Derivation And Propagation Of Human Induced Pluripotent Stem Cells On Human Feeder Cells

Generating of human induced pluripotent stem cells (hiPSCs) from adult human somatic cells is extremely exciting. It provides a new way to obtain patient-and disease-specific pluripotent stem cells without destroying human embryos. These cells can potentially be used for cell therapy, studies on human diseases and development, as well as drug screening and toxicology. hiPSCs are typically derived and propagated through coculture with primary mouse embryonic fibroblasts (MEFs) as feeder cells. However, the use of animal feeder cells may transfer exogenous antigens and viruses to hiPSCs, which limits their clinical use. It is necessary to devise optimal systems for culturing undifferentiated hiPSC at clinically applicable culture conditions. We focused on the use of suitable human feeder cells for hiPSC derivation and propagation.Chapter1:Human Bone Marrow Mesenchymal Stem Cells Support the Derivation and Propagation of Human Induced Pluripotent Stem Cells in CultureHuman mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from bone marrow (BM), adipose tissue, umbilical cord blood, the umbilical cord, placenta, muscle, liver, and so on, and can replicate as undifferentiated cells in vitro. The unrelated hMSCs did not generate alloreactive T lymphocytes in vitro and avoided normal alloresponses in vivo. Studies have shown that culture-expanded hMSCs fully support prolonged hESCs expansion in cultures; these expanded hESCs maintained their pluripotency and normal diploid karyotype. Thus, we determined whether hMSCs could be used as feeder layers to support the derivation and propagation of hiPSCs.Our results first show that inactivated hMSCs from different donors could support the derivation and expansion of hiPSCs. The human foreskin fibroblasts (HFF) were infected with the retroviral vectors. Six days after transduction, the cells were harvested by trypsinization and plated onto inactivated hMSC feeder cells. After approximately3weeks, well-defined colonies were picked up and cultured continuously on inactivated hMSC feeder cells with hESC medium. The cells formed typical hESC colonies.Some of two hiPSCs lines were analyzed for the expression of hESC-specific surface antigens. By AP staining and immunofluorescence analysis, the hiPSC colonies were strongly positive for AP, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81and NANOG By RT-PCR, many undifferentiated hESC marker genes were detected in the hiPSCs cultured on hMSCs. The markers included endo-KLF4, endo-SOX2, endo-cMyc, endo-OCT4, NANOQ DNMT3B, DPPA4, hTERT, NODAL, REX1, and TDGF1.The hiPSCs derived and propagated on hMSC feeder cells could be differentiated into derivatives of all the three germ layers in vitro and in vivo. And after more than14passages, standard G-banding chromosome analysis revealed that both hiPSC lines retained the same normal karyotype (46, XY), which suggests expansion hiPSCs on hMSC feeder cells retained a normal chromosomal karyotype.These findings suggest that hMSCs can be used as feeder cells to derive and maintain hiPSCs. This novel animal cell-free culture system provides another clinically feasible method for generating and expanding hiPSCs.Chapter2:Auto-fibroblasts Support the Derivation and Propagation of Human Induced Pluripotent Stem Cells in CultureIn the course of our test, we also noticed that after transduction most of the HFFs kept their original morphology and their proliferation ability. After inactivated via mitomycin C treatment, MEF lost their proliferation ability. One week after that the infected fibroblasts were plated onto inactivated feeder layers, it was hard to find out any MEF in the culture system. We speculated that in the later of reprogramming, infected HFFs who did not reprogram supported the reprogramming of other HFFs. In this study we wanted to study if auto-HFFs could support the derivation and propagation of hiPSCs.After infected with retroviral vectors, HFFs were cultured without collection by trypsinization and planting onto other feeder cells. About3weeks later, well-defined colonies appeared, picked up and cultured continuously on inactivated auto-HFF feeder cells. The cells formed typical hESC coloies. One of the hiPSCs lines was analyzed for the expression of hESC-specific surface antigens. By immunofluorescence analysis, the hiPSC colonies were strongly positive SSEA-4, TRA-1-60, TRA-1-81and NANOG By RT-PCR, many undifferentiated hESC marker genes were detected in the hiPSCs cultured on auto-HFF feeder cells. The markers included endo-KLF4, endo-SOX2, endo-cMyc, endo-OCT4, NANOG, DNMT3B, hTERT, DPPA4and NODAL. The hiPSCs derived and propagated on auto-HFF feeder cells could be differentiated into derivatives of all the three germ layers in vitro and in vivo. And after more than21passages, standard G-banding chromosome analysis revealed that the hiPSC line retained the same normal karyotype (46, XY), which suggests expansion hiPSCs on auto-HFF feeder cells retained a normal chromosomal karyotype.Our results first show that auto-HFFs who did not reprogram could supported the reprogramming of other HFFs and the expansion of hiPSCs. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder cells of hiPSCs. It is another important step toward the establishment of clinical grade hiPSCsChapter3:Generation Efficiency and Growth Kinetics of hiPSCs on Different Type of Feeder Cells Feeder cell layers can be viewed as a niche to support somatic cell reprogramming and maintain hiPSC self-renewal and pluripotency. In this study we wanted to study the influence of different type of feeder cells on the generation efficiency and growth kinetics of hiPSCs.To compare directly the generation efficiency of hiPSCs on hMSC feeder cells, auto HFF feeder cells with that on MEF feeder cells, all the obtained well-defined, hESCs-like colonies were counted on the three types of feeder layers. After three independent experiments, the rates of hiPSC generation on hMSC and auto-HFF feeder cells were7.26%±2.09%(P<0.05) and37.08%±5.63%(P<0.05) respectively compared to that on MEF feeder cells. Even used the MEF conditioned medium, the rates of hiPSC generation on hMSC and auto-HFF feeder cells didn’t increased. The calculated expansion efficiencies of the hiPSCs on hMSC, auto-HFF, MEF feeder cells were2.31±0.12(P<0.05),2.66±0.17(P<0.05) and4.77±0.64respectively. Also, hiPSC expansion on hMSC, auto-HFF feeder cells tended to differentiate. We founded out that the undifferentiated clonings significantly reduced cultured on hMSC, auto-HFF feeder cells after5days, while on MEF feeder cells only a litter clonings differentiated. The expression of TRA-1-60on hiPSCs on hMSC, auto-HFF feeder cells was also lower than that on MEF feeder cells.Our study first indicated that both the generation efficiency and the expansion efficiency of hiPSCs on hMSC, auto-HFF feeder cells was lower than that on MEF feeder cells. hiPSC expansion on hMSC, auto-HFF feeder cells tended to differentiate. We should also determine the molecular mechanism of reprogramming, hiPSC self-renewal and growth, and develop a preferable clinical culture condition for hiPSCs.