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(12) Patent Application Publication (10) Pub. No.: US 2014/0038291 A1 Ahlfors Et Al US 20140.038291A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0038291 A1 Ahlfors et al. (43) Pub. Date: Feb. 6, 2014 (54) METHODS FOR REPROGRAMMING CELLS Publication Classification AND USES THEREOF (51) Int. Cl. (71) Applicant: New World Laboratories Inc., Laval CI2N 15/85 (2006.01) (CA) (52) U.S. Cl. CPC ...................................... CI2N 15/85 (2013.01) (72) Inventors: Jan-Eric Ahlfors, Laval, CA (US); USPC ............................ 435/.441; 435/455; 435/325 Rouwayda Elayoubi, Laval, CA (US) (73) Assignee: New World Laboratories Inc., Laval (57) ABSTRACT (CA) (21) Appl. No.: 13/843,713 Described herein are reprogrammed cells, and methods for cell dedifferentiation, transformation and eukaryotic cell (22) Filed: Mar 15, 2013 reprogramming. Also descried are cells, cell lines, and tissues that can be transplanted in a patient after steps of in vitro Related U.S. Application Data dedifferentiation and in vitro reprogramming. In particular (63) Continuation-in-part of application No. 13/464,987, embodiments the cells are Stem-Like Cell s (SLCs), including filed on May 5, 2012, which is a continuation-in-part Neural Stem-Like Cells (NSLCs), Cardiac Stem-Like Cells of applicationy -No. 13/504,988,s filed on Apr. 30, 2012, (CSLC), Hematopoietic Stem-Like Cells (HSLC), Pancreatic filed as application No. PCT/CA10/01727 on Nov. 1, Progenitor-Like Cells, and Mesendoderm-like Cells. Also 2010 described are methods for generating these cells from human Somatic cells and other types of cells. Also provided are (60) Provisional application No. 61/256,967, filed on Oct. compositions and methods of using of the cells so generated 31, 2009. in human therapy and in other areas. Patent Application Publication Feb. 6, 2014 Sheet 1 of 28 US 2014/0038291 A1 Figure 1 NCAM Day 6 firslf in Patent Application Publication Feb. 6, 2014 Sheet 2 of 28 US 2014/0038291 A1 Figure 3 Patent Application Publication Feb. 6, 2014 Sheet 3 of 28 US 2014/0038291 A1 (A) ( B) Hoechst fill-tubulin Musashi NPC NSLC T Hoechs O4 NGFrec NPC NSC Hoechst Co133 Neun NPC NSC Hoechst Nestin NoC NSLC Figure 4 Patent Application Publication Feb. 6, 2014 Sheet 4 of 28 US 2014/0038291 A1 Utransfected x GFAP Fibroretin Fibroblasparker fibroblastices Figure 5 Patent Application Publication Feb. 6, 2014 Sheet 5 of 28 US 2014/0038291 A1 Hoechst (no Sox2) Sox2 3-tubulin Week Week 2 Week Figure 6 Patent Application Publication Feb. 6, 2014 Sheet 6 of 28 US 2014/0038291 A1 Hoechst Sox2 GFA HFF untransfected Nestin Sox2 GFAP HFF transfected Keratinocytes CD34 Figure 7 Patent Application Publication Feb. 6, 2014 Sheet 7 of 28 US 2014/0038291 A1 Figure 8 Transfected HFF with Msi1/Ngm2 Figure 9 Patent Application Publication Feb. 6, 2014 Sheet 8 of 28 US 2014/0038291 A1 Ngn2 Msl1/Ngn2 Figure 10 Figure 11 Patent Application Publication Feb. 6, 2014 Sheet 9 of 28 US 2014/0038291 A1 Os all: No Positive Castul: No Positive (Psti: No Positive Figure 12 Patent Application Publication Feb. 6, 2014 Sheet 10 of 28 US 2014/0038291 A1 3.3 3. 2. 2 --control DMso ses s sonic -o-controlcdosporine 3 is so is is 7 is 23 s 2 2s 3 33 3s 37 as 4 3 days post innurnization Figure 13 NAblation 25 Rotarod 20RPM ONo Treatment Treatment with Aiginate Treatment with Aiginate NPC Treatment with Matrix +NSCs 20 OTreatment with Aiginate - NSLCs Treatment with Matrix Only Treatment with Fibrin Girl 5 O O ^8.................. (sssssssss Figure 14 Patent Application Publication Feb. 6, 2014 Sheet 11 of 28 US 2014/0038291 A1 Walking beam No Aation --Notreatment --Treatment with Aginate O-Treatment with Aginate + NPC Treatment with Matra 4 NSLs OTreatment with Aginate 4 NSLCs Treatment with Matru Only Treatment with fibringe 4. a-a-a-a-a-a-a-a-a-a-a- Figure 15 Patent Application Publication Feb. 6, 2014 Sheet 12 of 28 US 2014/0038291 A1 Octa AP Figure 16 Untransfected ADSCs Untransfected ADSCs ADSC transfected with Rexl/Oct4 ADSC transfected with Rexl/Klf4 Figure 17 Patent Application Publication Feb. 6, 2014 Sheet 13 of 28 US 2014/0038291 A1 Figure 18 Patent Application Publication Feb. 6, 2014 Sheet 14 of 28 US 2014/0038291 A1 +ve control Transfected HFFINo inhibitornON3N6 Me2 hES cells -ye control UntransfectedHFF Figure 19 Patent Application Publication Feb. 6, 2014 Sheet 15 of 28 US 2014/0038291 A1 Brightfield TRA-1-81 SSEA-4s BgQ1NS, day15 NSLC, day 15 Figure 20 Patent Application Publication Feb. 6, 2014 Sheet 16 of 28 US 2014/0038291 A1 Figure 21 GFAP 8IIl-tubulin oechst B-tubulin Figure 22 Patent Application Publication Feb. 6, 2014 Sheet 17 of 28 US 2014/0038291 A1 Set 1: NSLCWS, HFF Set 2: NSLC vs. hNPC E. L -E E ES us E SE E R EL S CE E E E C E. E.R of arge if 2 iFaldichargeafculateds fogiaea Figure 23 Patent Application Publication Feb. 6, 2014 Sheet 18 of 28 US 2014/0038291 A1 Adiposeserved Sten Cell Neural Stem-like Ce Figure 24 Hoechst Hoechst, TRA1-60 Hoechst, RA1.81 Hoechst s Day 15 Eoechst, set- nanog Hoechst, Stx2 E-cadherin Day 15 echst, it inning Hoechst, Sox2 E-cadherin Day 32 Figure 25 Patent Application Publication Feb. 6, 2014 Sheet 19 of 28 US 2014/0038291 A1 Day 28 Ectodermal Mesodermal 2 Endodernal M Figure 26 Patent Application Publication Feb. 6, 2014 Sheet 20 of 28 US 2014/0038291 A1 ° S SC L es5' O. O.6' v S NSCL NP FF Nt2 . NTC 300 200 Figure 27a c N c N SS. SS && &8 NSCL : NP FF is Nt2 NTC : Figure 27b. Patent Application Publication Feb. 6, 2014 Sheet 21 of 28 US 2014/0038291 A1 NSCL NP FF Nt2 NTC NSCL NP FF NTC Figure 27d Patent Application Publication Feb. 6, 2014 Sheet 22 of 28 US 2014/0038291 A1 850 50 NSCL S00 400 850 NP 50 400 850 50 FF 400 850 50 850 50 NTC : 400 ATCB Figure 27g Patent Application Publication Feb. 6, 2014 Sheet 23 of 28 US 2014/0038291 A1 red filter red + green filters green filter red + phase filters phase filter red + green + phase filters Figure 28 Patent Application Publication Feb. 6, 2014 Sheet 24 of 28 US 2014/0038291 A1 MAP2/Synaptotagmin/Hoechst Figure 29 Patent Application Publication Feb. 6, 2014 Sheet 25 of 28 US 2014/0038291 A1 Figure 30 Patent Application Publication Feb. 6, 2014 Sheet 26 of 28 US 2014/0038291 A1 Figure 31 is a panel illustration of Nestin second intron CpG methylation in HFF, Keratinocytes, CD34+ cells before and after reprogramming into Neural Stem-Like Cells (NSLC). NPC serve as a positive control. Open circles indicate unmethylated CpG dinucleotides). FF: FF-S F: OXXXXX XXX s OCCCXXCXXCXC (XXXXX XXXC OOOOOOOXCO OXCXXCXCXCXXC) OCXCOOXOXOXOXO RS CCXXXXXXX) (CXXXCXCXCXCXCXC CO3: O34"-NSLC: OOXXXXXXX) OCCCXCXCXXCXC (CCCCXCXCXCXCXC CCCXCCXCXCXCXC OOOOOOOOO OOCOXOXOXCXC) featinocyte: Keratinocyte-iSLC: Ooooooooo CCXXCOXOXOXOXO. CCCCCXCXCXCXC ser {OOOOOOXCXC) E (COXXXXXXX) Figure 32 is a panel of photomicrographs showing all the chromosomesas of NSLC derived from CD34" cells (left) and HFF cells (right) showing that NSLC have a normal Karyotype. (2n=46, XY metaphase spread). Patent Application Publication Feb. 6, 2014 Sheet 27 of 28 US 2014/0038291 A1 Figure 33 is a panel of photomicrographs of Cardiac Stem-Like Cells (CSLC) taken with a Cellomics' ArrayScan V' HCS System at 10x showing Gata4, Nkx2.5, CXCR4, Brachyury, and Troponin1 positive cells at Day 15. Hoescht (blue) was used to stain all cell nuclei. The pCMV6-XL4-NkX-2.5, pCMV6-XL5-Mesp1, pCMV6-XL5-brachyury (Figure 3 b) transfected cells showed better expression of all markers than pCMV6-XL4 Tbx5, pCMV6-XL5-Mesp1, pCMV6-XL5-brachyury (Figure 3 a) transfected cells, as well as cells grown on the Laminin-211 Coated plates than on 0.1% gelatin-coated plated. Figure 33a): GAA4 Nix's CXCR4 rathyury Triporin Transfied Celsink. TESC. (LAN21) Figure 33b): GAA. Nexts XCR4 Brachyury Troponin Track Gokstin. Tad. LAN21) Patent Application Publication Feb. 6, 2014 Sheet 28 of 28 US 2014/0038291 A1 Figure 34 is a panel of photomicrographs of CSLC-cardiospheres taken with a light microscope at 20X. CSLC were completely dissociated into single cell suspensions and the single CSLC were monitored over time. The single cells formed individual cardiospheres that continuously grew in size and volume (followed for 55 days). SS . Figure 35 is a panel of photomicrographs taken with a Cellomics ArrayScan WHCS System at 10X showing early Cardiomyocyte-Like Cells differentiated from CSLC over 20 days and staining positive for Brachyury, NKX2.5, Troponin T, Troponin I, and Connexin 43. Hoescht (blue) was used to stain all cell nuclei. Art N Tripnin Fix US 2014/0038291 A1 Feb. 6, 2014 METHODS FOR REPROGRAMMING CELLS 0007 (iii) For a stem cell to efficiently permanently AND USES THEREOF graft (in a functional but not competitive manner) into the patient's tissue, the stem cell generally has to be CROSS REFERENCE TO RELATED autologous (the patient’s own). The main exception to APPLICATIONS this is hematopoietic stem cell grafts (bone marrow transplants) due to the changes in the immune and other 0001. This application is a continuation-in-part of U.S. systems in the body caused by these specific Somatic application Ser. No. 13/464,987, filed May 5, 2012, which is stem cells. a continuation-in-part of U.S. application Ser. No. 13/504, 988, which is the U.S. National Phase Application of Inter 0008 Thus there is a need for an efficient method to create national Appl. No.: PCT/CA2010/001727, filed Nov.
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