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Genetic and cellular aspects of the establishment of histocompatible stem cells: information gained from an animal model.

Lim JM, Gong SP - BMC Proc (2011)

Bottom Line: As an initial step, we succeeded in establishing histocompatible stem cells using preantral follicle cultures and subsequent parthenogenetic activation.However, more progress regarding the establishment and elucidation on origination of established cell lines is necessary to use this genetic manipulation-free procedure.Nevertheless, relevant information on the process will help to stimulate preclinical research on cell transformation into differentiated, undifferentiated, and even cancerous cells, as well as clinical studies on the application of induced pluripotent cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: WCU Biomodulation Program, Seoul National University, Seoul 151-742, Korea. limjm@snu.ac.kr.

ABSTRACT
The establishment of patient-specific histocompatible stem cells may be an alternative for overcoming current limitations in stem cell engineering. We are developing an animal model to assist the establishment of histocompatible, autologous stem cells. In this process, we obtained valuable information on establishing and characterizing stem cells. As an initial step, we succeeded in establishing histocompatible stem cells using preantral follicle cultures and subsequent parthenogenetic activation. The gene expression profile of the established stem cells was similar to that of embryonic stem cells (ESCs) derived from normal fertilization. On the other hand, we propose a way to derive histocompatible, ESC-like cells by co-culturing ovarian stromal cells with feeder fibroblasts, which may allow the derivation of stem cells from somatic tissue. However, more progress regarding the establishment and elucidation on origination of established cell lines is necessary to use this genetic manipulation-free procedure. Nevertheless, relevant information on the process will help to stimulate preclinical research on cell transformation into differentiated, undifferentiated, and even cancerous cells, as well as clinical studies on the application of induced pluripotent cells.

No MeSH data available.


Related in: MedlinePlus

Analyses of the origin of ovarian colony-forming cells (OCCs). (A) Single nucleotide polymorphism (SNP) genotyping of OCCs and control cells. The heterozygosity or homozygosity of SNP loci of OCC-1 and OCC-2 of B6D2F1 strain was compared with that of B6D2F1 embryonic stem cells (ESCs), somatic fibroblasts of DBA2 and C57BL6 mice, and parthenogenetic ESCs (pESCs). Both homozygosity and heterozygosity were concomitantly detected in the OCC line. ESCs of F1 strain showed heterozygosity alone, and only homozygotic SNP loci were detected in the fibroblasts of the inbred strain. The pESC line possessed both homozygotic and heterozygotic chromosomes. (B). Methylation status of OCCs, ESCs, and pESCs. Genomic DNA isolated from these cells was subjected to bisulfite genomic sequencing analysis. The methylation levels of the promoter regions of stemness-related genes (Oct-4 and Nanog) and imprinted genes expressed differentially after parthenogenetic activation (H19, Peg3, Snrpn, and Gtl2) were compared. The PCR products were cloned, and 10 plasmid clones were sequenced for each sample. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Stemness-related genes were demethylated in all cell lines, whereas the expression of other genes differed markedly among the cell lines. The methylation in OCCs was significantly different from that in ESCs or pESCs; OCCs had more methylated H19 and Gtl2 compared with pESCs and less methylated Peg3 and Snrpn compared with ESCs. (Reprinted with permission from Gong et al., 2010; 93:2564-601).
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Figure 2: Analyses of the origin of ovarian colony-forming cells (OCCs). (A) Single nucleotide polymorphism (SNP) genotyping of OCCs and control cells. The heterozygosity or homozygosity of SNP loci of OCC-1 and OCC-2 of B6D2F1 strain was compared with that of B6D2F1 embryonic stem cells (ESCs), somatic fibroblasts of DBA2 and C57BL6 mice, and parthenogenetic ESCs (pESCs). Both homozygosity and heterozygosity were concomitantly detected in the OCC line. ESCs of F1 strain showed heterozygosity alone, and only homozygotic SNP loci were detected in the fibroblasts of the inbred strain. The pESC line possessed both homozygotic and heterozygotic chromosomes. (B). Methylation status of OCCs, ESCs, and pESCs. Genomic DNA isolated from these cells was subjected to bisulfite genomic sequencing analysis. The methylation levels of the promoter regions of stemness-related genes (Oct-4 and Nanog) and imprinted genes expressed differentially after parthenogenetic activation (H19, Peg3, Snrpn, and Gtl2) were compared. The PCR products were cloned, and 10 plasmid clones were sequenced for each sample. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Stemness-related genes were demethylated in all cell lines, whereas the expression of other genes differed markedly among the cell lines. The methylation in OCCs was significantly different from that in ESCs or pESCs; OCCs had more methylated H19 and Gtl2 compared with pESCs and less methylated Peg3 and Snrpn compared with ESCs. (Reprinted with permission from Gong et al., 2010; 93:2564-601).

Mentions: To further understand the origin and genetic characteristics of the two colony-forming cell lines, we conducted a short tandem repeat microsatellite analysis, which showed that the two colony-forming cell lines were an identical match to the ovary donor. Furthermore, we performed single nucleotide polymorphism genotyping and methylation analysis of the cell lines, to determine their origin. Homozygotic and heterozygotic chromosome recombinations were detected in both the colony-forming cell lines and the parthenogenetic ESC lines. The ESCs of the F1 strain possessed only heterozygotic loci, whereas homozygotic recombination was detected in the fibroblasts of both inbred strains. In the methylation analysis, No difference in the methylation of stem cell-related genes (Nanog and Oct-4) was seen among the parthenogenetic ESCs, normally fertilized ESCs, and ovarian colony-forming cells, although there was a significant difference in the methylation of several imprinted genes between ovarian colony-forming cells and parthenogenetic ESCs (Fig. 2). Based on these results, we have identified and characterized a novel colony-forming cell population that appears to be derived from either cell transformation accompanying somatic cell reprogramming or from tissue-specific stem cell-progenitor cell isolation. Nevertheless, we cannot completely exclude the tumorigenesis of ovarian somatic cells or parthenogenetic cells as possible origins. Further research is necessary to obtain conclusive results regarding cell origin and to evaluate the feasibility of clinical and industrial applications.


Genetic and cellular aspects of the establishment of histocompatible stem cells: information gained from an animal model.

Lim JM, Gong SP - BMC Proc (2011)

Analyses of the origin of ovarian colony-forming cells (OCCs). (A) Single nucleotide polymorphism (SNP) genotyping of OCCs and control cells. The heterozygosity or homozygosity of SNP loci of OCC-1 and OCC-2 of B6D2F1 strain was compared with that of B6D2F1 embryonic stem cells (ESCs), somatic fibroblasts of DBA2 and C57BL6 mice, and parthenogenetic ESCs (pESCs). Both homozygosity and heterozygosity were concomitantly detected in the OCC line. ESCs of F1 strain showed heterozygosity alone, and only homozygotic SNP loci were detected in the fibroblasts of the inbred strain. The pESC line possessed both homozygotic and heterozygotic chromosomes. (B). Methylation status of OCCs, ESCs, and pESCs. Genomic DNA isolated from these cells was subjected to bisulfite genomic sequencing analysis. The methylation levels of the promoter regions of stemness-related genes (Oct-4 and Nanog) and imprinted genes expressed differentially after parthenogenetic activation (H19, Peg3, Snrpn, and Gtl2) were compared. The PCR products were cloned, and 10 plasmid clones were sequenced for each sample. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Stemness-related genes were demethylated in all cell lines, whereas the expression of other genes differed markedly among the cell lines. The methylation in OCCs was significantly different from that in ESCs or pESCs; OCCs had more methylated H19 and Gtl2 compared with pESCs and less methylated Peg3 and Snrpn compared with ESCs. (Reprinted with permission from Gong et al., 2010; 93:2564-601).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108227&req=5

Figure 2: Analyses of the origin of ovarian colony-forming cells (OCCs). (A) Single nucleotide polymorphism (SNP) genotyping of OCCs and control cells. The heterozygosity or homozygosity of SNP loci of OCC-1 and OCC-2 of B6D2F1 strain was compared with that of B6D2F1 embryonic stem cells (ESCs), somatic fibroblasts of DBA2 and C57BL6 mice, and parthenogenetic ESCs (pESCs). Both homozygosity and heterozygosity were concomitantly detected in the OCC line. ESCs of F1 strain showed heterozygosity alone, and only homozygotic SNP loci were detected in the fibroblasts of the inbred strain. The pESC line possessed both homozygotic and heterozygotic chromosomes. (B). Methylation status of OCCs, ESCs, and pESCs. Genomic DNA isolated from these cells was subjected to bisulfite genomic sequencing analysis. The methylation levels of the promoter regions of stemness-related genes (Oct-4 and Nanog) and imprinted genes expressed differentially after parthenogenetic activation (H19, Peg3, Snrpn, and Gtl2) were compared. The PCR products were cloned, and 10 plasmid clones were sequenced for each sample. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Stemness-related genes were demethylated in all cell lines, whereas the expression of other genes differed markedly among the cell lines. The methylation in OCCs was significantly different from that in ESCs or pESCs; OCCs had more methylated H19 and Gtl2 compared with pESCs and less methylated Peg3 and Snrpn compared with ESCs. (Reprinted with permission from Gong et al., 2010; 93:2564-601).
Mentions: To further understand the origin and genetic characteristics of the two colony-forming cell lines, we conducted a short tandem repeat microsatellite analysis, which showed that the two colony-forming cell lines were an identical match to the ovary donor. Furthermore, we performed single nucleotide polymorphism genotyping and methylation analysis of the cell lines, to determine their origin. Homozygotic and heterozygotic chromosome recombinations were detected in both the colony-forming cell lines and the parthenogenetic ESC lines. The ESCs of the F1 strain possessed only heterozygotic loci, whereas homozygotic recombination was detected in the fibroblasts of both inbred strains. In the methylation analysis, No difference in the methylation of stem cell-related genes (Nanog and Oct-4) was seen among the parthenogenetic ESCs, normally fertilized ESCs, and ovarian colony-forming cells, although there was a significant difference in the methylation of several imprinted genes between ovarian colony-forming cells and parthenogenetic ESCs (Fig. 2). Based on these results, we have identified and characterized a novel colony-forming cell population that appears to be derived from either cell transformation accompanying somatic cell reprogramming or from tissue-specific stem cell-progenitor cell isolation. Nevertheless, we cannot completely exclude the tumorigenesis of ovarian somatic cells or parthenogenetic cells as possible origins. Further research is necessary to obtain conclusive results regarding cell origin and to evaluate the feasibility of clinical and industrial applications.

Bottom Line: As an initial step, we succeeded in establishing histocompatible stem cells using preantral follicle cultures and subsequent parthenogenetic activation.However, more progress regarding the establishment and elucidation on origination of established cell lines is necessary to use this genetic manipulation-free procedure.Nevertheless, relevant information on the process will help to stimulate preclinical research on cell transformation into differentiated, undifferentiated, and even cancerous cells, as well as clinical studies on the application of induced pluripotent cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: WCU Biomodulation Program, Seoul National University, Seoul 151-742, Korea. limjm@snu.ac.kr.

ABSTRACT
The establishment of patient-specific histocompatible stem cells may be an alternative for overcoming current limitations in stem cell engineering. We are developing an animal model to assist the establishment of histocompatible, autologous stem cells. In this process, we obtained valuable information on establishing and characterizing stem cells. As an initial step, we succeeded in establishing histocompatible stem cells using preantral follicle cultures and subsequent parthenogenetic activation. The gene expression profile of the established stem cells was similar to that of embryonic stem cells (ESCs) derived from normal fertilization. On the other hand, we propose a way to derive histocompatible, ESC-like cells by co-culturing ovarian stromal cells with feeder fibroblasts, which may allow the derivation of stem cells from somatic tissue. However, more progress regarding the establishment and elucidation on origination of established cell lines is necessary to use this genetic manipulation-free procedure. Nevertheless, relevant information on the process will help to stimulate preclinical research on cell transformation into differentiated, undifferentiated, and even cancerous cells, as well as clinical studies on the application of induced pluripotent cells.

No MeSH data available.


Related in: MedlinePlus