSC homing receptor CXCR4, which is typically not
present in hematopoietic progenitors in traditional hESC
differentiation cultures.sup.42. DLL4 is expressed by HE underlying
intra-aortic hematopoietic clusters in the AGM.sup.47 and recent
mouse studies have revealed significant enrichment in pre-HSCs in
the DLL4.sup.+ fraction of AGM HE.sup.48. Thus, our in vitro
findings correlate with in vivo observation and suggest that
induction of HE arterialization is critical to mimic the proper
specification of definitive hematopoiesis and HSC formation from
hPSCs in vitro.
[0129] Discovering the important role of arterial programming in
lymphoid development from PSCs, allowed us to significantly improve
T cell progenitor production in defined conditions by applying
small molecules to enhance formation of arterial type HE. Scalable
T cell production is essential to advance translation of iPSC-based
immunotherapies into the clinic. However, in vivo studies from
ETS1-induced cultures have failed to show the evidence of long-term
engraftable cells (data not shown). Molecular profiling and
functional studies of PSC-derived phenotypical HSCs in human and
mouse system have revealed multiple pathway deficiencies in in
vitro generated cells as compared to their in vivo counterparts,
including lacking of Notch-signaling signature, and deficiency of
HOXA and AP-1 complex genes that are functioning independently of
arterial programming.sup.1, 42, 49-51. In addition, studies in
zebra fish revealed that HSC specification is also regulated by
mechanisms uncoupled from arterial patterning.sup.21,52,53. Thus,
arterial specification of HE per se may not be sufficient for HSC
formation. Further exploration of the interplay between mechanisms
coupled and uncoupled with arterial specification, and deciphering
kernels for the gene regulatory network required for HSC
development, will be essential for further advancing HSC generation
for clinical purposes.
[0130] Experimental Procedures
[0131] hESC Lines Maintenance and Hematopoietic Differentiation
[0132] H1 hESCs were obtained from WiCell Research Institute
(Madison, Wis.). H1 hESC line, the iETS1 H1 hESC line and the
tdTomato H1 hESCs line were maintained on Matrigel.TM. in
mTeSR1.TM. medium. Cells were passaged every 3-4 days using 0.5 mM
EDTA in PBS. The hESC lines were differentiated on ColIV coated
plate as previously described in details.sup.26.
[0133] Construction of Vectors and Generation of iETS1 and tdTomato
H1 hESC Lines
[0134] Human ETS1 cDNA was cloned into PiggyBac transposon vector
(Transposagen) downstream of TREtight promoter of
pTRE-P2A-Venus-EF1.alpha.-Zeo plasmid, and then co-electroporated
with pEF1.alpha.-M2rtTA-T2A-Puro and transposase plasmid into H1
hESCs using Amaxa.RTM. human stem cell nucleofector kit 2 (Lonza)
(FIG. 8). The colonies were selected in Zeocin (0.5-1 .mu.g/ml,
Invitrogen) and Puromycin (0.5-1 .mu.g/ml, Sigma) for 10-15 days
and the resistant clones were screened by Venus expression under a
fluorescence microscope with DOX treatment. The tdTomato cDNA was
cloned downstream of EF1.alpha. promoter of a pRMCE-EF1.alpha.-Zeo
plasmid and into H1 hESCs. 3 days after electroporation, cells were
treated with Zeocin (0.5-1 .mu.g/ml, Invitrogen). After 10-15 days,
tdTomato positive surviving colonies were picked out and expanded
in each well of a 12 well plate.
[0135] Hemangioblast (HB)-CFC and Hematopoietic CFC Assay
[0136] HB-CFCs were detected as described previously.sup.54.
HB-CFCs were detected using the semisolid colony-forming serum-free
medium (CF-SFM) containing 40% ES-Cult M3120 methylcellulose (2.5%
solution in IMDM, Stem Cell Technologies), 25% StemSpan.TM.
serum-free expansion medium (SFEM, Stem Cell Technologies), 25%
human endothelial serum-free medium (ESFM, Invitrogen), 10% BIT
9500 supplement (Stem Cell Technologies), GlutaMAX.TM. (1/100
dilution, Invitrogen), Ex-Cyte.TM. (1/1000 dilution, Millipore),
100 .mu.M MTG, 50 .mu.g/ml ascorbic acid and 20 ng/ml FGF
(Peprotech). Hematopoietic CFCs were detected using serum
containing H4435 MethoCult with FGF, SCF, IL-3, IL-6 and EPO (Stem
Cell Technologies).
[0137] Assessment of Hematopoietic Potential of DLL4.sup.- and
DLL4.sup.+CXCR4.sup.+/- HE.
[0138] The iETS1 DOX-treated cells from day 5 of culture were
dissociated into single cells by treatment with 1.times.TrypLE and
stained with DLL4-PE, CD144-PerCPVio700, CD43-APC and CD73-BV421
antibodies and then sorted using a FACSAria II cell sorter (BD
Biosciences) for isolation of DLL4.sup.+ and DLL4.sup.- HE. For
isolation of CXCR4.sup.+ and CXCR4.sup.-DLL4.sup.+ HE, cells were
stained with DLL4-PE, CD144-PerCPVio700, CXCR4-PEVio770, CD73-APC
and CD43-APCVio770 antibodies and sorted using a FACSAria cell
sorter (BD Biosciences). Isolated day 5
DLL4.sup.+CD144.sup.+CD73.sup.-CD43.sup.- and
DLL4.sup.-CD144.sup.+CD73.sup.-CD43.sup.- HE, or
CXCR4.sup.+DLL4.sup.+CD144.sup.+CD73.sup.-CD43.sup.- and
CXCR4.sup.-DLL4.sup.+CD144.sup.+CD73.sup.-CD43.sup.- HE were
cultured at a concentration of 4.times.10.sup.4 cells per well on a
monolayer of Mitomycin C (Cayman Chemicals)-pretreated OP9 cells
expressing human DLL4 (OP9-DLL4) in medium with SCF (50 ng/ml), TPO
(50 ng/ml), IL-3 (long/ml) and IL-6 (20 ng/ml, all cytokines from
PeproTech) in 6-well plates as we described previously.sup.55.
After 5 days of cultures on OP9-DLL4, cells were harvested and
analyzed by flow cytometry. The floating CD43.sup.+ cells were
collected for T cell or RBC differentiation.
[0139] T and B Cell Differentiation
[0140] T cell differentiation of hESC-derived hematopoietic
precursors was performed on the OP9-DLL4 in T cell differentiated
medium consisting of .alpha.-MEM (Gibco) supplemented with 20% FBS
(Hyclone), 5 ng/ml FLT3L, 5 ng/ml IL-7 and 10 ng/ml SCF (all from
PeproTech) as described previously.sup.26. For B cell
differentiation, sorted DLL4.sup.+ and DLL4.sup.- HE cells were
cocultured on OP9 for 4 weeks in .alpha.MEM medium containing 20%
FBS, FLT3L (5 ng/ml, PerproTech) and IL7 (5 ng/ml, PerproTech).
Cultures were fed with complete media changes weekly. Presence of B
cells was confirmed via staining with CD19 APC (Miltenyl Biotech)
and CD10 PE (BD Biosciences) antibodies.
[0141] Limiting Dilution Assay to Determine Frequency of T Cell
Progenitors
[0142] Limiting Dilution Assays were conducted with the floating
cells (CD43.sup.+) collected from day 5+5 cultures (DLL4.sup.+ and
DLL4.sup.- HE or CXCR.sup.+ and CXCR.sup.-DLL4.sup.+ HE) on
OP9-DLL4. Row A of a 96-well plate received 500 cells/well, and
each subsequent row afterwards received half the previous row (e.g.
Row B contained 250, Row C contained 125, etc, until eventually Row
H contained 3-4 cells). Wells were assessed 2 weeks later under
microscope for presence of floating blood cells and by
flow-cytometry for CD5.sup.+CD7.sup.+ expressing cells. Extreme
limiting dilution analysis was conducted using a previously
established algorithm.sup.56.
[0143] RBC Differentiation
[0144] Floating CD43.sup.+ cells at day 9 of differentiation were
collected and cultured in RBC differentiated medium consisting of
SFEM (serum free expansion medium, Stem Cell Technologies)
supplemented with 0.3% Ex-Cyte (Millipore), 1 mg/ml
Holo-Transferrin (Sigma), 10 .mu.M dexamethasone, 20 ng/ml insulin
(Sigma), 2 U/ml EPO, 50 ng/ml SCF, 50 ng/ml TPO, 5 ng/ml IL-3 and
long/ml IL-6 on ultra-low attachment 6 well plate (Corning). After
2 days, cells were cultured on OP9 cells using the same medium
without TPO, IL-3 and IL-6 for 20 days with weekly passage. Media
changes were performed every 2 days as described
previously.sup.57.
[0145] Flow Cytometry
[0146] Cells were analyzed using MACSQuant Analyzer (Miltenyi
Biotec) and FlowJo software (Tree star). Cell surface staining
utilized the antibodies listed in FIG. 12.
[0147] DAPT and DLL4 Antibody Treatment
[0148] Notch signaling was blocked by DAPT (.gamma.-secretase
inhibitor/GSI, 10 .mu.M, Cayman Chemicals) or DLL4 blocking
antibody (10 .mu.g/ml, Creative BioLabs)
[0149] LY294002 and U0126 Treatment
[0150] MAPK/ERK pathway was activated using LY294002 (2 .mu.M,
Cayman Chemicals) and was inhibited using U0126 (1 .mu.M, Cayman
Chemicals)
[0151] Quantitative Real Time PCR
[0152] Total RNA was isolated using the RNeasy.RTM. Plus Micro Kit
(Qiagen). RNA was reverse transcribed into cDNA using Oligo(dT)
with ImProm-II Reverse Transcriptase (Promega). Real time
quantitative PCR was performed in triplicates using SYBR Advantage
qPCR Premix (Clontech) on Mastercycler.RTM. ep realplex
(Eppendorf). Gene expression was evaluated as DeltaCt relative to
the RPL13A gene. Primer sequences are listed in FIG. 13.
[0153] Western Blot
[0154] Cells were suspended in lysis buffer containing 17 mM Tris
pH 8.0, 50 mM NaCl, 0.3% Triton X-100, 0.3% NP-40 and a protease
inhibitor cocktail tablet (Roche, Switzerland). ETS1 and GAPDH were
detected with anti-ETS1 (Santa Cruz Biotechnology, sc-55581) and
anti-GAPDH (Santa Cruz Biotechnology) antibodies, respectively.
[0155] Low Level RNAseq Data Processing.
[0156] Day 4 KDR.sup.+CD144.sup.+ or day 5 DLL4.sup.+ and
DLL4.sup.- RE cells were isolated from DOX treated and untreated
cultures as described above. Total RNA was prepared with RNeasy
Plus Micro Kit (Qian). RNA purity and integrity was evaluated by
capillary electrophoresis on the Bioanalyzer 2100 (Agilent
Technologies, Santa Clara, Calif.). Samples were then prepared for
sequencing using the Ligation Mediated Sequencing (LM-Seq)
protocol, according to the published guidelines.sup.58. Final
sample libraries were quantitated with the Life Technologies Qubit
fluorometer and sequenced on the Illumina HiSeq 3000
(SY-401-1003-PRE). Base-calling and demultiplexing were completed
with the Illumina Genome Analyzer Casava Software, version 1.8.2.
Following quality assessment and filtering for adapter molecules
and other sequencing artifacts, the remaining sequence reads were
aligned to transcript sequences corresponding to hg19 human genome
annotation. Bowtie v 1.1.2 was used, allowing two mismatches in a
25 bp seed, and excluding reads with more than 200
alignments.sup.66. RSEM v 1.3.0 was used to estimate isoform or
gene relative expression levels in units of "transcripts per
million" (tpm), as well as posterior mean estimate of the "expected
counts" (the non-normalized absolute number of reads assigned by
RSEM to each isoform/gene).sup.10,67. R statistical environment (R
core team, 2014) was used throughout all of the stages of
downstream data analysis.
[0157] Downstream RNAseq Bioinformatics Analysis: Testing for
Differential Expression
[0158] R statistical environment (R core team, 2014) was used for
all stages of downstream data analysis. The entire set of libraries
were pre-normalized as a pool to equilibrate 65th percentile of the
counts distribution, using the quantile scaling routine from EBSeq
package.sup.59. For each gene, maximal counts across all the
samples were plotted and the genes representing the lower mode of
the distribution were filtered out (only genes that have at least
40 counts in at least 1 sample were retained), restricting the set
of genomic features to 12,898. Additional median scaling was
applied to the pre-filtered set of genes. Differential expression
was called using EBSeq with 10 iterations. The EBSeq's default
procedure of filtering low-expressed genes was suppressed by
setting the QtrmCut parameter to zero. Genes with an assigned value
of Posterior Probability of Differential Expression above 0.95 were
preliminarily selected. Genes that passed two additional filters
were selected for downstream analysis: 1) fold change cutoff of 1.5
and 2) expression level should exceed 20.sup.th percentile of
genome-wide distribution of expression values in libraries
representing the condition with a larger mean expression of that
gene.
[0159] Visualization of Transcriptional Network
[0160] Using the known transcription-target relationships obtained
by combining largely complementary data from HTRIdb.sup.60 and
CellNet.sup.61, we generated combined sets of targets for 950
transcriptional regulators that involve 130,855 individual
transcription factor (TF)-target interactions, for regulon
analysis. To visualize the cascades of transcriptional regulation
that involve influence of active TFs on TF-encoding genes, we
restricted the overall regulatory network to TF-target
relationships that involve TF-encoding target genes. The resulting
"transcriptional backbone" network has 837 regulators and reduced
by over an order of magnitude (12,372) individual TF-target
relationships. To isolate the relevant part of this network, we
selected 8 transcription factors that demonstrated their activation
in response to Dox according to our regulon analysis, and
restricted the network to edges that have either outgoing or
incoming connections related to the 8 selected factors. The
resulting subnetwork had 59 nodes and 175 edges. Accession codes:
The RNAseq data has been deposited in Gene Expression Omnibus under
accession number GSE96815. Access code while in private status:
ktwzakaohxsbryv.
Example 2: NOTCH Activation at the Definitive Mesoderm Stage
Facilitates Efficient Generation of T Cells with High Proliferation
Potential from Human Pluripotent Stem Cells
[0161] Adoptive T cell therapies show promise in the treatment of
several types of blood cancers. Developing off-the-shelf T cell
products will further advance immunotherapies to the clinic and
broaden their application. Human pluripotent stem cells (hPSCs)
offer the potential to serve as a versatile and scalable source of
T cells for immunotherapies, which could be coupled with genetic
engineering technologies to meet specific clinical needs. However,
production and expansion of T cells from hPSCs remains inefficient.
In order to improve T cell production from hPSCs it is essential to
identify cell subsets that are highly enriched in T cell
progenitors, and those stages of development at which NOTCH
activation induces the most potent T cells. Previously, we have
developed both OP9-based and chemically defined systems for
hematopoietic differentiation from iPSCs (Vodyanik et al., 2006 and
Uenishi et. al, 2014). In these differentiation systems, hPSCs
undergo stepwise progression towards APLNR+PDGFR.alpha.+ mesoderm
with hemangioblast colony forming cells (HB-CFCs) that reflect
primitive hematopoiesis (day 3 of differentiation),
KDRbrightPDGFRalow/- hematovascular mesodermal progenitors (HVMP)
with definitive hematopoietic potential, VE-cadherin
(VEC)+CD43-CD73- HE with definitive hematopoietic potential (day
4-5 of differentiation) and CD43+ hematopoietic progenitors,
including CD235+CD41+ erythromegakaryocytic progenitors (E-MkP) and
CD235a/41a-CD45+/- multipotent hematopoietic progenitors (MHP) that
have lin-CD34+CD90+CD38-CD45RA- hematopoietic stem progenitor cells
(HSPC) phenotype (days 6-8 of differentiation) (FIG. 14).
[0162] To assess the stage at which NOTCH activation induces the
most potent T cells, we isolated the aforementioned blood forming
populations and cultured them in T cell conditions on OP9-DLL4.
This example shows that Day 3 APLNR+PDGFRa+ primitive posterior
mesodermal cells did not produce T cells, while all downstream
subsets except CD235a+CD41a+CD45-cells do produce T cells when
cultured on OP9-DLL4. As determined by limiting dilution assay, the
highest frequency of T cell precursors was detected from day 4 HVMP
(1 in 14 HVMP). The frequency of T cell precursors in day 5 HE and
day 8 HPs was 1 in 16 HEs and 1 in 20 MHPs, respectively (FIG.
15).
[0163] In addition, this example demonstrates that T cells
generated from HVMPs have the capacity to proliferate for 8 weeks,
in comparison to HEs and MHPs subsets, which could only be expanded
for 4-5 weeks (FIG. 16). T cell differentiation from hPSCs
proceeded through a CD5+CD7+ progenitor stage that eventually
transitions into CD8+CD4+ double positive cells (.about.90%),
CD3+TCRa/b+ and CD3+TCRg/d+ cells. To confirm T cell development,
the genomic DNA of the hematopoietic cells from OP9-DLL4 cultures
was analyzed for the presence of T cell receptor (TCR)
rearrangements. This analysis demonstrated the presence of multiple
PCR products of random V-J and D-J rearrangements at the .beta.
locus and V-J rearrangements at the .gamma. locus, indicative of a
polyclonal T lineage repertoire. In vitro generated T-cells were
functionally active and proliferated upon stimulation with PMA and
IL-2 (FIG. 17). Upon activation, the cells express
CD25+CD69+(.about.73%) markers, cytokines (IFN-.gamma..about.87%,
TNFa.about.22%, IL2.about.34.5%) and cytolytic proteins
(Perforin.about.37%). This Example also demonstrated that CD5+CD7+
T cell progenitors can be genetically modified to express CD19 CARs
and eventually differentiate into CAR T cells with significant
cytotoxic effect on Raji cells (FIG. 18). The methods may be used
for protocols for the efficient off-the shelf production and
expansion of PSC-derived CAR T cells for treating hematologic
malignancies.
[0164] Each publication, patent, and patent publication cited in
this disclosure is incorporated by reference herein in its
entirety. The present invention is not intended to be limited to
the foregoing examples, but encompasses all such modifications and
variations as come within the scope of the appended claims.
REFERENCES
[0165] 1. Sugimura, R. et al. Haematopoietic stem and progenitor
cells from human pluripotent stem cells. Nature 545, 432-438
(2017). [0166] 2. Rahman, N. et al. Engineering the haemogenic
niche mitigates endogenous inhibitory signals and controls
pluripotent stem cell-derived blood emergence. Nat Commun 8, 15380
(2017). [0167] 3. Ledran, M. H. et al. Efficient hematopoietic
differentiation of human embryonic stem cells on stromal cells
derived from hematopoietic niches. Cell Stem Cell 3, 85-98. (2008).
[0168] 4. Wang, L. et al. Generation of hematopoietic repopulating
cells from human embryonic stem cells independent of ectopic HOXB4
expression. J Exp Med 201, 1603-1614. Epub 2005 May 1609. (2005).
[0169] 5. Vizcardo, R. et al. Regeneration of human tumor
antigen-specific T cells from iPSCs derived from mature CD8(+) T
cells. Cell Stem Cell 12, 31-36 (2013). [0170] 6. Lin, Y., Yoder,
M. C. & Yoshimoto, M. Lymphoid progenitor emergence in the
murine embryo and yolk sac precedes stem cell detection. Stem Cells
Dev 23, 1168-1177 (2014). [0171] 7. Medvinsky, A., Rybtsov, S.
& Taoudi, S. Embryonic origin of the adult hematopoietic
system: advances and questions. Development 138, 1017-1031 (2011).
[0172] 8. Tober, J., Maijenburg, M. W. & Speck, N. A. Taking
the Leap: Runx1 in the Formation of Blood from Endothelium. Curr
Top Dev Biol 118, 113-162 (2016). [0173] 9. Goldie, L. C., Lucitti,
J. L., Dickinson, M. E. & Hirschi, K. K. Cell signaling
directing the formation and function of hemogenic endothelium
during murine embryogenesis. Blood 112, 3194-3204 (2008). [0174]
10. Li, W., Ferkowicz, M. J., Johnson, S. A., Shelley, W. C. &
Yoder, M. C. Endothelial cells in the early murine yolk sac give
rise to CD41-expressing hematopoietic cells. Stem Cells Dev 14,
44-54. (2005). [0175] 11. Frame, J. M., Fegan, K. H., Conway, S.
J., McGrath, K. E. & Palis, J. Definitive Hematopoiesis in the
Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium
Independently of Circulation and Arterial Identity. Stem Cells 34,
431-444 (2016). [0176] 12. Rybtsov, S., Ivanovs, A., Zhao, S. &
Medvinsky, A. Concealed expansion of immature precursors underpins
acute burst of adult HSC activity in foetal liver. Development 143,
1284-1289 (2016). [0177] 13. de Bruijn, M. F., Speck, N. A.,
Peeters, M. C. & Dzierzak, E. Definitive hematopoietic stem
cells first develop within the major arterial regions of the mouse
embryo. The EMBO journal 19, 2465-2474 (2000). [0178] 14. North, T.
et al. Cbfa2 is required for the formation of intra-aortic
hematopoietic clusters. Development 126, 2563-2575 (1999). [0179]
15. Yzaguirre, A. D. & Speck, N. A. Insights into blood cell
formation from hemogenic endothelium in lesser-known anatomic
sites. Dev Dyn (2016). [0180] 16. Gordon-Keylock, S., Sobiesiak,
M., Rybtsov, S., Moore, K. & Medvinsky, A. Mouse extraembryonic
arterial vessels harbor precursors capable of maturing into
definitive HSCs. Blood 122, 2338-2345 (2013). [0181] 17. Lawson, N.
D., Vogel, A. M. & Weinstein, B. M. sonic hedgehog and vascular
endothelial growth factor act upstream of the Notch pathway during
arterial endothelial differentiation. Dev Cell 3, 127-136 (2002).
[0182] 18. Lawson, N. D. et al. Notch signaling is required for
arterial-venous differentiation during embryonic vascular
development. Development 128, 3675-3683 (2001). [0183] 19. Gering,
M. & Patient, R. Hedgehog signaling is required for adult blood
stem cell formation in zebrafish embryos. Dev Cell 8, 389-400
(2005). [0184] 20. Kim, P. G. et al. Signaling axis involving
Hedgehog, Notch, and Scl promotes the embryonic
endothelial-to-hematopoietic transition. Proc Natl Acad Sci USA
110, E 141-150 (2013). [0185] 21. Burns, C. E. et al. A genetic
screen in zebrafish defines a hierarchical network of pathways
required for hematopoietic stem cell emergence. Blood 113,
5776-5782 (2009). [0186] 22. Ditadi, A. et al. Human definitive
haemogenic endothelium and arterial vascular endothelium represent
distinct lineages. Nat Cell Biol 17, 580-591 (2015). [0187] 23.
Chong, D. C., Koo, Y., Xu, K., Fu, S. & Cleaver, O. Stepwise
arteriovenous fate acquisition during mammalian vasculogenesis. Dev
Dyn 240, 2153-2165 (2011). [0188] 24. Sacilotto, N. et al. Analysis
of D114 regulation reveals a combinatorial role for Sox and Notch
in arterial development. Proc Natl Acad Sci USA 110, 11893-11898
(2013). [0189] 25. Wythe, J. D. et al. ETS factors regulate
Vegf-dependent arterial specification. Dev Cell 26, 45-58 (2013).
[0190] 26. Uenishi, G. et al. Tenascin C promotes hematoendothelial
development and T lymphoid commitment from human pluripotent stem
cells in chemically defined conditions. Stem cell reports 3,
1073-1084 (2014). [0191] 27. Yamamizu, K. et al. Convergence of
Notch and beta-catenin signaling induces arterial fate in vascular
progenitors. J Cell Biol 189, 325-338 (2010). [0192] 28. Bertrand,
J. Y. et al. Characterization of purified intraembryonic
hematopoietic stem cells as a tool to define their site of origin.
Proc Natl Acad Sci USA 102, 134-139. Epub 24 Dec. 2027. (2005).
[0193] 29. Gale, N. W. et al. Haploinsufficiency of delta-like 4
ligand results in embryonic lethality due to major defects in
arterial and vascular development. Proc Natl Acad Sci USA 101,
15949-15954 (2004). [0194] 30. Duarte, A. et al. Dosage-sensitive
requirement for mouse D114 in artery development. Genes Dev 18,
2474-2478 (2004). [0195] 31. Kim, I., Saunders, T. L. &
Morrison, S. J. Sox17 dependence distinguishes the transcriptional
regulation of fetal from adult hematopoietic stem cells. Cell 130,
470-483. Epub 27 Jul. 2026. (2007). [0196] 32. Yurugi-Kobayashi, T.
et al. Adrenomedullin/cyclic AMP pathway induces Notch activation
and differentiation of arterial endothelial cells from vascular
progenitors. Arterioscler Thromb Vasc Biol 26, 1977-1984 (2006).
[0197] 33. Corada, M. et al. Sox17 is indispensable for acquisition
and maintenance of arterial identity. Nat Commun 4, 2609 (2013).
[0198] 34. Villa, N. et al. Vascular expression of Notch pathway
receptors and ligands is restricted to arterial vessels. Mech Dev
108, 161-164 (2001). [0199] 35. Choi, K. et al. Hematopoietic and
endothelial differentiation of human induced pluripotent stem
cells. Stem Cells 27, 559-567 (2009). [0200] 36. Vodyanik, M. A.,
Thomson, J. A. & Slukvin, II Leukosialin (CD43) defines
hematopoietic progenitors in human embryonic stem cell
differentiation cultures. Blood 108, 2095-2105 (2006). [0201] 37.
Choi, K.-D. et al. Identification of the Hemogenic Endothelial
Progenitor and Its Direct Precursor in Human Pluripotent Stem Cell
Differentiation Cultures. Cell Rep 2, 553-567 (2012). [0202] 38.
Kennedy, M., D'Souza, S. L., Lynch-Kattman, M., Schwantz, S. &
Keller, G. Development of the hemangioblast defines the onset of
hematopoiesis in human ES cell differentiation cultures. Blood 109,
2679-2687. (2007). [0203] 39. Kennedy, M. et al. T lymphocyte
potential marks the emergence of definitive hematopoietic
progenitors in human pluripotent stem cell differentiation
cultures. Cell Rep 2, 1722-1735 (2012). [0204] 40. Sankaran, V. G.
et al. Human fetal hemoglobin expression is regulated by the
developmental stage-specific repressor BCL11A. Science 322,
1839-1842 (2008). [0205] 41. Ayllon, V. et al. The Notch ligand
DLL4 specifically marks human hematoendothelial progenitors and
regulates their hematopoietic fate. Leukemia 29, 1741-1753 (2015).
[0206] 42. Salvagiotto, G. et al. Molecular profiling reveals
similarities and differences between primitive subsets of
hematopoietic cells generated in vitro from human embryonic stem
cells and in vivo during embryogenesis. Experimental Hematology 36,
1377-1389 (2008). [0207] 43. Hong, C. C., Peterson, Q. P., Hong, J.
Y. & Peterson, R. T. Artery/vein specification is governed by
opposing phosphatidylinositol-3 kinase and MAP kinase/ERK
signaling. Curr Biol 16, 1366-1372 (2006). [0208] 44. Herbert, S.
P. et al. Arterial-venous segregation by selective cell sprouting:
an alternative mode of blood vessel formation. Science 326, 294-298
(2009). [0209] 45. Espin-Palazon, R. et al. Proinflammatory
signaling regulates hematopoietic stem cell emergence. Cell 159,
1070-1085 (2014). [0210] 46. Li, Y. et al. Inflammatory signaling
regulates embryonic hematopoietic stem and progenitor cell
production. Genes Dev 28, 2597-2612 (2014). [0211] 47. Richard, C.
et al. Endothelio-mesenchymal interaction controls runx1 expression
and modulates the notch pathway to initiate aortic hematopoiesis.
Dev Cell 24, 600-611 (2013). [0212] 48. Hadland, B. K. et al. A
Common Origin for B-1a and B-2 Lymphocytes in Clonal
Pre-Hematopoietic Stem Cells. Stem cell reports 8, 1563-1572
(2017). [0213] 49. Ng, E. S. et al. Differentiation of human
embryonic stem cells to HOXA+ hemogenic vasculature that resembles
the aorta-gonad-mesonephros. Nat Biotechnol (2016). [0214] 50. Dou,
D. R. et al. Medial HOXA genes demarcate haematopoietic stem cell
fate during human development. Nat Cell Biol 18, 595-606 (2016).
[0215] 51. McKinney-Freeman, S. et al. The transcriptional
landscape of hematopoietic stem cell ontogeny. Cell Stem Cell 11,
701-714 (2012). [0216] 52. Monteiro, R. et al. Transforming Growth
Factor beta Drives Hemogenic Endothelium Programming and the
Transition to Hematopoietic Stem Cells. Dev Cell (2016). [0217] 53.
Robert-Moreno, A. et al. Impaired embryonic haematopoiesis yet
normal arterial development in the absence of the Notch ligand
Jagged1. EMBO J 27, 1886-1895 (2008). [0218] 54. Vodyanik, M. A. et
al. A mesoderm-derived precursor for mesenchymal stem and
endothelial cells. Cell Stem Cell 7, 718-729 (2010). [0219] 55.
Choi, K. D. et al. Identification of the hemogenic endothelial
progenitor and its direct precursor in human pluripotent stem cell
differentiation cultures. Cell Rep 2, 553-567 (2012). [0220] 56.
Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis
for comparing depleted and enriched populations in stem cell and
other assays. J Immunol Methods 347, 70-78 (2009). [0221] 57. Dias,
J. et al. Generation of red blood cells from human induced
pluripotent stem cells. Stem Cells Dev 20, 1639-1647 (2011). [0222]
58. Hou, Z. et al. A cost-effective RNA sequencing protocol for
large-scale gene expression studies. Sci Rep 5, 9570 (2015). [0223]
59. Leng, N. et al. EBSeq: an empirical Bayes hierarchical model
for inference in RNA-seq experiments. Bioinformatics 29, 1035-1043
(2013). [0224] 60. Bovolenta, L. A., Acencio, M. L. & Lemke, N.
HTRIdb: an open-access database for experimentally verified human
transcriptional regulation interactions. BMC Genomics 13, 405
(2012). [0225] 61. Cahan, P. et al. CellNet: network biology
applied to stem cell engineering. Cell 158, 903-915 (2014). [0226]
62. Shannon, P. et al. Cytoscape: a software environment for
integrated models of biomolecular interaction networks. Genome Res
13, 2498-2504 (2003). [0227] 63. Slukvin, II (2013). Hematopoietic
specification from human pluripotent stem cells: current advances
and challenges toward de novo generation of hematopoietic stem
cells. Blood 122, 4035-4046. [0228] 64. Slukvin, II (2016).
Generating human hematopoietic stem cells in vitro-exploring
endothelial to hematopoietic transition as a portal for stemness
acquisition. FEBS Lett. [0229] 65. Vo, L. T., and Daley, G. Q.
(2015). De novo generation of HSCs from somatic and pluripotent
stem cell sources. Blood 125, 2641-2648. [0230] 66. Langmead B,
Trapnell C, Pop M, Salzberg S L. Ultrafast and memory-efficient
alignment of short DNA sequences to the human genome. Genome
Biology. 2009; 10(3):R25. doi:10.1186/gb-2009-10-3-r25. [0231] 67.
Li B, Dewey C N. RSEM: accurate transcript quantification from
RNA-Seq data with or without a reference genome. BMC
Bioinformatics. 2011; 12:323. doi:10.1186/1471-2105-12-323.
Sequence CWU 1
1
18119DNAArtificial Sequencesynthetic 1catccacaag acagcgggg
19219DNAArtificial Sequencesynthetic 2ctcgtcggca tctggcttg
19322DNAArtificial Sequencesynthetic 3ccagcctcaa aatcgtggcc cg
22422DNAArtificial Sequencesynthetic 4tttgatggcc cgaagccact cg
22520DNAArtificial Sequencesynthetic 5agaatccaga cctgcacaac
20619DNAArtificial Sequencesynthetic 6gccggtactt gtagttggg
19722DNAArtificial Sequencesynthetic 7caatgtggat gccgcagttg tg
22821DNAArtificial Sequencesynthetic 8cagcaccttg gcggtctcgt a
21918DNAArtificial Sequencesynthetic 9ggcacctttg ccacactg
181020DNAArtificial Sequencesynthetic 10cactggtggg gtgaattctt
201120DNAArtificial Sequencesynthetic 11gcctgtggag caagatgaat
201216DNAArtificial Sequencesynthetic 12gcgggcttga ggttgt
161321DNAArtificial Sequencesynthetic 13cttcaagctc ctgggaaatg t
211425DNAArtificial Sequencesynthetic 14gcagaataaa gcctatcctt gaaag
251520DNAArtificial Sequencesynthetic 15aaccccagca cttaagcaaa
201620DNAArtificial Sequencesynthetic 16ggaggtcatg atccccttct
201723DNAArtificial Sequencesynthetic 17cctggaggag aagaggaaag aga
231825DNAArtificial Sequencesynthetic 18ttgaggacct ctgtgtattt gtcaa
25
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