Anja S. Knaupp

A predictive computational framework for direct reprogramming between human cell types

Transdifferentiation, the process of converting from one cell type to another without going through a pluripotent state, has great promise for regenerative medicine. The identification of key transcription factors for reprogramming is currently limited by the cost of exhaustive experimental testing of plausible sets of factors, an approach that is inefficient and unscalable. Here we present a predictive system (Mogrify) that combines gene expression data with regulatory network information to predict the reprogramming factors necessary to induce cell conversion. We have applied Mogrify to 173 human cell types and 134 tissues, defining an atlas of cellular reprogramming. Mogrify correctly predicts the transcription factors used in known transdifferentiations. Furthermore, we validated two new transdifferentiations predicted by Mogrify. We provide a practical and efficient mechanism for systematically implementing novel cell conversions, facilitating the generalization of reprogramming of human cells. Predictions are made available to help rapidly further the field of cell conversion.

Serpin polymerization and its role in disease—The molecular basis of α1‐antitrypsin deficiency

Protein aggregation is the cause of several human diseases. Understanding the molecular mechanisms involved in protein aggregation requires knowledge of the kinetics and structures populated during the reaction. Arguably, the best structurally characterized misfolding reaction is that of α1‐antitrypsin. α1‐Antitrypsin misfolding leads to both liver disease and emphysema and affect approximately 1 in 2000 of the population. This review will focus on the mechanism of α1‐antitrypsin misfolding and the development of potential therapeutic strategies.

The Roles of Helix I and Strand 5A in the Folding, Function and Misfolding of α1-Antitrypsin

α1-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that α1-Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change. Both strand 5 and helix I are proposed to adopt different conformations when forming the various polymers, and possess a number of highly conserved residues however their role in the folding and misfolding of α1-Antitrypsin has never been examined. We have therefore created a range of α1Antitypsin variants in order to explore the role of these conserved residues in serpin folding, misfolding, stability and function. Our data suggest that key residues in helix I mediate efficient folding from the folding intermediate and residues in strand 5A ensure native state stability in order to prevent misfolding. Additionally, our data indicate that helix I is involved in the inhibitory process and that both structural elements undergo differing conformational rearrangements during unfolding and misfolding. These findings suggest that the ability of α1-Antitrypsin to adopt different types of polymers under different denaturing conditions may be due to subtle conformational differences in the transiently populated structures adopted prior to the I and M* states.

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.

A Versatile Strategy for Isolating a Highly Enriched Population of Intestinal Stem Cells.

Summary The isolation of pure populations of mouse intestinal stem cells (ISCs) is essential to facilitate functional studies of tissue homeostasis, tissue regeneration, and intestinal diseases. However, the purification of ISCs has relied predominantly on the use of transgenic reporter alleles in mice. Here, we introduce a combinational cell surface marker-mediated strategy that allows the isolation of an ISC population transcriptionally and functionally equivalent to the gold standard Lgr5-GFP ISCs. Used on reporter-free mice, this strategy allows the isolation of functional, transcriptionally distinct ISCs uncompromised by Lgr5 haploinsufficiency.

Cell Type of Origin Dictates the Route to Pluripotency

Our current understanding of induced pluripotent stem cell (iPSC) generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By characterizing and profiling the reprogramming pathways of fibroblasts, neutrophils, and keratinocytes, we unveil that key events of the process, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion, and reactivation of the pluripotency network, are to a large degree cell-type specific. Thus, we reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources.

An improved reprogrammable mouse model harbouring the reverse tetracycline-controlled transcriptional transactivator 3.

Reprogrammable mouse models engineered to conditionally express Oct-4, Klf-4, Sox-2 and c-Myc (OKSM) have been instrumental in dissecting molecular events underpinning the generation of induced pluripotent stem cells. However, until now these models have been reported in the context of the m2 reverse tetracycline-controlled transactivator, which results in low reprogramming efficiency and consequently limits the number of reprogramming intermediates that can be isolated for downstream profiling. Here, we describe an improved OKSM mouse model in the context of the reverse tetracycline-controlled transactivator 3 with enhanced reprogramming efficiency (>9-fold) and increased numbers of reprogramming intermediate cells albeit with similar kinetics, which we believe will facilitate mechanistic studies of the reprogramming process.

SRSF3 promotes pluripotency through Nanog mRNA export and coordination of the pluripotency gene expression program

The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.

Fine Tuning of Canonical Wnt Stimulation Enhances Differentiation of Pluripotent Stem Cells Independent of β‐Catenin‐Mediated T‐Cell Factor Signaling

The canonical Wnt/β‐catenin pathway is crucial for early embryonic patterning, tissue homeostasis, and regeneration. While canonical Wnt/β‐catenin stimulation has been used extensively to modulate pluripotency and differentiation of pluripotent stem cells (PSCs), the mechanism of these two seemingly opposing roles has not been fully characterized and is currently largely attributed to activation of nuclear Wnt target genes. Here, we show that low levels of Wnt stimulation via ectopic expression of Wnt1 or administration of glycogen synthase kinase‐3 inhibitor CHIR99021 significantly increases PSC differentiation into neurons, cardiomyocytes and early endodermal intermediates. Our data indicate that enhanced differentiation outcomes are not mediated through activation of traditional Wnt target genes but by β‐catenins secondary role as a binding partner of membrane bound cadherins ultimately leading to the activation of developmental genes. In summary, fine‐tuning of Wnt signaling to subthreshold levels for detectable nuclear β‐catenin function appears to act as a switch to enhance differentiation of PSCs into multiple lineages. Our observations highlight a mechanism by which Wnt/β‐catenin signaling can achieve dosage dependent dual roles in regulating self‐renewal and differentiation. Stem Cells 2018;36:822–833