Aline Fiebig-Comyn

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.

Identification of Drugs Including a Dopamine Receptor Antagonist that Selectively Target Cancer Stem Cells

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

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.

Niche displacement of human leukemic stem cells uniquely allows their competitive replacement with healthy HSPCs

Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is currently the leading strategy to manage acute myeloid leukemia (AML). However, treatment-related morbidity limits the patient generalizability of HSCT use, and the survival of leukemic stem cells (LSCs) within protective areas of the bone marrow (BM) continues to lead to high relapse rates. Despite growing appreciation for the significance of the LSC microenvironment, it has remained unresolved whether LSCs preferentially situate within normal HSC niches or whether their niche requirements are more promiscuous. Here, we provide functional evidence that the spatial localization of phenotypically primitive human AML cells is restricted to niche elements shared with their normal counterparts, and that their intrinsic ability to initiate and retain occupancy of these niches can be rivaled by healthy hematopoietic stem and progenitor cells (HSPCs). When challenged in competitive BM repopulation assays, primary human leukemia-initiating cells (L-ICs) can be consistently outperformed by HSPCs for BM niche occupancy in a cell dose-dependent manner that ultimately compromises long-term L-IC renewal and subsequent leukemia-initiating capacity. The effectiveness of this approach could be demonstrated using cytokine-induced mobilization of established leukemia from the BM that facilitated the replacement of BM niches with transplanted HSPCs. These findings identify a functional vulnerability of primitive leukemia cells, and suggest that clinical development of these novel transplantation techniques should focus on the dissociation of L-IC-niche interactions to improve competitive replacement with healthy HSPCs during HSCT toward increased survival of patients.

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.

In vivo generation of neural tumors from neoplastic pluripotent stem cells models early human pediatric brain tumor formation.

Recent studies have identified gene signatures in malignant tumors that are associated with human embryonic stem cells, suggesting a molecular relationship between aggressive cancers and pluripotency. Here, we characterize neural precursors (NPs) derived from transformed human embryonic stem cells (N‐t‐hESCs) that exhibit neoplastic features of human brain tumors. NPs derived from t‐hESCs have enhanced cell proliferation and an inability to mature toward the astrocytic lineage, compared with progeny derived from normal human embryonic stem cells (N‐hESCs) independent of adherent or neurosphere outgrowth. Intracranial transplantation of NPs derived from N‐t‐hESCs and N‐hESCs into NOD SCID mice revealed development of neuroectoderm tumors exclusively from the N‐t‐hESCs NPs and not from normal N‐hESCs. These tumors infiltrated the ventricles and the cerebellum of recipient mice and displayed morphological, phenotypic, and molecular features associated with classic medulloblastoma including retention of a pluripotent signature. Importantly, N‐t‐hESCs did not exhibit cytogenetic changes associated with medulloblastoma, suggesting that aberrant cellular and molecular properties precede the acquisition of karyotypic changes thus underscoring the value of this model system of human medulloblastoma. Our study demonstrates that NPs from a starting population of neoplastic human pluripotent parent cells possess brain tumor‐initiating cell capacity, thereby providing a model system to investigate initiation and progression of primitive human neural cancers that are difficult to assess using somatic sources. STEM CELLS 2012;30:392–404

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.

Expansive Generation of Functional Airway Epithelium From Human Embryonic Stem Cells

Production of human embryonic stem cell (hESC)‐derived lung progenitors has broad applicability for drug screening and cell therapy; however, this is complicated by limitations in demarcating phenotypic changes with functional validation of airway cell types. In this paper, we reveal the potential of hESCs to produce multipotent lung progenitors using a combined growth factor and physical culture approach, guided by the use of novel markers LIFRα and NRP1. Lung specification of hESCs was achieved by priming differentiation via matrix‐specific support, followed by air‐liquid interface to allow generation of lung progenitors capable of in vitro maturation into airway epithelial cell types, resulting in functional characteristics such as secretion of pulmonary surfactant, ciliation, polarization, and acquisition of innate immune activity. This approach provided a robust expansion of lung progenitors, allowing in vivo assessment, which demonstrated that only fully differentiated hESC‐derived airway cells were retained in the distal airway, where they aided in physiological recovery in immunocompromised mice receiving airway injury. Our study provides a basis for translational applications of hESCs for lung diseases.

Lineage-Specific Differentiation Is Influenced by State of Human Pluripotency

Human pluripotent stem cells (hPSCs) have been reported in naive and primed states. However, the ability to generate mature cell types remains the imperative property for utility of hPSCs. Here, we reveal that the naive state enhances self-renewal while restricting lineage differentiation inxa0vitro to neural default fate. Molecular analyses indicate expression of multiple lineage-associated transcripts in naive hPSCs that failed to predict biased functional differentiation capacity. Naive hPSCs can be converted to primed state over long-term serial passage that permits recovery of multi-germ layer differentiation. Suppression of OCT4 but not NANOG allows immediate recovery directly from naive state. To this end, we identified chemical inhibitors of OCT4 that restore naive hPSC differentiation. Our study reveals unique cell-fate restrictions in human pluripotent states andxa0provides an approach to overcome these barriers that harness both efficient naive hPSC growth whilexa0maintaining inxa0vitro differentiation essential for hPSC applications.

Overcoming mcr-1 mediated colistin resistance with colistin in combination with other antibiotics

Plasmid-borne colistin resistance mediated by mcr-1 may contribute to the dissemination of pan-resistant Gram-negative bacteria. Here, we show that mcr-1 confers resistance to colistin-induced lysis and bacterial cell death, but provides minimal protection from the ability of colistin to disrupt the Gram-negative outer membrane. Indeed, for colistin-resistant strains of Enterobacteriaceae expressing plasmid-borne mcr-1, clinically relevant concentrations of colistin potentiate the action of antibiotics that, by themselves, are not active against Gram-negative bacteria. The result is that several antibiotics, in combination with colistin, display growth-inhibition at levels below their corresponding clinical breakpoints. Furthermore, colistin and clarithromycin combination therapy displays efficacy against mcr-1-positive Klebsiella pneumoniae in murine thigh and bacteremia infection models at clinically relevant doses. Altogether, these data suggest that the use of colistin in combination with antibiotics that are typically active against Gram-positive bacteria poses a viable therapeutic alternative for highly drug-resistant Gram-negative pathogens expressing mcr-1.The plasmid-borne mcr-1 gene confers resistance to the antibiotic colistin. Here, MacNair et al. show that mcr-1 positive bacteria are however susceptible to colistin-mediated disruption of the outer membrane, and can be killed in vitro and in vivo by combining colistin with other antibiotics.