Michiko Hirose

Generation of Induced Pluripotent Stem Cells in Rabbits: POTENTIAL EXPERIMENTAL MODELS FOR HUMAN REGENERATIVE MEDICINE*

Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2, and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of basic FGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4, and NANOG, whereas the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm, and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state, showing high similarity to their embryonic stem cell counterparts we generated recently. However, their global gene expression analysis revealed a slight but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and embryonic stem cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans.

Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer

In general, cloning undifferentiated preimplantation embryos (blastomeres) or embryonic stem cells is more efficient than cloning differentiated somatic cells. Therefore, there has been an assumption that tissue-specific stem cells might serve as efficient donors for nuclear transfer because of the undifferentiated state of their genome. Here, we show that this is not the case with adult hematopoietic stem cells (HSCs). Although we have demonstrated for the first time that mouse HSCs can be cloned to generate offspring, the birth rates (0-0.7%) were lowest among the clones tested (cumulus, immature Sertoli and fibroblast cells). Only 6% of reconstructed embryos reached the morula or blastocyst stage in vitro (versus 46% for cumulus clones; P<5×10-10). Transcription and gene expression analyses of HSC clone embryos revealed that they initiated zygotic gene activation (ZGA) at the appropriate timing, but failed to activate five out of six important embryonic genes examined, including Hdac1 (encoding histone deacetylase 1), a key regulator of subsequent ZGA. These results suggest that the HSC genome has less plasticity than we imagined, at least in terms of reprogrammability in the ooplasm after nuclear transfer.

Isolation, characterization, and in vitro and in vivo differentiation of putative thecal stem cells

Although ovarian theca cells play an indispensable role in folliculogenesis by providing follicular structural integrity and steroid substrates for estrogen production, little information is available about their recruitment, growth, and differentiation because their immature forms have not been identified. We have isolated putative thecal stem cells with the ability to self-renew and differentiate in vivo and in vitro. They are similar to fibroblasts in morphology and proliferate in vitro as round colonies with a homogenous cell population. They were induced to differentiate into early precursors and steroidogenic cells in a stepwise manner after treatment with serum, luteinizing hormone, and paracrine factors from granulosa cells. At each differentiation step, these cells displayed appropriate gene expression and morphological markers and later secreted androstenedione. The fully mature morphology was achieved by coculture with isolated granulosa cells. When transplanted into the ovaries, the putative thecal stem cells colonized exclusively in the ovarian interstitium and the thecal layer of follicles as differentiated cells. Thus, thecal stem cells appear to be present in neonatal ovaries and can be isolated, purified, and induced to differentiate in vitro. Thecal stem cells could provide an invaluable in vitro experimental system to study interactions among the oocytes, granulosa cells, and theca cells during normal folliculogenesis and to study ovarian pathology caused by theca cell dysfunction.

BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin.

Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast.

Basic FGF and Activin/Nodal but not LIF signaling sustain undifferentiated status of rabbit embryonic stem cells.

Recently, we proposed that rabbit embryonic stem (ES) cells can be stable mammalian ES cells and can be a small animal model for human ES cell research. However, the signaling pathways controlling rabbit ES cell pluripotency remain largely unknown. Here we report that bFGF can maintain the undifferentiated status of rabbit ES cells and found that Activin/Nodal signaling through Smad2/3 activation is necessary to maintain the pluripotent status of rabbit ES cells. We further show that in spite of STAT3 in rabbit ES cells, LIF is dispensable for maintenance of undifferentiated status in rabbit ES cells. Although phosphorylation of Janus Kinase signal transducer and activator (JAK/STAT) disappeared after JAK-inhibitor treatment, OCT4 is constantly produced. When rabbit ES cells were cultured for more than 40 passages in the absence of LIF, expression of stem cell markers and teratoma formation were observed. Additionally, treatment with Rho-associated kinase (ROCK) inhibitor, Y27632, to rabbit ES cells significantly enhanced cell growth. These findings suggest that molecular mechanisms underlying rabbit ES cell self-renewal and pluripotency are similar to primate ES cells. Rabbit ES cells may provide a translational research model for the study of human diseases in vitro and applications to transplantation therapy.

Stable embryonic stem cell lines in rabbits: potential small animal models for human research

Although embryonic stem (ES) cell lines derived from mice and primates are used extensively, the development of such lines from other mammals is extremely difficult because of their rapid decline in proliferation potential and pluripotency after several passages. This study describes the establishment of rabbit ES cell lines with indefinite proliferation potential. It was found that the feeder cell density determines the fate of rabbit ES cells, and that maximum proliferation potential was obtained when they were cultured on a feeder cell density of one-sixth of the density at confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Under optimized conditions, rabbit ES cells were passaged 50 times, after which they still possessed high telomerase activity. This culture system enabled efficient gene transduction and clonal expansion from single cells. During culture, rabbit ES cells exhibited flattened monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo respectively. These ES cell lines can be safely cryopreserved for later use. Thus, rabbit ES cells can be added to the list of stable mammalian ES cells, enabling the rabbit to be used as a small animal model for the study of human cell transplantation therapy.

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.

Regulation of pluripotency in male germline stem cells by Dmrt1

Spermatogonial stem cells (SSCs) present the potential to acquire pluripotency under specific culture conditions. However, the frequency of pluripotent cell derivation is low, and the mechanism of SSC reprogramming remains unknown. In this study, we report that induction of global DNA hypomethylation in germline stem (GS) cells (cultured SSCs) induces pluripotent cell derivation. When DNA demethylation was triggered by Dnmt1 depletion, GS cells underwent apoptosis. However, GS cells were converted into embryonic stem (ES)-like cells by double knockdown of Dnmt1 and p53. This treatment down-regulated Dmrt1, a gene involved in sexual differentiation, meiosis, and pluripotency. Dmrt1 depletion caused apoptosis of GS cells, but a combination of Dmrt1 and p53 depletion also induced pluripotency. Functional screening of putative Dmrt1 target genes revealed that Dmrt1 depletion up-regulates Sox2. Sox2 transfection up-regulated Oct4 and produced pluripotent cells. This conversion was enhanced by Oct1 depletion, suggesting that the balance of Oct proteins maintains SSC identity. These results suggest that spontaneous SSC reprogramming is caused by unstable DNA methylation and that a Dmrt1-Sox2 cascade is critical for regulating pluripotency in SSCs.

A high-speed congenic strategy using first-wave male germ cells.

Background In laboratory mice and rats, congenic breeding is essential for analyzing the genes of interest on specific genetic backgrounds and for analyzing quantitative trait loci. However, in theory it takes about 3–4 years to achieve a strain carrying about 99% of the recipient genome at the tenth backcrossing (N10). Even with marker-assisted selection, the so-called ‘speed congenic strategy’, it takes more than a year at N4 or N5. Methodology/Principal Findings Here we describe a new high-speed congenic system using round spermatids retrieved from immature males (22–25 days of age). We applied the technique to three genetically modified strains of mice: transgenic (TG), knockin (KI) and N-ethyl-N-nitrosourea (ENU)-induced mutants. The donor mice had mixed genetic backgrounds of C57BL/6 (B6)∶DBA/2 or B6∶129 strains. At each generation, males used for backcrossing were selected based on polymorphic marker analysis and their round spermatids were injected into B6 strain oocytes. Backcrossing was repeated until N4 or N5. For the TG and ENU-mutant strains, the N5 generation was achieved on days 188 and 190 and the proportion of B6-homozygous loci was 100% (74 markers) and 97.7% (172/176 markers), respectively. For the KI strain, N4 was achieved on day 151, all the 86 markers being B6-homozygous as early as on day 106 at N3. The carrier males at the final generation were all fertile and propagated the modified genes. Thus, three congenic strains were established through rapid generation turnover between 41 and 44 days. Conclusions/Significance This new high-speed breeding strategy enables us to produce congenic strains within about half a year. It should provide the fastest protocol for precise definition of the phenotypic effects of genes of interest on desired genetic backgrounds.

Naive-like conversion overcomes the limited differentiation capacity of induced pluripotent stem cells

Background: The quality of induced pluripotent stem (iPS) cells might be inherently worse than embryonic stem cells. Results: Although the differentiation capacity of iPS cells is limited, it can be enhanced. Conclusion: Improving their level of pluripotency alleviated the limited capacity of iPS cells. Significance: This report offers an effective strategy for the development of iPS cell-based research. Although induced pluripotent stem (iPS) cells are indistinguishable from ES cells in their expression of pluripotent markers, their differentiation into targeted cells is often limited. Here, we examined whether the limited capacity of iPS cells to differentiate into neural lineage cells could be mitigated by improving their base-line level of pluripotency, i.e. by converting them into the so-called “naive” state. In this study, we used rabbit iPS and ES cells because of the easy availability of both cell types and their typical primed state characters. Repeated passages of the iPS cells permitted their differentiation into early neural cell types (neural stem cells, neurons, and glial astrocytes) with efficiencies similar to ES cells. However, unlike ES cells, their ability to differentiate later into neural cells (oligodendrocytes) was severely compromised. In contrast, after these iPS cells had been converted to a naive-like state, they readily differentiated into mature oligodendrocytes developing characteristic ramified branches, which could not be attained even with ES cells. These results suggest that the naive-like conversion of iPS cells might endow them with a higher differentiation capacity.