Method to reduce oncogenic potential of induced pluripotent stem cells from aged donors

Abstract

Disclosed are findings that: (a) induced pluripotent stem cells derived from aged donors (A-iPSC) show increased genomic instability, a defect in apoptosis, a defect in glucose metabolism, and a blunted DNA damage response are compared to those derived from young donors (Y-iPSC); and (b) inhibition of excessive glutathione-mediated H202 scavenging activity, found to be associated with A-iPSC and in turn inhibiting DNA damage response and apoptosis, substantially rescues these defects and reduces the oncogenic potential of A-iPSC. Supplementation of pluripotency factor ZSCAN 10 (shown to be poorly activated in A-iPSC and to act upstream of glutathione involvement), e.g., by expression as an adjunct to the four Yamanaka iPSC reprogramming factors, led to substantial recovery of genomic stability, DNA damage response, and apoptosis in A-iPSC through enhancing GLUT3 and normalizing homeostasis of glutathione/H202; GLUT3 (a pluripotent stem cell-specific glucose transporter acting upstream of glutathione and also poorly activated in A-iPSC) has similar effects, indicating that inhibition of glutathione/H202 notably through delivery of ZSCAN 10 and/or GLUT3 and/or an exosome subunit will be clinically useful, resulting in A-iPSC of improved properties and reduced oncogenic potential.

1 .- 39 . (canceled) 
     
     
         40 . A method for producing induced pluripotent stem cells generated from somatic cells of aged donors (A-iPSCs), the method comprising: supplementing somatic cells of aged donors prior to the initiation of reprogramming, during reprogramming, and/or after reprogramming of the somatic cells with an effective amount of GLUT3, an exosome subunit, or a combination thereof, thereby producing A-iPSCs with at least one of DNA damage response, apoptosis response, glucose metabolism, and genomic stability levels approximating those of induced pluripotent stem cells from young donors (Y-iPSCs), wherein the supplementation is carried out by adding GLUT3, an exosome subunit, or a combination thereof to a culture medium in which the somatic cells are maintained or by transfecting the somatic cells with a vector harboring a nucleic acid sequence encoding GLUT3, an exosome subunit, or a combination thereof. 
     
     
         41 . The method of  claim 40 , further comprising reducing expression of A-iPSC glutathione peroxidase 2 (GPX2) or glutathione synthase (GSS) by
 supplementing the A-iPSC with an effective amount of GLUT3, an exosome subunit, or a combination thereof,   thereby rescuing one or more of DNA damage response, apoptosis, and genomic stability in the A-iPSC.   
     
     
         42 . The method of  claim 40 , wherein the supplementation:
 is sufficient to restore GLUT3, exosome subunit, or a combination thereof levels in the A-iPSCs to about 50% or more of the respective levels of embryonic stem cells (ESCs);   is sufficient to reduce oxidation capacity of glutathione in the A-iPSCs to about 80% to about 120% of that of ESCs;   is sufficient to restore genomic stability of the A-iPSCs to approximately that of Y-iPSCs;   is sufficient to restore apoptosis rate of the A-iPSCs to approximately that of Y-iPSCs;   is sufficient to restore DNA damage response of the A-iPSCs to approximately that of Y-iPSCs;   is sufficient to reduce oxidation capacity of glutathione in the A-iPSCs to approximately that of Y-iPSCs; and/or   is sufficient to reduce GPX2 levels in the A-iPSCs to approximately those of Y-iPSCs.   
     
     
         43 . The method of  claim 42 , wherein the genomic stability is measured by incidence of aneuploid clones; the apoptosis rate is measured by DNA fragmentation assay in response to a DNA damaging agent; and the DNA damage response is measured by ATM or H2AX phosphorylation in response to a DNA damaging agent. 
     
     
         44 . An A-iPSC produced by the method of  claim 40 , wherein the A-iPSC in the absence of GLUT3, an exosome subunit, or a combination thereof supplementation was first deficient in GLUT3, exosome subunit, or a combination thereof expression, expressing either no GLUT3, exosome subunit, or a combination thereof or a level of GLUT3, exosome subunit, or a combination thereof substantially lower than that of a control iPSC derived from a healthy young donor (Y-iPSC) or embryo (ESC), and wherein the A-iPSC comprises a vector harboring nucleic acid for GLUT3, exosome subunit, or a combination thereof. 
     
     
         45 . An A-iPSC produced by the method of  claim 40 , wherein the A-iPSC is characterized by GLUT3, exosome subunit, or a combination thereof expression levels, oncogenic potential, GPX2 expression levels, and/or GSS expression levels comparable to those of a control Y-iPSC or embryonic stem cell (ESC). 
     
     
         46 . The method of  claim 40 , wherein the reprogramming of the somatic cells is carried out with Yamanaka factors OCT4, SOX2, KLF4, and MYC. 
     
     
         47 . The method of  claim 40 , wherein the reprogramming of the somatic cells is carried out with pluripotency factors selected from the group of those of Yamanaka wherein one or more of OCT4, SOX2, KLF4 and MYC are replaced as follows: Nanog and Lin28 replace Klf4 and MYC; esrb replaces Klf4; SV40 LT (T) replaces Klf4, MYC lin28 and Nanog; BIX-01294 replaces SOX2, OCT4; VPA replaces Klf4 and MYC. 
     
     
         48 . A method for reducing the oncogenic potential of induced pluripotent stem cells (iPSCs) or A-iPSCs, the cells having one or more of genomic instability, a defect in apoptosis, a defect in DNA damage response, and a defect in glucose metabolism, and exhibiting excessive glutathione-mediated H 2 O 2  scavenging activity compared to embryonic stem cells (ESCs) or induced pluripotent stem cells from young donors (Y-iPSCs), the method comprising
 supplementing iPSCs or A-iPSCs with pluripotency factor GLUT3, an exosome subunit, or a combination thereof as an adjunct to reprogramming to substantially restore the at least one of DNA damage response, apoptosis response, glucose metabolism and genomic stability to levels substantially the same as those of Y-iPSC or ESC, wherein the supplementation is carried out by adding GLUT3, an exosome subunit, or a combination thereof to a culture medium in which the iPSCs or A-iPSCs are maintained, or by transfecting the iPSCs or A-iPSCs with a vector harboring a nucleic acid sequence encoding GLUT3, an exosome subunit, or a combination thereof.   
     
     
         49 . The method of  claim 48 , wherein the supplementation:
 is sufficient to restore GLUT3, exosome subunit, or a combination thereof levels in the iPSCs or A-iPSCs to about 50% or more of the respective levels of embryonic stem cells (ESCs);   is sufficient to reduce oxidation capacity of glutathione in the iPSCs or A-iPSCs to about 80% to about 120% of that of ESCs;   is sufficient to restore genomic stability of the iPSCs or A-iPSCs to approximately that of Y-iPSCs;   is sufficient to restore apoptosis rate of the iPSCs or A-iPSCs to approximately that of Y-iPSCs; and/or   is sufficient to restore DNA damage response of the iPSCs or A-iPSCs to approximately that of Y-iPSCs.   
     
     
         50 . The method of  claim 49 , wherein the genomic stability is measured by incidence of aneuploid clones; the apoptosis rate is measured by DNA fragmentation assay in response to a DNA damaging agent; and the DNA damage response is measured by ATM or H2AX phosphorylation in response to a DNA damaging agent. 
     
     
         51 . An iPSC or A-iPSC produced by the method of  claim 44 , wherein the iPSC or A-iPSC exhibits increased GLUT3, exosome subunit, or a combination thereof expression relative to an untreated control iPSC or A-iPSC, and wherein the iPSC or A-iPSC comprises a vector harboring nucleic acid for GLUT3, exosome subunit, or a combination thereof. 
     
     
         52 . The iPSC or A-iPSC of  claim 51 , wherein the iPSC or A-iPSC further exhibits decreased GPX2 expression relative to an untreated control iPSC or A-iPSC. 
     
     
         53 . An iPSC derived from a somatic cell of an aged donor (A-iPSC) where the A-iPSC has been engineered to express GLUT3, an exosome subunit, or a combination thereof by transfecting the iPSC with a vector harboring nucleic acid for GLUT3, an exosome subunit, or a combination thereof at levels comparable to an iPSC derived from a healthy young donor (Y-iPSC) or an embryonic stem cell (ESC), wherein the iPSC comprises the vector harboring nucleic acid for GLUT3, an exosome subunit, or a combination thereof, and wherein the iPSC originally displayed reduced GLUT3, exosome subunit, or a combination thereof expression levels compared to a Y-iPSC or ESC control. 
     
     
         54 . The iPSC of  claim 53 , wherein the iPSC in the absence of GLUT3, an exosome subunit, or a combination thereof supplementation was first deficient in GLUT3, exosome subunit, or a combination thereof expression, expressing either no GLUT3, exosome subunit, or a combination thereof or a level of GLUT3, exosome subunit, or a combination thereof substantially lower than that of a control iPSC derived from a healthy young donor (Y-iPSC) or embryo (ESC).