In vitro and in vivo differentiation of induced pluripotent stem cells into male germ cells.
The introduction of induced pluripotent stem cell (iPSC) lines has been a breakthrough in the field of stem cell research. However, the extent of pluripotency among those cell lines tends to be variable due to their different epigenetic signatures. Mouse iPS cell line 4.1 has been established via retroviral transfer of human transcription factors Oct4, Sox2, Klf4, and c-Myc; the germline competence of this line has not been determined. In the present study, we induced the differentiation of miPS-4.1 cells into male germ cells, in vivo and in vitro. In the in vitro model, the behavior of miPS-4.1 cells was identical to that of differentiating mouse embryonic stem cells (ESCs). We obtained primordial germ cell-like cells (PGC-LC) that were positive for alkaline phosphatase (AP) activity. In continuous culture, these cells expressed pluripotent marker Oct4 and male germline markers C-kit and MVH. For our in vivo model, miPS-4.1 cells were co-transplanted with neonatal testicular cell suspension. We observed ectopically reconstituted seminiferous tubule structures, in which the miPS-4.1 cells were homing and developing. In conclusion, we successfully induced the differentiation of miPS-4.1 cells into male germ cells, albeit their epigenetic characteristics. Our study provides a system to examine the mechanisms of male germ cell development and might help to supply an effective treatment for male infertility in the future.
Cai H
，Xia X
，Wang L
，Liu Y
，He Z
，Guo Q
，Xu C
... -
《-》
A novel model of urinary tract differentiation, tissue regeneration, and disease: reprogramming human prostate and bladder cells into induced pluripotent stem cells.
Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.
To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.
Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.
The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.
Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.
IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.
Moad M
，Pal D
，Hepburn AC
，Williamson SC
，Wilson L
，Lako M
，Armstrong L
，Hayward SW
，Franco OE
，Cates JM
，Fordham SE
，Przyborski S
，Carr-Wilkinson J
，Robson CN
，Heer R
... -
《-》
Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4.
The development of human induced pluripotent stem cells (iPSCs) holds great promise for regenerative medicine. However the iPSC induction efficiency is still very low and with lengthy reprogramming process. We utilized the highly potent transactivation domain (TAD) of MYC protein to engineer the human OCT4 fusion proteins. Applying the MYC-TAD-OCT4 fusion proteins in mouse iPSC generation leads to shorter reprogramming dynamics, with earlier activation of pluripotent markers in reprogrammed cells than wild type OCT4 (wt-OCT4). Dramatic enhancement of iPSC colony induction efficiency and shortened reprogramming dynamics were observed when these MYC-TAD-OCT4 fusion proteins were used to reprogram primary human cells. The OCT4 fusion proteins induced human iPSCs are pluripotent. We further show that the MYC Box I (MBI) is dispensable while both MBII and the linking region between MBI/II are essential for the enhanced reprogramming activity of MYC-TAD-OCT4 fusion protein. Consistent with an enhanced transcription activity, the engineered OCT4 significantly stimulated the expression of genes specifically targeted by OCT4-alone, OCT4/SOX2, and OCT4/SOX2/KLF4 during human iPSC induction, compared with the wt-OCT4. The MYC-TAD-OCT4 fusion proteins we generated will be valuable tools for studying the reprogramming mechanisms and for efficient iPSC generation for humans as well as for other species.
Wang L
，Huang D
，Huang C
，Yin Y
，Vali K
，Zhang M
，Tang Y
... -
《-》
Induced pluripotent stem cells from goat fibroblasts.
Embryonic stem cells (ESCs) are a powerful model for genetic engineering, studying developmental biology, and modeling disease. To date, ESCs have been established from the mouse (Evans and Kaufman, 1981, Nature 292:154-156), non-human primates (Thomson et al., , Proc Nat Acad Sci USA 92:7844-7848), humans (Thomson et al., 1998, Science 282:1145-1147), and rats (Buehr et al., , Cell 135:1287-1298); however, the derivation of ESCs from domesticated ungulates such as goats, sheep, cattle, and pigs have not been successful. Alternatively, induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors (OCT3/4, SOX2, KLF4, cMYC, LIN28, and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs (Ren et al., 2011, Cell Res 21:849-853). The objectives of this study were to generate goat iPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human transcription factors: OCT4, SOX2, KLF4, and cMYC. The goat iPSCs were successfully generated by co-culture with mitomycin C-treated mouse embryonic fibroblasts using medium supplemented with knockout serum replacement and human basic fibroblast growth factor. The goat iPSCs colonies are flat, compact, and closely resemble human iPSCs. They have a normal karyotype; stain positive for alkaline phosphatase, OCT4, and NANOG; express endogenous pluripotency genes (OCT4, SOX2, cMYC, and NANOG); and can spontaneously differentiate into three germ layers in vitro and in vivo.
Song H
，Li H
，Huang M
，Xu D
，Gu C
，Wang Z
，Dong F
，Wang F
... -
《-》
ResearchGate
(全网免费下载)
钛学术
(全网免费下载)
