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Pluripotent Stem Cells

Differentiation Efficiency of Induced Pluripotent Stem Cells Depends on the Number of Reprogramming Factors

From Stem Cells
By Stuart P. Atkinson

Multiple studies in the last few years have attempted the production of induced pluripotent stem cells (iPSCs) with a reduced set of reprogramming factors (from OCT4, SOX2, KLF4, MYC or OCT4, SOX2, LIN28 and NANOG) in order that the process becomes streamlined in the hope that this may reduce potential mutation load in the resultant cells. This has been accomplished by many groups, resulting in iPSC formation at a lower efficiency, but the effect of a reduction in reprogramming factors on the subsequent differentiation capacity has not been fully explored. A report in the March edition of Stem Cells, from the laboratory of Alexander Storch now addresses this point, and finds that a reduction of reprogramming factors not only reduces reprogramming efficiency but negatively affects differentiation (Löhle et al).

Elevated Coding Mutation Rate During the Reprogramming of Human Somatic Cells into Induced Pluripotent Stem Cells

From Stem Cells
By Stuart P. Atkinson

Recent studies in the field of induced pluripotent stem cells (iPSCs) through karyotypic (Taapken et al) and meta-analysis of gene expression data (Mayshar et al) have revealed aneuploidy, and also copy number analysis has detected large-scale sub-chromosomal aberrations (Laurent­­ et al and Martins-Taylor et al) that arise upon prolonged passaging. Further, it is known that mutations not present in the parental cell of reprogramming arise during the reprogramming process (Gore et al and Hussein et al), but the proportion of mutations in iPSCs acquired due to the reprogramming process is unknown.

New Human Embryonic Stem Cell Line Added to U.S. Registry

The University of Michigan's first human embryonic stem cell line will be placed on the U.S. National Institutes of Health's registry, making the cells available for federally funded research. The university is among just a handful of U.S. universities creating human embryonic stem cell lines. There are only 147 stem cell lines currently available on the registry.

The line, known as UM4-6, is a genetically normal line, derived in October 2010 from a cluster of about 30 cells removed from a donated five-day-old embryo.

"This is significant, because acceptance of these cells on the registry demonstrates our attention to details of proper oversight, consenting and following of NIH guidelines established in 2009," says Gary Smith, Ph.D., who derived the line and also is co-director of the University of Michigan (UM) Consortium for Stem Cell Therapies, part of the A. Alfred Taubman Medical Research Institute. "It now makes the line available to researchers who can apply for federal funding to use it in their work; this is an important step."

The line was made possible by Michigan voters' November 2008 approval of a state constitutional amendment permitting scientists in Michigan to derive embryonic stem cell lines using surplus embryos from fertility clinics or embryos with genetic abnormalities and not suitable for implantation.

"We envision in the future that investigators will be able to use the genetically normal embryonic stem cell lines like UM4-6, together with disease-specific embryonic stem cell lines, as a model system to investigate what causes these diseases and come up with treatments," says Sue O'Shea, professor of cell and developmental biology and co-director of the Consortium for Stem Cell Therapies.

UM also has submitted two other human embryonic stem cells lines to the national registry. Both are disease-specific, the first carrying the genetic defect that causes hemophilia B and the other carrying the gene responsible for Charcot-Marie-Tooth disease, a hereditary neurological disorder.

Smith expects to soon submit eight additional human embryonic stem lines for consideration on the national registry, including three genetically normal and five new disease-specific lines.

Learn more:
www.taubmaninstitute.org
www.umich.edu/stemcell

Control of Ground-state Pluripotency by Allelic Regulation of Nanog

The attainment of pluripotency during development has been linked to genome-wide epigenetic changes, such as DNA methylation and histone modifications, and also to the expression of key genes, such as Oct4 and Nanog (Chambers et al, Mitsui et al, Nichols et al and Reik). However, details on how such epigenetic changes affect key pluripotency-associated gene expression in pre-and post-implantation embryo’s and also in embryonic stem cells (ESCs) are relatively scarce. Now, in a report published in Nature, Miyanari and Torres-Padilla from the IGBMC, Universitaire de Strasbourg, France, Nanog has been shown to undergo a switch from mono-allelic expression in early pre-implantation embryos to bi-allelic expression during the transition towards ground-state pluripotency in the naive epiblast of the late blastocyst, controlled in part by histone modifications.

Attenuation of extrinsic signaling reveals the importance of matrix remodeling on maintenance of ESC self-renewal

From PNAS
By Stuart P. Atkinson

The mammalian embryo requires properly controlled extrinsic signalling for normal development. Autocrine and paracrine signals are also important in blastocyst-derived embryonic stem cell (ESC) self-renewal (Bendall et al), growth (Mittal and Voldman) and differentiation (Kunath et al and Peerani et al). Understanding the range of cell-secreted factors that mediate autocrine and paracrine signalling which are important for self-renewal would enhance our comprehension of early embryonic fate choices and for exploiting the therapeutic potential of these cells, but only a few factors, which are saturated in culture by exogenous addition, are actually known. To this end Przybylaa and Voldman from the Hospital for Sick Children, University of Toronto, have studied mouse ESC (mESC) growth using a microfluidic perfusion system, in which cell-secreted diffusible molecules can be removed by flow, establishing culture conditions in which signaling pathways are not obscured by cell-secreted signals.

iPSCs Prove Worthy for Alzheimer’s Modelling: Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells

From Nature
By Stuart P. Atkinson

Alzheimer’s disease has been associated with certain molecular events, such as the presence of amyloid-β containing plaques (Citron et al and Scheuner et al), neurofibrillary tangles of hyperphosphorylated Tau (MAPT), a microtubule-associated protein (Ballatore et al and Arriagada et al), the co-localisation of Tau with the kinase GSK3b (Cho and Johnson), and the accumulation of large RAB5+ early endosomes in neurons (Cataldo et al). Induced pluripotent stem cell (iPSC) technology has allowed the modelling of several neurological diseases in vitro (Ebert et al, Nguyen et al and Qiang et al) but a full investigation of Alzheimer’s disease and its different forms has yet to fully appreciated. Now, researchers from the laboratory of Lawrence S. B. Goldstein at the Department of Cellular and Molecular Medicine, University of California present data in a report published in Nature regarding the production of iPSC cells from both familiar and sporadic Alzheimer’s disease patients and their differentiation to neurons which carry the phenotype of the disease making them useful for modelling the disease in vitro (Israel et al).

Generation of Chimeric Rhesus Monkeys

From Cell
By Stuart P. Atkinson

A recent research article in Cell from the laboratory of Shoukhrat Mitalipov at the Oregon National Primate Research Center at the Oregon Health & Science University has reported the generation of the first chimaeras from a non-human primate (Tachibana et al). In mouse, the ability to contribute to chimeric animals upon re-introduction into host embryos is the key feature of totipotent and pluripotent cells, and while chimaeric animals have been produced in other mammals (rats, rabbits, sheep and cattle) this had not been extended to non-human primates. Further as relatively little is known about human and non-human primate embryo development and lineage specification and how closely the mouse development reflects primates, research such as this promises to assess the usefulness of mouse models and mouse embryonic stem cells (mESCs) to human embryonic stem cell (hESC) biology

The Living Dead (of the iPSC world): Autopsy donor-derived iPSCs

From Neuroscience Letters
Commentary by Carla B. Mellough

In vitro disease modelling approaches largely involve the use of immortalised cell lines that have been genetically altered in order to induce a disease phenotype. While these systems provide valuable information, such models are unable to represent complex human disease aetiology and are therefore not always physiologically relevant. As induced pluripotent stem cells (iPSCs) retain the genetic profile of the somatic donor cell of origin, iPSCs represent an important additional option for disease modelling in vitro. This approach offers many advantages over previous methods, for example the non-invasive study of neurological or neurodegenerative conditions that are ordinarily impossible premortem, or certainly risk some cognitive or functional impairment if undertaken. Yet one major complication of this approach is that the effectiveness of iPSC disease models in vitro relies entirely upon the accuracy of the premortem diagnosis. In fact, most premortem diagnoses of neurological disease made from clinical criteria are not definite and can only be confirmed following postmortem histopathological analysis, so it would be advantageous if we could generate iPSC from post-mortem tissues. A study from Arizona, USA, by Hjelm et al.1 has demonstrated that iPSCs can indeed be generated from autopsy-derived fibroblasts. Creating iPSC lines from dead human tissues may be viewed by some as a little macabre, hence our reference to the 1974 film “The living dead” in the title, but in reality this development opens another avenue for iPSC-based disease modelling following a definite postmortem diagnosis.

Skeletogenic phenotype of human Marfan ESCs faithfully phenocopied by patient-specific iPSCs

From PNAS
By Stuart P. Atkinson

Marfan syndrome (MFS) is a heritable dominant disorder caused by mutations in the FBN1 gene (Dietz et al and Pereira et al) affecting the skeletal, ocular and cardiovascular systems. FBN1 itself is an extracellular matrix (ECM) glycoprotein and although the molecular pathogenesis was originally thought to be due to resultant defects in the ECM, other results suggested that altered TGFβ signalling may be the main cause of pathogenic abnormalities in MFS (Dietz et al and Liu et al). Now, researchers from the laboratory of Michael Longlaker at the Standford University School of Medicine have succeeded in deriving FBN1 mutant human embryonic stem cells (hESCs) and also producing human induced pluripotent stem cells (hiPSCs) from FBN1 mutant fibroblasts (Quarto et al). Importantly, during this study of FBN1 mutations in hESCs, they also found that mutant hESCs and hiPSCs give rise to differentiated cells which demonstrate the same phenotype, demonstrating that iPSCs can provide complementary and powerful tools to gain further insights into human molecular pathogenesis.

ESCs JNK their way to Neurogenesis – A chromatin-modifying function of JNK during stem cell differentiation

From Nature Genetics
By Stuart P. Atkinson

c-Jun N-terminal kinases (JNKs) are a subgroup of mitogen-activated protein kinases (MAPKs), and have been suggested to mediate transcriptional changes through their downstream effectors (Bogoyevitch and Kobe and Pearson et al), such as AP-1 (a heterodimeric transcription factor composed of proteins belonging to the c-Fos, c-Jun, ATF and JDP families). Additionally, some signal-activated kinases have been suggested to bind to chromatin at certain genes. Mouse embryonic stem cells (mESCs) lacking JNK1 (Mapk8) show dysregulation of lineage-commitment genes (Amura et al) and fail to undergo neuronal differentiation, suggesting a vital role for JNK1 in the lineage specific differentiation of mESCs. Researchers from the group of Christian Beisel from the Eidgenössische Technische Hochschule (ETH) Zürich, Basel, Switzerland have now undertaken a detailed analysis of the JNK proteins in mESCs during self renewal and differentiation, finding that gene specific JNK-mediated chromatin modification is critical for neuronal differentiation (Tiwari et al).

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