Michaela Patterson

Directed differentiation of human induced pluripotent stem cells generates active motor neurons

The potential for directed differentiation of human‐induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS‐derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS‐derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS‐derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. STEM CELLS 2009;27:806–811

Female Human iPSCs Retain an Inactive X Chromosome

Generating induced pluripotent stem cells (iPSCs) requires massive epigenome reorganization. It is unclear whether reprogramming of female human cells reactivates the inactive X chromosome (Xi), as in mouse. Here we establish that human (h)iPSCs derived from several female fibroblasts under standard culture conditions carry an Xi. Despite the lack of reactivation, the Xi undergoes defined chromatin changes, and expansion of hiPSCs can lead to partial loss of XIST RNA. These results indicate that hiPSCs are epigenetically dynamic and do not display a pristine state of X inactivation with two active Xs as found in some female human embryonic stem cell lines. Furthermore, whereas fibroblasts are mosaic for the Xi, hiPSCs are clonal. This nonrandom pattern of X chromosome inactivation in female hiPSCs, which is maintained upon differentiation, has critical implications for clinical applications and disease modeling, and could be exploited for a unique form of gene therapy for X-linked diseases.

Defining the nature of human pluripotent stem cell progeny

While it is clear that human pluripotent stem cells (hPSCs) can differentiate to generate a panoply of various cell types, it is unknown how closely in vitro development mirrors that which occurs in vivo. To determine whether human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) make equivalent progeny, and whether either makes cells that are analogous to tissue-derived cells, we performed comprehensive transcriptome profiling of purified PSC derivatives and their tissue-derived counterparts. Expression profiling demonstrated that hESCs and hiPSCs make nearly identical progeny for the neural, hepatic, and mesenchymal lineages, and an absence of re-expression from exogenous reprogramming factors in hiPSC progeny. However, when compared to a tissue-derived counterpart, the progeny of both hESCs and hiPSCs maintained expression of a subset of genes normally associated with early mammalian development, regardless of the type of cell generated. While pluripotent genes (OCT4, SOX2, REX1, and NANOG) appeared to be silenced immediately upon differentiation from hPSCs, genes normally unique to early embryos (LIN28A, LIN28B, DPPA4, and others) were not fully silenced in hPSC derivatives. These data and evidence from expression patterns in early human fetal tissue (3-16 weeks of development) suggest that the differentiated progeny of hPSCs are reflective of very early human development (< 6 weeks). These findings provide support for the idea that hPSCs can serve as useful in vitro models of early human development, but also raise important issues for disease modeling and the clinical application of hPSC derivatives.

Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation

Linker histones are essential components of chromatin, but the distributions and functions of many during cellular differentiation are not well understood. Here, we show that H1.5 binds to genic and intergenic regions, forming blocks of enrichment, in differentiated human cells from all three embryonic germ layers but not in embryonic stem cells. In differentiated cells, H1.5, but not H1.3, binds preferentially to genes that encode membrane and membrane-related proteins. Strikingly, 37% of H1.5 target genes belong to gene family clusters, groups of homologous genes that are located in proximity to each other on chromosomes. H1.5 binding is associated with gene repression and is required for SIRT1 binding, H3K9me2 enrichment, and chromatin compaction. Depletion of H1.5 results in loss of SIRT1 and H3K9me2, increased chromatin accessibility, deregulation of gene expression, and decreased cell growth. Our data reveal for the first time a specific and novel function for linker histone subtype H1.5 in maintenance of condensed chromatin at defined gene families in differentiated human cells.

Adipogenesis and epicardial adipose tissue: A novel fate of the epicardium induced by mesenchymal transformation and PPARγ activation

Significance A layer of fat surrounds the heart in most mammals, including humans. The biology of this tissue has been speculated for centuries, but never subjected to experimental analysis because common experimental model species are thought to not have this tissue. In this study, we show that rodents have cardiac fat, albeit in a very specific location in the heart. We implicate the origin of this tissue from the epicardium (the outer epithelium of the heart) and the underlying mechanisms that account for its derivation. By comparing human and mouse epicardial cells, we provide an explanation for the prominent species differences in the presence and amount of cardiac adipose tissue. The hearts of many mammalian species are surrounded by an extensive layer of fat called epicardial adipose tissue (EAT). The lineage origins and determinative mechanisms of EAT development are unclear, in part because mice and other experimentally tractable model organisms are thought to not have this tissue. In this study, we show that mouse hearts have EAT, localized to a specific region in the atrial–ventricular groove. Lineage analysis indicates that this adipose tissue originates from the epicardium, a multipotent epithelium that until now is only established to normally generate cardiac fibroblasts and coronary smooth muscle cells. We show that adoption of the adipocyte fate in vivo requires activation of the peroxisome proliferator activated receptor gamma (PPARγ) pathway, and that this fate can be ectopically induced in mouse ventricular epicardium, either in embryonic or adult stages, by expression and activation of PPARγ at times of epicardium–mesenchymal transformation. Human embryonic ventricular epicardial cells natively express PPARγ, which explains the abundant presence of fat seen in human hearts at birth and throughout life.

let-7 miRNAs Can Act through Notch to Regulate Human Gliogenesis

Summary It is clear that neural differentiation from human pluripotent stem cells generates cells that are developmentally immature. Here, we show that the let-7 plays a functional role in the developmental decision making of human neural progenitors, controlling whether these cells make neurons or glia. Through gain- and loss-of-function studies on both tissue and pluripotent derived cells, our data show that let-7 specifically regulates decision making in this context by regulation of a key chromatin-associated protein, HMGA2. Furthermore, we provide evidence that the let-7/HMGA2 circuit acts on HES5, a NOTCH effector and well-established node that regulates fate decisions in the nervous system. These data link the let-7 circuit to NOTCH signaling and suggest that this interaction serves to regulate human developmental progression.

From Skin Biopsy to Neurons through a Pluripotent Intermediate Under Good Manufacturing Practice Protocols

The clinical application of human‐induced pluripotent stem cells (hiPSCs) requires not only the production of Good Manufacturing Practice‐grade (GMP‐grade) hiPSCs but also the derivation of specified cell types for transplantation under GMP conditions. Previous reports have suggested that hiPSCs can be produced in the absence of animal‐derived reagents (xenobiotics) to ease the transition to production under GMP standards. However, to facilitate the use of hiPSCs in cell‐based therapeutics, their progeny should be produced not only in the absence of xenobiotics but also under GMP conditions requiring extensive standardization of protocols, documentation, and reproducibility of methods and product. Here, we present a successful framework to produce GMP‐grade derivatives of hiPSCs that are free of xenobiotic exposure from the collection of patient fibroblasts, through reprogramming, maintenance of hiPSCs, identification of reprogramming vector integration sites (nrLAM‐PCR), and finally specification and terminal differentiation of clinically relevant cells. Furthermore, we developed a primary set of Standard Operating Procedures for the GMP‐grade derivation and differentiation of these cells as a resource to facilitate widespread adoption of these practices.

Physically Associated Synthetic Hydrogels with Long-Term Covalent Stabilization for Cell Culture and Stem Cell Transplantation

Although the natural extracellular matrix (ECM) is primarily a self-assembled structure, a large emphasis has been placed in recent years on the development of covalently crosslinked hydrogel scaffolds for tissue regeneration and stem cell transplantation. These hydrogels allow for cell spreading and migration through cell-mediated protease degradation [1, 2] or hydrolytic hydrogel degradation [3, 4]. However, in vivo cells do not always migrate and spread following matrix degradation; cells are able to navigate through protein fibers [5, 6]. Further, the requirement of matrix degradation to achieve cell migration and spreading results in a change in the mechanical properties of these hydrogels with time, which makes it challenging to decouple chemical cues from mechanical cues. Hydrogel materials that can mimic non-protease mediated cell spreading and migration and do not require active matrix degradation to achieve these cellular processes can offer an alternative system to study stem cell differentiation in vitro and overcome some of the current limitations of purely covalently crosslinked hydrogels.

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.

Nipah Virus Envelope-Pseudotyped Lentiviruses Efficiently Target ephrinB2-Positive Stem Cell Populations In Vitro and Bypass the Liver Sink When Administered In Vivo

ABSTRACT Sophisticated retargeting systems for lentiviral vectors have been developed in recent years. Most seek to suppress the viral envelopes natural tropism while modifying the receptor-binding domain such that its tropism is determined by the specificity of the engineered ligand-binding motif. Here we took advantage of the natural tropism of Nipah virus (NiV), whose attachment envelope glycoprotein has picomolar affinity for ephrinB2, a molecule proposed as a molecular marker of “stemness” (present on embryonic, hematopoietic, and neural stem cells) as well as being implicated in tumorigenesis of specific cancers. NiV entry requires both the fusion (F) and attachment (G) glycoproteins. Truncation of the NiV-F cytoplasmic tail (T5F) alone, combined with full-length NiV-G, resulted in optimal titers of NiV-pseudotyped particles (NiVpp) (∼106 IU/ml), even without ultracentrifugation. To further enhance the infectivity of NiVpp, we engineered a hyperfusogenic NiV-F protein lacking an N-linked glycosylation site (T5FΔN3). T5FΔN3/wt G particles exhibited enhanced infectivity on less permissive cell lines and efficiently targeted ephrinB2+ cells even in a 1,000-fold excess of ephrinB2-negative cells, all without any loss of specificity, as entry was abrogated by soluble ephrinB2. NiVpp also transduced human embryonic, hematopoietic, and neural stem cell populations in an ephrinB2-dependent manner. Finally, intravenous administration of the luciferase reporter NiVpp-T5FΔN3/G to mice resulted in signals being detected in the spleen and lung but not in the liver. Bypassing the liver sink is a critical barrier for targeted gene therapy. The extraordinary specificity of NiV-G for ephrinB2 holds promise for targeting specific ephrinB2+ populations in vivo or in vitro.