Ryan R. Mitchell

Direct conversion of human fibroblasts to multilineage blood progenitors

As is the case for embryo-derived stem cells, application of reprogrammed human induced pluripotent stem cells is limited by our understanding of lineage specification. Here we demonstrate the ability to generate progenitors and mature cells of the haematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. Ectopic expression of OCT4 (also called POU5F1)-activated haematopoietic transcription factors, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages, and demonstrated in vivo engraftment capacity. We note that adult haematopoietic programs are activated, consistent with bypassing the pluripotent state to generate blood fate: this is distinct from haematopoiesis involving pluripotent stem cells, where embryonic programs are activated. These findings demonstrate restoration of multipotency from human fibroblasts, and suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human pluripotent stem cells.

Structure of the endonuclease domain of MutL: unlicensed to cut

DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn(2+)-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.

Activation of neural cell fate programs toward direct conversion of adult human fibroblasts into tri-potent neural progenitors using OCT-4.

Several transcription factors and methods have been used to convert fibroblasts directly to neural fate and have provided insights into molecular mechanisms as to how each of these required factors orchestrate neural fate conversion. Here, we provide evidence and detailed characterization of the direct conversion process of primary adult human fibroblasts (hFib) to neural progenitor cells (NPC) using OCT4 alone. Factors previously associated with neural cell fate conversion were induced during hFib-NPC(OCT-4) generation, where OCT-4 alone was sufficient to induce neural fate conversion without the use of promiscuous small-molecule manipulation. Human Fib-NPC(OCT-4) proliferate, express neural stem/progenitor markers, and possess developmental potential that gives rise to all three major subtypes of neural cells: astrocytes, oligodendrocytes, and neurons with functional capacity. We propose a de-convoluted reprogramming approach for neural fate conversion in which OCT4 is sufficient for inducing neural conversion from hFib for disease modeling as well as the fundamental study of early neural fate induction.

Single Transcription Factor Conversion of Human Blood Fate to NPCs with CNS and PNS Developmental Capacity

The clinical applicability of direct cell fate conversion depends on obtaining tissue from patients that is easy to harvest, store, and manipulate for reprogramming. Here, we generate induced neural progenitor cells (iNPCs) from neonatal and adult peripheral blood using single-factor OCT4 reprogramming. Unlike fibroblasts that share molecular hallmarks of neural crest, OCT4 reprogramming of blood was facilitated by SMAD+GSK-3 inhibition to overcome restrictions on neural fate conversion. Blood-derived (BD) iNPCs differentiate in vivo and respond to guided differentiation in vitro, producing glia (astrocytes and oligodendrocytes) and multiple neuronal subtypes, including dopaminergic (CNS related) and nociceptive neurons (peripheral nervous system [PNS]). Furthermore, nociceptive neurons phenocopy chemotherapy-induced neurotoxicity in a system suitable for high-throughput drug screening. Our findings provide an easily accessible approach for generating human NPCs that harbor extensive developmental potential, enabling the study of clinically relevant neural diseases directly from patient cohorts.

Molecular Evidence for OCT4‐Induced Plasticity in Adult Human Fibroblasts Required for Direct Cell Fate Conversion to Lineage Specific Progenitors

Here we characterize the molecular and biological requirements for OCT4 plasticity induction in human skin derived fibroblasts (hFibs) that allows direct conversion of cell fate without iPSC formation. Our results indicate that adult hFibs not only require OCT4 but also short‐term exposure to reprogramming media (RM) to successfully undergo direct conversion to early hematopoietic and neural progenitor fates. RM was found to be essential in this process and allowed for unique changes in global gene expression specific to the combined effects of OCT4 and treatment with reprogramming media to establish a plastic state. This molecular state of hFib plasticity was distinct from transient expression of a full complement of iPSC reprogramming factors consistent with a lack in molecular hallmarks of iPSC formation. Human Fib‐derived OCT4 plastic cells display elevated levels of developmentally related genes associated with multiple lineages, but not those associated with pluripotency. In response to changes in the extracellular environment, plastic OCT4‐expressing hFibs further activate genes involved in hematopoietic as well as tripotent neural progenitor biology that allow cell fate conversion. Our study provides a working definition of hFib‐induced plasticity using OCT4 and a deconvoluted system to elucidate the process of direct cell fate reprogramming. Stem Cells 2014;32:2178–2187

Somatic transcriptome priming gates lineage-specific differentiation potential of human-induced pluripotent stem cell states

Human-induced pluripotent stem cells (hiPSCs) provide an invaluable source for regenerative medicine, but are limited by proficient lineage-specific differentiation. Here we reveal that hiPSCs derived from human fibroblasts (Fibs) versus human cord blood (CB) exhibit indistinguishable pluripotency, but harbour biased propensities for differentiation. Genes associated with germ layer specification were identical in Fib- or CB-derived iPSCs, whereas lineage-specific marks emerge upon differentiation induction of hiPSCs that were correlated to the cell of origin. Differentiation propensities come at the expense of other lineages and cannot be overcome with stimuli for alternative cell fates. Although incomplete DNA methylation and distinct histone modifications of lineage-specific loci correlate to lineage-specific transcriptome priming, transitioning hiPSCs into naive state of pluripotency removes iPSC-memorized transcriptome. Upon re-entry to the primed state, transcriptome memory is restored, indicating a human-specific phenomenon whereby lineage gated developmental potential is not permanently erased, but can be modulated by the pluripotent state.

Sam68 Allows Selective Targeting of Human Cancer Stem Cells

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/β-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/β-catenin signaling within CSCs. Disruption of CBP-β-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

Acquisition of pluripotency through continued environmental influence on OCT4-induced plastic human fibroblasts

The combination of OCT4 expression and short-term exposure to reprogramming media induces a state of transcriptional plasticity in human fibroblasts, capable of responding to changes in the extracellular environment that facilitate direct cell fate conversion toward lineage specific progenitors. Here we reveal that continued exposure of OCT4-induced plastic human fibroblasts to reprogramming media (RM) is sufficient to induce pluripotency. OCT4-derived induced pluripotent stem cell (iPSC(OCT4)) colonies emerged after prolonged culture in RM, and formed independently of lineage specific progenitors. Human iPSC(OCT4) are morphologically indistinguishable from conventionally derived iPSCs and express core proteins involved in maintenance of pluripotency. iPSC(OCT4) display in vivo functional pluripotency as measured by teratoma formation consisting of the three germ layers, and are capable of targeted in vitro differentiation. Our study indicates that acquisition of pluripotency is one of multiple cell fate choices that can be facilitated through environmental stimulation of OCT4-induced plasticity, and suggests the role of other reprogramming factors to induce pluripotency can be substituted by prolonged culture of plastic fibroblasts.

Derivation of human induced pluripotent stem cells through continued exposure of OCT4-induced plastic human fibroblasts to reprogramming media

The combination of OCT4 expression and short-term exposure to reprogramming media induces a state of transcriptional plasticity in human fibroblasts, capable of responding to changes in the extracellular environment. Here we provide characterization of iPSCs established through continued culture of OCT4-induced plastic human fibroblasts in pluripotent-supportive reprogramming media. Human iPSC(OCT4) are morphologically indistinguishable from conventionally derived iPSCs and express core proteins involved in maintenance of pluripotency. iPSC(OCT4) display bona fide functional pluripotency as measured by in vivo teratoma formation consisting of the three germ layers.

Author Correction: Direct conversion of human fibroblasts to multilineage blood progenitors

In this Article, there were duplicated empty lanes in Supplementary Figs. 2e and 3b. The corrected figures are presented in the Supplementary Information to the accompanying Amendment. The original Article has not been corrected.