# Patent

## Title
ETS2 and MESP1 generate cardiac progenitors from fibroblasts

## Abstract

A method for modulating cell differentiation capabilities using heterologous gene expression. Some embodiments of the invention relate to a method for inducing a cardiac progenitor cell by delivering a reprogramming factor to the cell, wherein the reprogramming factor comprises ETS2 or a combination of ETS2 and Mesp1.

## Background
This application claims priority to U.S. Provisional Patent Application Ser. No. 61/339,509, filed on Mar. 5, 2010, entitled ETS2 AND MESP1 GENERATE CARDIAC PROGENITORS FROM FIBROBLASTS, the entire content of which is hereby incorporated by reference.


FIELD OF THE INVENTION
One aspect of the present invention relates generally to the field of cell differentiation, and more specifically to a strategy for cardiovascular tissue regeneration via the isolation, renewal, and directed differentiation, of fibroblasts into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.
BACKGROUND
Damage to mammalian heart tissue frequently results in the loss of large numbers of cardiac cells, including mature cardiac cells, pacemaker cells, smooth muscle, and endothelial cells. Although there is some indication that cardiac cells can be regenerated in humans (Bergmann et al., 2009), the mechanism is not well understood and the process does not appear to proceed rapidly enough to repair common types of cardiac damage such as ischemia, infarction, trauma, or injury due to toxins or viral infections. Therefore, a central goal of experimental cardiac medicine has been the development of a means for regenerating cardiac cells which have been lost due to cardiac damage. Studies of the mechanisms behind the embryonic cardiogenesis have been conducted, with the aim of replicating cardiogenesis in vitro or in vivo for the purposes of regenerating damaged tissue.
Recent research has identified multipotent (Isl1+) cardiovascular progenitor (MICP) cells, which are capable of differentiating to form mature cardiac tissue. MICP cells derived from embryonic stem (ES) cells which can give rise to endothelial, cardiac, and smooth muscle cells, have been isolated (Moretti et al., 2006). Genetic studies have shown that these MICP cells express Isl1, Nkx2.5 and Flk1.
Model systems for investigating cardiogenesis include the ascidian Ciona intestinalis (Beh et al., 2007). Lineage studies have shown that the adult Ciona heart is derived from two founder cells that express Ci-Mesp, a basic helix-loop-helix (bHLH) transcription factor, and also Ci-Ets1/2 (Imai et al., 2004; Satou et al., 2004). In addition, ascidian orthologs of the conserved heart specification genes NK4 (tinman Nkx2.5), GATAa (pannier/GATA4/5/6), Hand and Hand-like (Imai et al., 2003; Davidson, 2007; Davidson and Levine, 2003; Satou et al., 2004) are expressed. Ci-Mesp-knockdown embryos did not develop heart primordia, and target inhibition of Ets1/2 activity also blocked heart specification and the expansion of the heart field. Similarly, murine homologues of Ci-Mesp, Mesp1 and Mesp2 are expressed in the early mesoderm fated to become cranio-cardiac mesoderm (Saga et al., 2000). Only the Mesp1/Mesp2 double-knockout mouse lacked any cardiac mesoderm (Saga et al., 1999; Kitajima et al., 2000), indicating a role for these genes in directing the appearance of cardiac progenitors in higher vertebrates. Redundancies of Mesp genes have made further study in embryos a daunting task.
What is needed in the art is a method of inducing cardiogenesis for the purpose of regenerating cardiac cells for the use in the treatment of damaged cardiac tissue. Reprogramming of human somatic cells into pluripotent cells by a limited number of transcriptional factors important for maintaining self renewal and pluripotency has been reported by Yamanaka's, Thomson's and Daley's groups (Takahashi et al., 2007; Yu et al., 2007; Park et al., 2008). One aspect of the present invention provides a means of reprogramming the somatic cells and directed differentiation into cardiac progenitor cells. Therefore, one embodiment of this application provides a way to test a unique regulatory paradigm that ETS2 and Mesp1 are transformative, and unlike NKX2.5 and ISL1, convert non-embryonic normal human dermal fibroblasts (NHDFs) into primary cardiac progenitors. Another aspect of the present application was to elucidate the role of Mesp1 in the regulatory hierarchy directing the appearance of cardiac progenitors.
SUMMARY
One embodiment of the present invention relates to the modulation of cell differentiation capabilities using heterologous gene expression. Some embodiments of the invention relate to a method for inducing a cardiac progenitor cell by delivering a reprogramming factor to the cell, wherein the reprogramming factor comprises ETS2 or a combination of ETS2 and Mesp1.
A further embodiment of the present invention provides a cardiac progenitor cell which has been induced by reprogramming a somatic cell, wherein reprogramming comprises delivery of a reprogramming factor comprising the ETS2 gene to the somatic cell. The somatic cell may be a normal human dermal fibroblast (NHDF), and the reprogramming factor may be ETS2 or Mesp1, or a combination thereof.
Still a further embodiment of the present invention provides a method of reprogramming a somatic cell to produce a cardiac progenitor cell, wherein reprogramming comprises delivery of a reprogramming factor comprising the ETS2 gene to the somatic cell. The somatic cell may be an NHDF, and the reprogramming factor may be ETS2 or Mesp1, or a combination thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 shows a schematic map of lentivirus with the insertion of ETS2 and ELK4 full length DNA coding sequences. Functional elements and abbreviations: constitutive Ef-1alpha promoter, multiple cloning sites (MCS), independent ribosome entry site (IRES) from human polio virus, coding sequence for enhanced green fluorescence protein (eGFP), Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), activation domain (AD), DNA-binding ETS domain. These plasmids were generated by standard recombinant DNA cloning techniques and then used to make lentiviruses to infect normal human dermal fibroblasts (NHDFs);
FIG. 2 shows (top panel) NHDFs treated with empty lentivirus (pWPI-GFP), pWPI-ELK4-GFP lentivirus, or pWPI-ETS2-GFP lentivirus, and (bottom panel) expression levels of GAPDH, NANOG, OCT3/4, SOX2 in cells infected with empty virus, virus carrying ETS2, virus carrying ELK4, or uninfected NHDF passage 3 (NHDF-P3);
FIG. 3 shows immunofluorescence staining with antibody to a stem cell marker NANOG in NHDF-P3 cells infected with lentivirus carrying ETS2, but not ELK4 or empty vector (top panel). A protein blot using anti-ETS2 antibody revealed expression of ETS2 in NHDFs infected with lentivirus carrying ETS2 for 4 weeks, while NHDFs infected with empty lentivirus showed no expression (bottom left panel). Immunofluorescence staining with anti-ETS2 antibody confirmed the induced expression in the cells infected with ETS2 lentivirus (bottom right panel);
FIG. 4 shows induction of stem cell markers NANOG, OCT3/4, SOX2, REX1 and c-MYC in stem cell-like colonies after 4 weeks in culture. The fluorescent colors (colors not shown in the figures) are as follows: DAPI (blue), GFP (green), NANOG and OCT3/4 (red), a mixture of all three colors is observed in the “merge” panels. Color fluorescent images are available upon request;
FIG. 5 shows flow cytometry demonstrating the percent of H9 human embryonic stem cells (A), uninfected NHDFs (B), or ETS2-infected cells (C) that stained for the stem cell surface marker SSEA-3. Negative controls, black lines; SSEA-3 staining, gray lines;
FIG. 6 shows flow cytometry demonstrating the percent of H9 human embryonic stem cells (A), uninfected NHDFs (B), or ETS2-infected cells (C) that stained for the stem cell surface marker Tra-1-81. Negative controls, black lines; Tra-1-81 staining, gray lines;
FIG. 7 shows images of EPS cells after infection with Mesp1 lentivirus. Cells were stained with DAPI to visualize nuclei (left panels) and with specific antibodies to ISL1, NKX2.5, GATA4, MEF2C, TNT and MHC3 to visualize indicated cardiac progenitor proteins (right panels). Panels in the middle show phase contrast images of cells. The fluorescent colors are as follows: DAPI (blue), protein-specific staining (red). Color fluorescent images are available upon request;
FIG. 8 shows that expression of NKX2.5, ISL1, GATA4, MEF2C, TBX5, MHC3, TNT, MLC2, CX43 and CX45, detected using RT-PCR, was only induced by the combination of ETS2 and Mesp1. Neither ETS2 or Mesp1 alone are capable of inducing these cardiogenic genes in NHDFs;
FIG. 9 shows induction of sequential de novo cardiac progenitor program. NHDFs were infected with ETS2 lentivirus, grown for 4 weeks, infected with Mesp1 lentivirus and cultured for 7 days, then aggregated by the hang-drop procedure and plated on a gelatin-coated dish;
FIG. 10 shows activation of cardiac progenitor program gene expression, measured by fluorescence of the reporter protein Red-Tomato which is expressed only when the cardiac progenitor factor NKX2.5 is expressed. The fluorescent colors (colors not shown in the figures) are as follows: GFP (green), Red (red), a mixture of the two colors is observed in the “merge” panel;
FIG. 11 shows flow cytometry of cardiac progenitor cells obtained from NHDFs by infection with ETS2 and Mesp1 lentivectors and sorted for either GFP or GFP and reporter protein Red-Tomato;
FIG. 12 shows display of endothelial and cardiac cell surface markers CD31, CD34 and CD144 in cardiac progenitor cells after 9 days in culture; and
FIG. 13 shows the data on rhythmic beating in reprogrammed cardiac progenitor cells. EPS cells were infected with Mesp1 lentivirus, as well as with virus carrying a myosin heavy chain promoter driving the puromycin resistance gene. To select cardiac progenitor cells resistant to antibiotic, cells were treated with 50 ug/ml puromycin. After 9 days, rhythmic beating in the cell cultures was observed and captured by video microscopy and converted into MPEG videos. Beats per cultured aggregate per dish were counted for 20 sec and then multiplied by 3, resulting in beats per one minute. Three separate measurements were done per aggregate in a tissue culture dish or well.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
One embodiment of the present invention relates to the modulation of cell differentiation using heterologous gene expression. Some embodiments of the invention relate to a method for inducing a cardiac progenitor cell by delivering a reprogramming factor to the cell, wherein the reprogramming factor comprises ETS2 or a combination of ETS2 and Mesp1.
An embodiment of the present invention provides a method for inducing a cardiac progenitor cell by reprogramming a somatic cell, wherein reprogramming comprises delivery of a reprogramming factor comprising a single heterologous gene to the somatic cell. The somatic cell may be a fibroblast, preferably a normal human dermal fibroblast. The heterologous gene may be ETS2. The heterologous gene may comprise the human ETS2 coding sequence (SEQ ID NO:9) or the ETS2 gene (SEQ ID NO:7), or the heterologous gene may encode the human ETS2 protein sequence (SEQ ID NO:8). The induced stem-like cell may exhibit cardiogenesis or other characteristics of cardiac progenitor cells as a result of programming, including the expression of cardiac progenitor factors such as NKX2.5, ISL1, MEF2C, dHAND and GATA4, or rhythmic beating.
Another embodiment of the present invention provides a method for inducing a cardiac progenitor cell by reprogramming a somatic cell, wherein reprogramming comprises delivery of a reprogramming factor comprising two heterologous genes to the somatic cell. The somatic cell may be a fibroblast, preferably a normal human dermal fibroblast. The heterologous genes may be ETS2 and Mesp1. The heterologous genes may comprise the human ETS2 coding sequence (SEQ ID NO:9), the ETS2 gene (SEQ ID NO:7), or a DNA sequence encoding the human ETS2 protein sequence (SEQ ID NO:8) and the mouse Mesp1 coding sequence (SEQ ID NO:6), the mouse Mesp1 gene (SEQ ID NO:4), or a DNA sequence encoding the mouse Mesp1 protein sequence (SEQ ID NO:5). The induced stem-like cell may exhibit cardiogenesis or other characteristics of cardiac progenitor cells as a result of programming, including the expression of cardiac progenitor factors such as NKX2.5, ISL1, MEF2C, dHAND and GATA4, or rhythmic beating.
Yet another embodiment of the present invention, reprogramming of a somatic cell, may be accomplished by delivering a reprogramming factor to the somatic cell using a recombinant vector. The reprogramming factor may also be delivered using a lentiviral transduction system to express the reprogramming factor in the somatic cell. In these embodiments, the reprogramming factor may be ETS2 and Mesp1.
A further embodiment of the present invention provides a somatic cell which has been reprogrammed, wherein reprogramming comprises delivery of a reprogramming factor comprising a single heterologous gene or multiple heterologous genes to the somatic cell. The somatic cell may be a fibroblast, preferably a normal human dermal fibroblast. The heterologous genes may be ETS2 or the multiple heterologous genes may be ETS2 and Mesp1. The induced stem-like cell may exhibit cardiogenesis or or other characteristics of cardiac progenitor cells as a result of programming, including the expression of cardiac progenitor factors such as NKX2.5, ISL1, MEF2C, dHAND and GATA4, or rhythmic beating.
Example 1
Selection of a Reprogramming Factor
It was noted that the ETS domain (FIG. 1), a highly conserved DNA-binding domain, is capable of binding to a 5′-GGA(A/T)-3′ DNA core motif found on the promoters of many stem cell marker genes. The expression of ETS2 is linked to immortalization of cells, mediation of oncogenesis, and enhancement of telomerase activity.
ETS2 and ELK4, an ETS family gene homologous to ETS2 in its DNA-binding region, were transduced using lentiviral vectors into NHDF-P3. Within one week, fibroblasts transduced with lentiviral vectors containing ETS2 were replaced with highly proliferative small rounded cells. These highly proliferative cells were not observed in controls transduced with empty lentivirus, or in the fibroblasts transduced with lentiviral vectors containing ELK4 (FIG. 2-3).
Example 2
Lentiviral Transduction System
FIG. 1 shows a schematic map of lentivirus with the insertion of ETS2 (SEQ ID NO:9) and ELK4 (SEQ ID NO:3) DNA coding sequences. Note that only ETS2 has a pointed domain. These plasmids were used to make lentiviruses to infect NHDFs. The ETS2 full-length sequence (SEQ ID NO:7) comprises an ETS2 coding sequence (SEQ ID NO:9) encoding an ETS2 protein sequence (SEQ ID NO:8). The ELK4 full-length sequence (SEQ ID NO:1) comprises an ELK4 coding sequence (SEQ ID NO:3) encoding a protein sequence (SEQ ID NO:2).
The empty lentivirus vector pWPI-eGFP was a gift from Dr. D. Trono (Ecole Polytechnique Fédérale de Lausanne, Switzerland). cDNA for cloning the human ETS2 and ELK4 genes (Clone IDs 3852274 and 4364006) were obtained from Open Biosystems, whereas the Mesp1 cDNA was a gift from Dr. Y. Saga (National Institute of Genetics, Mishima, Japan). The consensus Kozak sequence for initiation of protein translation and the epitope HA-tag were added respectively to the 5′- and 3′-ends of ETS2, ELK4 and Mesp1 coding sequences by PCR cloning.
Lentivirus packing and infection proceeded as follows: Seeded 293FT cells in 6-cm dishes were transfected with either pWPI-eGFP, or pWPI-ELK4-eGFP (human ELK4 coding sequence, SEQ ID NO:3), or pWPI-ETS2-eGFP (human ETS2 coding sequence, SEQ ID NO:9), or pWPI-Mesp1-eGFP (mouse MesP1 sequence, SEQ ID NO:6), or SMPU-alphaMHC/puro-Rex1/Blast (gift from Dr. M. Mercola, Burnham Institute for Medical Research, La Jolla, Calif.). 4.5 ug of either construct was mixed in a solution of 458 ul of serum-free Dulbecco-modified Eagle medium (DMEM) and 27.5 ul of Fugene (Roche), 2.8 ug of packing vector psPAX2 and 1.9 ug of envelope vector pMD2.G for 25 min at room temperature. Afterwards the mix was added to 293FT cells grown in DMEM, phenol red-free (Invitrogen) supplemented with 10% FBS (heat-inactivated), 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate and 6 mM L-glutamate. After 24-26 hrs in culture, medium with viral particles was collected for 3 days and used for infection.
Collected medium was used to infect NHDFs grown in Fibroblast Basal Medium (FBM, Lonza) until 80% confluency. Before transfection, cells were reseeded in 6-cm Petri dishes at a density of 2.5×106 cell/dish, the medium was changed to StemPro and the viral particles and polybrene (8 ug/ml final concentration) were added. To increase the efficiency of infection, the procedure was repeated within 48 hours. All cells were grown at 37° C. and 5% CO2.
Example 3
Gene Expression in Reprogrammed Cells
FIG. 2 shows that ETS2 lentivirus but not ELK4 lentivirus induced stem cell appearance and the induction of stem cell marker proteins, NANOG, OCT3/4 and SOX2 within 7 days of culture. The top panel of FIG. 2 shows NHDFs (Lonza, USA, cc-2509) grown under FBM, supplemented (supplements provided by Lonza) with hFGF-beta, insulin, gentamycin/amphotericin and 2% FBS, to a confluence of ca. 80% before viral infection. Empty pWPI-eGFP and pWI-ELK4-eGFP and pWPI-ETS2-eGFP lentiviruses were used to separately infect NHDF-P3. Infected and non-infected NHDF-P3 cells were grown under human induced pluripotent medium StemPro hES SFM (Invitrogen) over collagen-coated Petri dishes for 7 days. The green fluorescent protein cloned into the lentiviral vectors was expressed in the infected cells, as revealed by the green fluorescence microscopy. During this culture period, morphological changes were observed in which ETS2-infected NHDFs changed their appearance from elongated pleomorphic fibroblastic shapes to rounded “stem-like cells”. In comparison, NHDFs infected with an empty or ELK4 lentiviral vectors did not alter cell shape.
The bottom panel of FIG. 2 shows the reverse transcription PCR (RT-PCR) analysis of reprogrammed cells. Cells were washed in chilled PBS and RNA was isolated with Qiagen RNeasy Kit. RNA was transcribed using MMLV reverse transcriptase (Invitrogen), and PCR amplification (30 cycles) was performed for GAPDH, NANOG, OCT3/4, SOX2 (refer to Table 1 for primer sets) using LA16 polymerase mix. This enzyme mix was prepared using 15 ul of KlenTaq1 (25 units/ul, Ab Peptides. St Louis, Mo.) and 1 ul Pfu (2.5 units/ul, Stratagene, La Jolla, Calif.). Amplified DNA samples were then electrophoresed on 2% agarose gel and ethidium bromide staining revealed the induced expression of stem cell marker genes NANOG, OCT3/4 and SOX2 in ETS2-infected cells but not in NHDF-P3 or cells infected with either ELK4 or empty lentiviruses.







TABLE 1







Primers for Cardiac Progenitor Study











SEQ

Prod-



ID

uct


Primer
NO
Seq
size













GapdhFP crtl exp
11
TGTTGCCATCAATGACCCCTT
202


GapdhRP ctrl exp
12
CTCCACGACGTACTCAGCG



 


hNanogFP ctrl
13
CAGAAGGCCTCAGCACCTAC
225


exp





hNanogRP ctrl
14
TATAGAAGGGACTGTTCCAGGC



exp





 


hOct 3⁄4FP ctrl
15
CTTGAATCCCGAATGGAAAGGG
206


exp





hOct 3⁄4 RP ctrl
16
CCTTCCCAAATAGAACCCCCA



exp





 


Sox2 HPB F
17
TGGACAGTTACGCGCACAT
215


Sox2 HPB R
18
CGAGTAGGACATGCTGTAGGT



 


hRex1FP ctrl exp
19
GCTGACCACCAGCACACTAGGC
298


hRex1RPctrl exp
20
TTTCTGGTGTCTTGTCTTTGCCCG



 


c-Myc HPB F
21
AGGCGAACACACAACGTCTT



c-Myc HPB R
22
TTGGACGGACAGGATGTATGC



 


hKlf4FP ctrl exp
23
ATGGCTGTCAGCGACGCGCTGCTC
293


hKlf4RP ctrl exp
24
CGTTGAACTCCTCGGTCTCTCTCC



 


Nkx 2.5 FP
25
ccctgaccgatcccacctcaac
358


Nkx 2.5 RP
26
GGCGGGCGACGGCGAGATAGC



 


Mesp 1 FP
27
tcgaagtggttccttggcagac
162


Mesp 1 RP
28
CCTCCTGCTTGCCTCAAAGTGTC



 


Mesp 2 FP
29
CGCTGCGCCTGGCCATCCGCTACAT
113


Mesp 2 RP
30
GCCCCAAGGGGACCCCGCGAC



 


Mef2c FP
31
gcaccagtgcagggaacggg
202


Mef2c RP
32
GACTGAGCCGACTGGGAGTTA



 


Sox 17 FP
33
gcggcgcaagcaggtgaag
205


Sox 17 RP
34
ACTCTGGCAGTCGCGGTAGTGGC



 


FoxA2 FP
35
ctgaagccggaacaccactacgc
214


FoxA2 RP
36
TCCAGGCCCGTTTTGTTCGTGAC



 


FGF8 FP
37
agctcagccgccgcctcatccg
313


FGF8 RP
38
AGCCCTCGTACTTGGCATTCTGC



 


MyoD FP
39
AggggctaggttcagctttctcG
240


MyoD RP
40
CTCCTGCTCTGGCAAAGCAACTC



 


BMP2 HBP FP
41
ACCTTTATGGAGGGAAACCCA
201


BMP2 HBP RP
42
CCGGATCTGGTTCAAGCATGA



 


hTert HBP FP
43
AACCTTCCTCAGCTATGCCC
210


hTert HBP RP
44
GCGTGAAACCTGTACGCCT



 


Islet-1 HPB F
45
GTGGAGAGGGCCAGTCTAGG
250


Islet-1 HPB R
46
CCGTCATCTCTACCAGTTGCT



 


Troponin T HPB F
47
GAGTTGCAGGCGCTGATTG
229


Troponin T HPB R
48
TCTGGATGTAACCCCCAAAATG



 


Dhand HPB F
49
ATGAGTCTGGTAGGTGGTTTTCC
205


Dhand HPB R
50
CATACTCGGGGCTGTAGGACA



 


T HPB F
51
GATCACGCAGCTCAAGATTGC
230


T HPB R
52
TCTCTGGTGTGTTCCTAGACG



 


T5 HPB F
53
CACTTCTCCGCTCACTTCACC
210


T5 HPB R
54
TGGCACGCCATGAGAGTAGA



 


ETS-2 HPB F
55
AAAGCTACCTTCAGTGGCTTC
225


ETS-2 HPB R
56
AATGTCACCCACAAAGTCAGG



 


Dkk-1 FP
57
ATTCCAACGCTATCAAGAACC
384


Dkk-1 RP
58
CCAAGGTGCTATGATCATTACC



 


TBX 20 FP
59
tccagattctccttttaccg
190


TBX 20 RP
60
ttcagacttcaggttgagca



 


SM actin HBP F
61
CGGTGCTGTCTCTCTATGCC
156


SM actin HBP R
62
CACGCTCAGTCAGGATCTTCA



 


hGATA1 F
63
AGAAGCGCCTGATTGTCAGTA
229


hGATA1 R
64
AGAGACTTGGGTTGTCCAGAA



 


hGTAT2 F
65
GGCCCACTCTCTGTGTACC
243


hGATA2 R
66
CATCTTCATGCTCTCCGTCAG



 


TBX3-1 F
67
GTGTCTCGGGCCTGGATTC
164


TBX3-1 R
68
ACGTGTAGGGGTAAGGGAACA



 


TBX3-2 F
69
TTAAAGTGAGATGTTCTGGGCTG
298


TBX3-2 R
70
ACTATAATTCCCCTGCCACGTA



 


TBX4 F
71
TGACCATCGCTACAAGTTCTGT
163


TBX4 R
72
GGTGGTTGTTTGTCAGCTTCAG



 


TBX6 F
73
ACACCCCTAAACTGGATTGCT
229


TBX6 R
74
CCTCCCAGCTTTGGTGATGAT



 


TBX10 F
75
CCTCGGCATACTTGCACCC
208


TBX10 R
76
ATTCCTCCCACAGAGGCTTCA



 


TBX18 F
77
GCCCCTGCTGACTATTCTGC
227


TBX18 R
78
CTGCATGGATAAGCTGGTCTG



 


TBX19 F
79
AAGAATGGCAGACGGATGTTT
204


TBX19 R
80
CCGGGTGAATGTAGACGCAG



 


RUNX2 F
81
CGGCAAAATGAGCGACGTG
268


RunX2 R
82
CACCGAGCACAGGAAGTTG



 


LMO2 F
83
GGCCATCGAAAGGAAGAGCC
221


LMO2 R
84
GGCCCAGTTTGTAGTAGAGGC



 


TAU F
85
CCCCTGGAGTTCACGTTTCAC
240


TAU R
86
GCGAGCTTTGAGTTGAGGGA









Example 4
Stem Cell Marker Proteins in Reprogrammed Cells
FIG. 3 shows immunofluorescence staining of infected NHDF-P3 with antibody to the stem cell marker NANOG. Induced NANOG staining is revealed in ETS2-infected cells but not in ELK4- or empty vector-infected cells. Also, a protein blot using ETS2-specific antibody revealed its expression in cells infected with ETS2 lentivirus but not in NHDFs infected with empty lentivirus.
FIG. 4 shows ETS2 lentivirus-induced stem cell-like colonies and the induction of stem cell marker proteins NANOG, OCT3/4, SOX2, REX1 and c-MYC after 4 weeks in culture. The top left panel shows that NHDFs infected with ETS2 virus converted to colonies of small rounded cells highly reminiscent of cultured murine and human embryonic stem cells. Note that colonies were GFP-labeled through the infection with ETS2 lentivirus. Non-infected fibroblasts failed to round and did not stain for GFP or form cellular colonies.
The right top panel of FIG. 4 shows the expression of stem cell marker genes NANOG, OCT3/4, SOX2, REX1, KLF4 and c-MYC in ETS2-induced cellular colonies analyzed by RT-PCR. Underneath is graphic representation of quantitative RT-PCR for NANOG, OCT3/4, SOX2 and c-MYC in ETS2-infected cells.
The lower panels of FIG. 4 confirm the presence of NANOG and OCT3/4 proteins in ETS2-induced cellular colonies by immunofluorescence staining with specific antibodies.
Example 5
Characteristics of Cells Transduced with ETS2
FIG. 5 shows that approximately 97% of H9 human embryonic stem cells stained for the stem cell surface marker SSEA-3 (Panel A). Over 60% of ETS2-infected cells also displayed surface marker SSEA-3 (Panel C), in comparison to less than 2% staining of the NHDFs infected with empty lentivirus (Panel B). Thus, ETS2 efficiently converted NHDFs into cells with the SSEA-3 surface marker resembling human embryonic stem cells.
Flow cytometry was done using a BD Biosciences LSR II analyzer. Confluent colony-forming cells were dissociated by trypsin, washed with PBS and diluted to a concentration of 5×106 cells/ml PBS in 8 samples (100 ul each). Thereafter 10 ul of normal human serum was used for blocking for 5 min. Antibodies to SSEA-3-PE (Becton Dickinson) were diluted and added according to manufacturer's specifications, and incubated for 1 hr at 4° C. Then 400 ul PBS was added and the mixture was spun down and half of the supernatant was removed and 200 ul PBS was added and assayed by flow cytometry.
FIG. 6 shows flow cytometry of cells stained for the stem cell surface marker Tra-1-81 performed as for SSEA-3. Approximately 45% of H9 human embryonic stem cells stained for Tra-1-81 (Panel A). No Tra-1-81 staining (Panel B) was detected for NHDFs infected with empty lentivirus (not carrying a heterologous gene). Infection with ETS2 virus gave rise to approximately 39% cells displaying embryonic stem cell marker Tra-1-81 (Panel C). This indicates that ETS2 efficiently converted NHDFs into cells with the Tra-1-81 surface marker resembling human embryonic stem cells.
OCT3/4, NANOG and SOX2 gene transcripts were observed only after NHDF-P3 were transduced with lentiviral vector containing ETS2 but not after transduction with empty lentivector or vector containing ELK4. OCT3/4, NANOG and SOX2 transcripts were visualized (FIG. 2) and NANOG induction was visualized using immunofluorescence (FIG. 3).
Lentiviral transduction of HNDF-P3 cells with ETS2 resulted in whole populations which showed robust ETS2 expression over 4 weeks visualized by protein blots (FIG. 3). These ETS2-transduced cells formed large green fluorescent colonies similar to those of pluripotent ES and/or induced pluripotent stem (iPS) cells.
Reprogramming of fibroblasts with ETS2 resulted in strong expression of the pluripotent marker genes NANOG, OCT3/4, SOX2 and c-MYC measured by both RT-PCR and quantitative PCR and immunostaining. Additionally, flow cytometry shows that ETS2 efficiently converted NHDFs into cells with surface markers SSEA-3 and Tra-1-81 resembling human embryonic stem cells. Thus, these ETS2-treated human fibroblast cells resemble iPS cells in their ability to express pluripotent stem cell marker proteins. These cells were therefore named “EPS” cells.
Example 6
Combination of ETS2 and Mesp1 Induces De Novo Cardiac Progenitor Program in Fibroblasts
Next, EPS cells were subjected to lentiviral transduction with mouse Mesp1. The resulting EPS cells expressing Mesp1 could be induced to form embryoid bodies using protocols for forming embryoid bodies from ES cells. Plated cellular aggregates were further treated with activin and BMP4 for 4 days and then examined at 10 days. Constitutive expression of stem cell markers continued even after the transduction with Mesp1 and addition of growth factor morphogens.
Robust induction of the cardiac progenitor factors ISL1, NKX2.5, GATA4, MEF2C, TNT and MHC was observed by immunostaining only in the EPS cells infected with lentivirus expressing Mesp1. FIG. 7 shows cells stained with DAPI to visualize nuclei (left panels), phase contrast images (middle) and cells stained with specific antibodies to visualize indicated proteins (right panels).
FIG. 8 shows that the MesP1 infection of EPS cells induces de novo cardiac progenitor program in cell that were originally NHDFs. Cardiac progenitor cells post-aggregation were plated for a week and then taken for RNA isolation. RNA was transcribed using MMLV reverse transcriptase, and PCR amplification for 30 cycles was performed for GAPDH, NKX2.5, ISL1, GATA4, MEF2C, TBX5, MHC3, TNT, MLC2, CX43 and CX45 (refer to Table 1 for primer sets), using LA-16 polymerase mix.
FIG. 8 shows that in EPS cells grown for 4 weeks, aggregated and plated for 7 days, no transcripts were observed for markers of early heart development by RT-PCR. Similarly, no appreciable expression of the early heart development markers was detected after infection of NHDFs with Mesp1 alone. However, Mesp1 addition to EPS cells induced robust expression of cardiac mesoderm progenitor markers including NKX2.5, ISL1, GATA4, MEF2C, TBX5, and cardiac contractile protein gene expression including alpha MHC and troponin T.
FIG. 9 shows that the MesP1 infection of EPS cells induced sequential de novo cardiac progenitor program as determined by RT-PCR. NHDF-P3 cells were infected with ETS2 lentivirus, grown for 4 weeks, then infected with Mesp1 lentivirus, aggregated and plated for 7 days. RNA was isolated daily and analyzed by RT-PCR for expression of cardiogenic genes.
FIG. 10 shows that the MesP1 infection of EPS cells induced de novo cardiac program gene expression as shown by the appearance of Red Tomato fluorescence staining from the reporter construct NKX2.5-Red Tomato. NKX2.5/Smad/GATA enhancer, which is activated in cardiac progenitors, was linked to the minimal HSP68 promoter adjacent to the puromycin resistance gene and an IRES sequence and the powerful reporter td-Tomato (cDNA was a gift from Dr. R. Tsien, University of California, San Diego). As shown above, GFP staining resulted from ETS2 and Mesp1 lentivirus infection of NHDFs. The Red Tomato fluorescence is consistent with the ETS2/Mesp1 driven conversion to cardiac progenitors by the induction of NKX2.5 gene expression. Note the appearance of many triangular and rectangular appearing cells that are highly similar in shape to cardiac myocytes.
FIG. 11, Panel A shows a summary of FACS sorting of cardiac progenitor cells obtained from sequential treatment of NHDFs with ETS2 lentivirus and then of the resultant EPS cells with Mesp1 lentivirus. Flow cytometry was done using a BD Biosciences LSR II analyzer. Confluent colony-forming cells were dissociated by trypsin, washed with PBS and diluted to a concentration of 5×106 cells/ml PBS in 8 samples. Panel A shows GFP stained cells accounting for the total lentivirus infection, since each virus is GFP-tagged. Panel B shows that approximately 19% of the ETS2 and Mesp1 infected cells were both stained by GFP and Red-Tomato (shaded area). As evidenced by activation of a key cardiogenic reporter NKX2.5 Red-Tomato, ETS2 and Mesp1 efficiently convert NHDFs into cells with characteristics resembling cardiac progenitors.
FIG. 12 shows that approximately 10 to 40% of ETS2 and Mesp1 infected NHDF cells display endothelial and cardiac cell surface markers, CD31. CD34 and CD144 after 9 days in culture. This is an additional line of evidence that ETS2 and Mesp1 efficiently converted NHDFs into cells with characteristics resembling embryonic endothelial and cardiac myocytes.
Example 7
Cardiac Properties of Reprogrammed Cells
Lentiviral transduction of a puromycin selectable system using a lentiviral cardiac-specific alpha-myosin heavy chain (alpha-MHC) promoter and enhancer linked to the puromycin resistance gene resulted in enrichment of the cardiac progenitor cells and subsequent observation of a rhythmic beating of the transduced cells, similar to that observed in cardiac myocytes.
A myosin heavy chain prometer driving the puromycin selectable gene construct was transduced into NHDFs which were then sequentially transduced with ETS2 and Mesp1. Cellular aggregates obtained during hang-drop embryoid body formation were then treated with 50 ug/ml puromycin to select cells resistant to puromycin and therefore having the active cardiac specific alpha-MHC promoter.
After 9 days beating in the cell cultures was observed and captured using video microscopy and converted into MPEG videos. Beating per cultured aggregate per dish was counted for 20 sec and then multiplied by 3 for beats per one minute (FIG. 13). Three separate measurements were done per aggregate in a tissue culture dish or well.
Reprogramming of EPS cells with Mesp1 resulted in strong expression of cardiac progenitor genes as determined by RT-PCR and immunostaining. Additionally, flow cytometry showed that Mesp1 efficiently converted EPS cells into cells with surface markers CD31, CD34 and CD144 resembling human cardiac cells. Finally, rhythmic beating was observed in the cell cultures. This completed the conversion from skin fibroblasts to terminally differentiated cardiogenic cells.
REFERENCES CITED
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
U.S. PATENT DOCUMENTS


    U.S. Patent publication No. 2009/0227032 to S. Yamanaka.


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## Claims

1. A method for preparing a cardiac progenitor cell, the method comprising:
(a) transducing a somatic cell with an ETS2 reprogramming factor forming an ETS2 treated cell, wherein the ETS2 treated cell produces a pluripotent stem cell marker protein not present in the somatic cell; and
(b) transducing the ETS2 treated cell with a Mesp1 reprogramming factor forming a cardiac progenitor cell, wherein the cardiac progenitor cell produces a cardiac cell marker protein neither expressed in the somatic cell nor in the ETS2 treated cell;
wherein the somatic cell is a normal human dermal fibroblast cell.

2. The method of claim 1, wherein the ETS2 comprises the DNA sequence having SEQ ID NO:9.

3. The method of claim 1, wherein the Mespl comprises the DNA sequence having SEQ ID NO:6.