Paul J. Verma

Generation of Induced Pluripotent Stem Cell Lines from Friedreich Ataxia Patients

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterised by neurodegeneration and cardiomyopathy. It is caused by a trinucleotide (GAA) repeat expansion in the first intron of the FXN gene that results in reduced synthesis of FXN mRNA and its protein product, frataxin. We report the generation of induced pluripotent stem (iPS) cell lines derived from skin fibroblasts from two FRDA patients. Each of the patient-derived iPS (FA-iPS) cell lines maintain the GAA repeat expansion and the reduced FXN mRNA expression that are characteristic of the patient. The FA-iPS cells are pluripotent and form teratomas when injected into nude mice. We demonstrate that following in vitro differentiation the FA-iPS cells give rise to the two cell types primarily affected in FRDA, peripheral neurons and cardiomyocytes. The FA-iPS cell lines have the potential to provide valuable models to study the cellular pathology of FRDA and to develop high-throughput drug screening assays. We have previously demonstrated that stable insertion of a functional human BAC containing the intact FXN gene into stem cells results in the expression of frataxin protein in differentiated neurons. As such, iPS cell lines derived from FRDA patients, following correction of the mutated gene, could provide a useful source of immunocompatible cells for transplantation therapy.

NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts1

Since the first reports on isolation of pluripotent mouse embryonic stem (ES) cells 3 decades ago, there have been numerous attempts to derive ES cell lines from commercially important livestock species with limited success. The recent discovery that ectopic expression of a handful of stem cell-related genes was capable of inducing pluripotency in rodents and primates provided a novel approach to derivation of pluripotent stem cell lines. We used this approach in cattle and demonstrated that the ectopic expression of POU5F1 (also known as Oct4), SOX2, KLF4, and c-MYC alone was not sufficient for stable induction of pluripotency in bovine adult fibroblasts and that the additional expression of NANOG to the reprogramming cocktail was essential for the generation of stable bovine (b) induced pluripotent stem (iPS) cells. The resulting biPS cells were characterized by reverse-transcription PCR for a panel of ES marker genes. Immunocytochemical localization of POU5F1, SSEA-1, SSEA-4, and colorimetric alkaline phosphatase activity was measured in the iPS clones. The differentiation potential of the biPS cells was determined in vitro by expression of differentiation markers in embryoid bodies. Injection of biPS into immunocompromised mice resulted in teratomas containing cell types of the 3 germ lineages. This study reports the first generation of bovine induced pluripotent cell lines and paves the way for the use of biPS cells for biotechnological and agricultural purposes.

The efficient generation of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells.

Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 ± 2.62%, ADCs 70.61 ± 15.4%, NSCs, 68.72 ± 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 ± 4.10 colonies, 0.13 ± 0.06%) and ADCs (118.20 ± 38.28 colonies, 1.14 ± 0.77%) when compared with the mEF control (3.17 ± 0.29 colonies, 0.03 ± 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Flow characterization of a spinner flask for induced pluripotent stem cell culture application.

We present detailed quantitative measurement analyses for flow in a spinner flask with spinning rates between 20 to 45 RPM, utilizing the optical velocimetry measurement technique of Particle Image Velocimetry (PIV). A partial section of the impeller was immersed in the working fluid to reduce the shear forces induced on the cells cultured on microcarriers. Higher rotational speeds improved the mixing effect in the medium at the expense of a higher shear environment. It was found that the mouse induced pluripotent stem (iPS) cells achieved the optimum number of cells over 7 days in 25 RPM suspension culture. This condition translates to 0.0984 Pa of maximum shear stress caused by the interaction of the fluid flow with the bottom surface. However, inverse cell growth was obtained at 28 RPM culture condition. Such a narrow margin demonstrated that mouse iPS cells cultured on microcarriers are very sensitive to mechanical forces. This study provides insight to biomechanical parameters, specifically the shear stress distribution, for a commercially available spinner flask over a wide range of Reynolds number.

Generation and characterization of reprogrammed sheep induced pluripotent stem cells.

Embryonic stem cells (ESCs) from domestic species have numerous potential applications in agricultural and biomedical sciences; however, despite intensive efforts, derivation of ESCs from sheep remains elusive. The objective was to derive sheep induced pluripotent stem cells (iPSCs), as an alternative pluripotent cell type to ESCs, from sheep fibroblasts by ectopic expression of heterologous transcription factors OCT4, SOX2, KLF4, and cMYC. Sheep fibroblasts were infected with pantropic retroviruses coding the four transcription factors and reprogrammed to pluripotency at a rate of 0.002%. The sheep iPSCs (siPSCs) reactivated endogenous OCT4 and SOX2 genes assessed by qRT-PCR and immuno-cytochemistry, retained normal karyotyping, and more importantly, concurrently silenced all exogenous transgenes. The siPSCs were enzymatically dissociated to single cells, making them amenable to efficient transfection and fluorescent-activated cell sorting techniques. Further, the siPSCs differentiated in vitro to form embryoid bodies, and in vivo to form robust teratomas, containing cells representative of the three germ layers. Moreover, when injected into diploid or tetraploid sheep embryos, siPSCs contributed to the inner cell mass of resulting blastocysts, suggesting true pluripotential. These reprogrammed siPSCs may constitute a robust pluripotent alternative to elusive sheep ESCs, with great potential for use in agriculture and pharmaceutical biotechnology.

The effects of nuclear reprogramming on mitochondrial DNA replication

Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers.

Effect of DNA concentration on transgenesis rates in mice and pigs.

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10 ng/μl, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5 ng/μl and then decreased at 10 ng/μl for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10 ng/μl to maximise integration and efficiency rates in pigs.

Cardiogenesis of Embryonic Stem Cells with Liquid Marble Micro-Bioreactor

A liquid marble micro-bioreactor is prepared by placing a drop of murine embryonic stem cell (ESC) (Oct4B2-ESC) suspension onto a polytetrafluoroethylene (PTFE) particle bed. The Oct4B2-ESC aggregates to form embryoid bodies (EBs) with relatively uniform size and shape in a liquid marble within 3 d. For the first time, the feasibility of differentiating ESC into cardiac lineages within liquid marbles is being investigated. Without the addition of growth factors, suspended EBs from liquid marbles express various precardiac mesoderm markers including Flk-1, Gata4, and Nkx2.5. Some of the suspended EBs exhibit spontaneous contraction. These results indicate that the liquid marble provides a suitable microenvironment to induce EB formation and spontaneous cardiac mesoderm differentiation. Some of the EBs are subsequently plated onto gelatin-coated tissue culture dishes. Plated EBs express mature cardiac markers atrial myosin light chain 2a (MLC2a) and ventricular myosin light chain (MLC2v), and the cardiac structural marker α-actinin. More than 60% of the plated EBs exhibit spontaneous contraction and express mature cardiomyocyte marker cardiac troponin T (cTnT), indicating that these EBs have differentiated into functional cardiomyocytes. Together, these results demonstrate that the liquid-marble technique is an easily employed, cost effective, and efficient approach to generate EBs and facilitating their cardiogenesis.

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

The state of the art for pluripotent stem cells derivation in domestic ungulates

Since the successful isolation, characterization and long-term culture of embryonic stem cells (ESCs) from mice in the early 1980s and from humans a decade later, considerable effort has been made to establish ESCs lines from livestock. The derivation of validated ESCs lines is a necessary step if the generation of economically relevant transgenic animals is to be achieved. However, this is still elusive, as the isolation of true ESCs lines for livestock has not been accomplished to date. It has been demonstrated that by forced expression of a defined set of transcription factors, it is possible to reprogram somatic cells to cells that closely resemble an ES-like state. These cells were termed induced pluripotent stem cells (iPSCs). We introduce the basic concepts relating to stem cell biology and give an overview of the various attempts to isolate and generate pluripotent stem cells (PSCs) from species relevant to livestock production. Further, we point out the issues to be addressed and hurdles to be overcome to realize the promise of stem cells in agriculture.