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Concept: Induced pluripotent stem cell

247

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid, we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells, whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine.

Concepts: DNA, Gene, Genetics, Gene expression, Developmental biology, Stem cell, Transcription factor, Induced pluripotent stem cell

187

Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%-0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33(+)CD45(+)CD34(-) myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG(+)TRA-1-81(+) hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self-renewal and differentiation in both hematopoietic progenitors and ESC.

Concepts: Developmental biology, Stem cell, Stem cells, Cell biology, Cellular differentiation, Embryonic stem cell, Induced pluripotent stem cell, Pluripotency

180

The integumentary organ system is a complex system that plays important roles in waterproofing, cushioning, protecting deeper tissues, excreting waste, and thermoregulation. We developed a novel in vivo transplantation model designated as a clustering-dependent embryoid body transplantation method and generated a bioengineered three-dimensional (3D) integumentary organ system, including appendage organs such as hair follicles and sebaceous glands, from induced pluripotent stem cells. This bioengineered 3D integumentary organ system was fully functional following transplantation into nude mice and could be properly connected to surrounding host tissues, such as the epidermis, arrector pili muscles, and nerve fibers, without tumorigenesis. The bioengineered hair follicles in the 3D integumentary organ system also showed proper hair eruption and hair cycles, including the rearrangement of follicular stem cells and their niches. Potential applications of the 3D integumentary organ system include an in vitro assay system, an animal model alternative, and a bioengineered organ replacement therapy.

Concepts: Developmental biology, Stem cell, Stem cells, Organ, Skin, In vitro, Induced pluripotent stem cell, Sebaceous gland

177

Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.

Concepts: DNA, Developmental biology, Stem cell, Stem cells, Cell biology, Amyotrophic lateral sclerosis, Induced pluripotent stem cell, Pluripotency

172

In a recent study published in this journal it was claimed that the rate of publications from US-based authors in the human embryonic stem cell (hESC) research field was slowing or even declining from 2008 to 2010. It was assumed that this is the result of long-term effects of the Bush administration’s funding policy for hESC research and the uncertain policy environment of recent years. In the present study, we analyzed a pool of more than 1,700 original hESC research papers published world-wide from 2007 to 2011. In contrast to the previous study, our results do not support the hypothesis of a decline in the productivity of US-based research but rather confirm a nearly unchanged leading position of US research in the hESC field with respect to both publication numbers and impact of research. Moreover, we analyzed about 500 papers reporting original research involving human induced pluripotent stem cells (hiPSCs) published through 2011 and found a dominant position of US research in this research field as well.

Concepts: Stem cell, Stem cells, Cell biology, Academic publishing, Embryonic stem cell, Induced pluripotent stem cell, Pluripotency, Martin Evans

155

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Concepts: Cell, Developmental biology, Stem cell, Mammal, Human anatomy, Induced pluripotent stem cell, Pluripotency, Inner cell mass

155

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced upregulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time.

Concepts: DNA, Genetics, Gene expression, Organism, Developmental biology, Stem cell, Stem cells, Induced pluripotent stem cell

153

Fibrodysplasia ossificans progressiva (FOP) is a rare disease characterized by progressive ossification of soft tissues, for which there is no effective treatment. Mutations in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ACVR1/ALK2) are the main cause of FOP. We generated human induced pluripotent stem cells (hiPSCs) from FOP patients with the ALK2 R206H mutation. The mutant ALK2 gene changed differentiation efficiencies of hiPSCs into FOP bone-forming progenitors: endothelial cells (ECs) and pericytes. ECs from FOP hiPSCs showed reduced expression of vascular endothelial growth factor receptor 2 and could transform into mesenchymal cells through endothelial-mesenchymal transition. Increased mineralization of pericytes from FOP hiPSCs could be partly inhibited by the ALK2 kinase inhibitor LDN-212854. Thus, differentiated FOP hiPSCs recapitulate some aspects of the disease phenotype in vitro, and they could be instrumental in further elucidating underlying mechanisms of FOP and development of therapeutic drug candidates.

Concepts: DNA, Gene, Developmental biology, Stem cell, Stem cells, Cellular differentiation, Induced pluripotent stem cell, Fibrodysplasia ossificans progressiva

147

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.

Concepts: DNA, Protein, Gene expression, Developmental biology, Stem cell, Embryonic stem cell, Induced pluripotent stem cell, Podocyte

146

Frontotemporal dementia with parkinsonism linked to chromosome 17q21.2 (FTDP-17) is an autosomal-dominant neurodegenerative disorder. Mutations in the gene coding the microtubule-associated protein tau (MAPT) can cause FTDP-17 but the underlying mechanisms of the disease are still unknown. Induced pluripotent stem cells (iPSCs) hold great promise to model FTDP-17 as such cells can be differentiated in vitro to the required neuronal cell type. Here, we report the generation of iPSCs from a 44-year-old symptomatic woman carrying a S305I mutation in the MAPT-gene.

Concepts: DNA, Gene, Cell, Developmental biology, Stem cell, Cell biology, Induced pluripotent stem cell, Progressive supranuclear palsy