Efficient Feeder-Free Episomal Reprogramming with Small Molecules

Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3β inhibitor CHIR99021, TGF-β/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs

Identification of the Hemogenic Endothelial Progenitor and Its Direct Precursor in Human Pluripotent Stem Cell Differentiation Cultures

SummaryHemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential for enabling the investigation of the molecular determinants of HSC specification. Here, we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by a VE-cadherin+CD73−CD235a/CD43− phenotype. This phenotype discriminates true HEPs from VE-cadherin+CD73+ non-HEPs and VE-cadherin+CD235a+CD41a− early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post-primitive-streak stage of differentiation directly from VE-cadherin-negative KDRbrightAPLNR+PDGFRαlow/− hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR+PDGFRα+ mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal of facilitating the generation of HSCs from hPSCs

Cell-Engineered Nanovesicle as a Surrogate Inducer of Contact-Dependent Stimuli

Heterotypic interactions between cells are crucial in various biological phenomena. Particularly, stimuli that regulate embryonic stem cell (ESC) fate are often provided from neighboring cells. However, except for feeder cultures, no practical methods are identified that can provide ESCs with contact-dependent cell stimuli. To induce contact-dependent cell stimuli in the absence of living cells, a novel method that utilizes cell-engineered nano-vesicles (CNVs) that are made by extruding living cells through microporous membranes is described. Protein compositions of CNVs are similar to their originating cells, as well as freely diffusible and precisely scalable. Treatment of CNVs produced from three different stromal cells successfully induces the same effect as feeder cultures. The results suggest that the effects of CNVs are mainly mediated by membrane-associated components. The use of CNVs might constitute a novel and efficient tool for ESC research.11Nsciescopu

Transcriptionally and Functionally Distinct Mesenchymal Subpopulations Are Generated from Human Pluripotent Stem Cells

Summary: Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRβ), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies. : Crooks and colleagues demonstrated a previously underappreciated functional and molecular heterogeneity in mesenchyme generated from human pluripotent stem cells. Two mesenchymal subsets were distinguished by the reciprocal expression of CD146, CD73, and CD140a. CD146hiCD73hi mesenchyme supported self-renewing hematopoietic stem and progenitor cells (HSPCs), expressed markers of the HSPC niche, and shared a similar molecular signature with primary human adult pericytes. Keywords: pluripotent stem cell, mesenchyme, hematopoietic stem cell niche, pericyte biology, directed differentiation, mesoder

Human Pluripotent Stem Cells Differentiated in Fully Defined Medium Generate Hematopoietic CD34+ and CD34− Progenitors with Distinct Characteristics

Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells

Production of Embryonic and Fetal-Like Red Blood Cells from Human Induced Pluripotent Stem Cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications

СОВРЕМЕННАЯ ТЕХНОЛОГИЯ ПОДГОТОВКИ И ПРОВЕДЕНИЯ ЛУЧЕВОЙ ТЕРАПИИ БОЛЬНЫХ ПЛОСКОКЛЕТОЧНЫМ РАКОМ АНАЛЬНОГО КАНАЛА

Squamous-cell anal cancer is a rare disease that requires a comprehensive approach in treatment and skilled professionals. Modern diagnostics is important for rational choice of treatment tactics. Radiotherapy is the cornerstone of sphincter-sparing anal cancer treatment. Radiotherapy dose, volume and duration are the key factors affecting treatment efficacy and toxicity.3D-conformal radiotherapy is a priority treatment allowing exact reproduction of treatment conditions, controlled by OBI (on-board imager) and kV X-Ray and cone-beam CT analysis. Intensity-modulated radiation therapy (IMRT) is a next-generation treatment with improved technologies, allowing better protection of normal tissues.Our experience with 21 squamous-cell anal cancer patients treated with IMRT during Nov 2011 – March 2013 is presented in this article.Эпителиальный рак анального канала является редким заболеванием, требующим в лечении комплексного подхода и квалифицированных специалистов. Для выбора рациональной комбинации лечебных методов важно использовать необходимый комплекс современных диагностических методов исследования.Основой органосохраняющего комбинированного метода лечения больных плоскоклеточным раком анального канала является лучевая терапия (ЛТ). Прогностическими факторами, влияющими на эффективность и негативные последствия комбинированного лечения, являются доза, объем и длительность проведения ЛТ.Приоритетом при проведении ЛТ является объемное 3D-планирование и использование конформной ЛТ (3D-CRT), точность воспроизведения условий ЛТ, контролируемой с помощью системы портальной визуализации в мегавольтном пучке линейного ускорителя электронов – OBI (on-board imager) и использование конического киловольтного пучка рентгеновского излучения.ЛТ с модуляцией интенсивности (IMRT) является следующей ступенью и более усовершенствованным вариантом с технологическими инновациями в конформной ЛТ, позволяющей уменьшить объем облучения нормальных тканей.В работе представлены непосредственные результаты первого опыта использования курса IMRT в комбинированном консервативном лечении 21 больного эпителиальным раком анального канала, проведенного с 11.2011 по 03.2013 в радиологическом отделении отдела радиационной онкологии ФГБУ «РОНЦ им. Н.Н. Блохина» РАМН

A Defined, Feeder-Free, Serum-Free System to Generate In Vitro Hematopoietic Progenitors and Differentiated Blood Cells from hESCs and hiPSCs

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimentional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells

The HOXB4 Homeoprotein Promotes the Ex Vivo Enrichment of Functional Human Embryonic Stem Cell-Derived NK Cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34+CD45RA+ precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells