Heterologous Cell Types Research Articles

Single-cell analyses reveal suppressive tumor microenvironment of human colorectal cancer.

Profiling heterologous cell types within tumors is essential to decipher tumor microenvironment that shapes tumor progress and determines the outcome of therapeutic response. Here, we comprehensively characterized transcriptomes of 34,037 single cells obtained from 12 treatment‐naïve patients with colorectal cancer. Our comprehensive evaluation revealed attenuated B‐cell antigen presentation, distinct regulatory T‐cell clusters with different origin and novel polyfunctional tumor associated macrophages associated with CRC. Moreover, we identified expanded XCL1+ T‐cell clusters associated with tumor mutational burden high status. We further explored the underlying molecular mechanisms by profiling epigenetic landscape and inferring transcription factor motifs using single‐cell ATAC‐seq. Our dataset and analysis approaches herein provide a rich resource for further study of the impact of immune cells and translational research for human colorectal cancer.

Membrane-bottomed microwell array added to Transwell insert to facilitate non-contact co-culture of spermatogonial stem cell and STO feeder cell

In vivo cells express their characteristics in three-dimensional (3D) microenvironments via cell-cell interactions through autocrine, contact-dependent, paracrine, and synaptic signaling, often between heterologous cell types. Various in vitro 3D microwell-based culture methods have been proposed to further identify cellular characteristics by recreating cellular environments, typically in the form of spheroids and organoids, thereby realizing contact-based cell-cell interactions. However, in vivo cells generally exhibit multiple cellular interaction modes that have not been completely evaluated using existing microwell-based methods. This has led to a demand for more advanced and comprehensive methods. This study introduces a novel apparatus, the membrane-bottomed microwell (MBM) for non-contact co-cultures and 3D cell cultures. The MBM is a combination of a Transwell and a microwell array; these have previously been utilized to facilitate heterologous cell co-culturing and spheroid 3D cell culturing, respectively. In the Transwell insert, the lower part of the MBM is immersed in the culture media in which the cells are being two-dimensionally (2D) cultured, and the spheroids of the MBM are affected by the 2D cultured cells via the membrane at the bottom of the microwell. Here, we describe the methods for manufacturing the MBM in detail and elucidate the results of simulations of diffusion through the bottom of the membrane. We validate the proposed MBM for the spheroid culture of spermatogonial stem cells (SSCs), which had previously been 2D co-cultured with Sandos inbred mouse (SIM)-derived 6-thioguanine- and ouabain-resistant (STO; a mouse embryonic feeder cell line) feeder cells. The proposed system is shown to facilitate successful SSC spheroid culturing with paracrine signaling of STOs through an apparatus that simplifies both the loading and the evaluation processes; therefore, we believe that our findings will enable a more comprehensive understanding of SSCs and associated phenomena and that our system can be applied to various in vitro cell and tissue experiments.

YAP Non-cell-autonomously Promotes Pluripotency Induction in Mouse Cells.

SummaryYes-associated protein (YAP) is known to promote the stemness of multiple stem cell types, including pluripotent stem cells, while also antagonizing pluripotency during early embryogenesis. How YAP accomplishes these distinct functions remains unclear. Here, we report that, depending on the specific cells in which it is expressed, YAP could exhibit opposing effects on pluripotency induction from mouse somatic cells. Specifically, YAP inhibits pluripotency induction cell-autonomously but promotes it non-cell-autonomously. For its non-cell-autonomous role, YAP alters the expression of many secreted and matricellular proteins, including CYR61. YAP's non-cell-autonomous promoting effect could be recapitulated by recombinant CYR61 and abrogated by CYR61 depletion. Thus, we define a YAP-driven effect on enhancing pluripotency induction largely mediated by CYR61. Our work highlights the importance of considering the distinct contributions from heterologous cell types in deciphering cell fate control mechanisms and calls for careful re-examination of the co-existing bystander cells in complex cultures and tissues.

Sensing Sour: The OTOP1 Proton Channel from Function to Structure

The taste system evaluates chemicals to avoid toxins and identify nutrients. Among these is the ubiquitous hydrogen ion (H+), which when present at high concentrations elicits a sour taste sensation. In order to understand how taste receptor cells detect changes in H+ concentration (pH), we previously used patch clamp recording from sour-sensing taste receptor cells to identify a novel pH-sensitive proton-selective ionic current. To identify the gene that encodes this novel proton channel, we screened transcripts enriched in sour-sensing taste receptor cells for the ability to produce a proton current in a heterologous cell type. One gene, Otop1, encoding a protein (OTOP1) with twelve transmembrane domains, generated large inward currents in response to lowering extracellular pH. We showed that OTOP1 forms a proton channel, selective for H+ over other ions by a factor of a million-fold. To determine if Otop1 is required for sour taste, we generated and tested mice with an inactivating mutation of Otop1. In these mice, taste responses to acids were strongly attenuated, evidence that OTOP1 is a sour taste receptor. There are two murine homologs of Otop1, Otop2 and Otop3, which we find also encode proton channels. How OTOP channels are able to permeate H+ ions with such extraordinary selectivity is not known. The CryoEM structures of zebrafish OTOP1 and chicken OTOP3 show that the channel is a dimer, in which each subunit contains two domains (N and C) assembled so that the channel adopts a pseudotetrameric structure. However, unlike voltage-gated ion channels, the central cavity of OTOP channels is filled with cholesterol. OTOP channel isoforms are expressed in a diverse array of tissues, including in adipose tissue and the gastrointestinal tract, where they may be involved in pH regulation, ion transport or cellular signaling.

A Protocol for Non-biased Identification of RNAs Transferred Between Heterologous Mammalian Cell Types Using RNA Tagging, Cell Sorting, and Sequencing.

Intercellular communication is a major hallmark of multicellular organisms and is responsible for coordinating cell and tissue differentiation, immune responses, synaptic transmission, and both paracrine and endocrine signaling, for example. Small molecules, peptides, and proteins have all been studied extensively as mediators of intercellular communication; however, RNAs have also been shown recently to transfer between cells. In mammalian cells, microRNAs, tRNAs, short noncoding RNAs, mRNA fragments, as well as full-length mRNAs have all been shown to transfer between cells either by exosomes or by membrane nanotubes. We have previously described nanotube-mediated cell-cell transfer of specific mRNAs between heterologous mammalian cell types cultured in vitro. Here, we describe a simple method for the unbiased and quantitative identification of the complete range of transferred mRNAs (i.e., the mRNA transferome) in one population of mammalian cells following co-culture with another population. After co-culture, the individual cell populations are sorted by magnetic bead-mediated cell sorting and the transferred RNAs are then identified by downstream analysis methods, such as RNA sequencing. Application of this technique not only allows for determination of the mRNA transferome, but can also reveal changes in the native transcriptome of a cell population after co-culture. This can indicate the effect that co-culture and intercellular transfer of mRNA have upon cell physiology.

Modeling Neurodegenerative Spinocerebellar Ataxia Type 13 in Zebrafish Using a Purkinje Neuron Specific Tunable Coexpression System.

Purkinje cells (PCs) are primarily affected in neurodegenerative spinocerebellar ataxias (SCAs). For generating animal models for SCAs, genetic regulatory elements specifically targeting PCs are required, thereby linking pathological molecular effects with impaired function and organismic behavior. Because cerebellar anatomy and function are evolutionary conserved, zebrafish represent an excellent model to study SCAs in vivo We have isolated a 258 bp cross-species PC-specific enhancer element that can be used in a bidirectional manner for bioimaging of transgene-expressing PCs in zebrafish (both sexes) with variable copy numbers for tuning expression strength. Emerging ectopic expression at high copy numbers can be further eliminated by repurposing microRNA-mediated posttranslational mRNA regulation.Subsequently, we generated a transgenic SCA type 13 (SCA13) model, using a zebrafish-variant mimicking a human pathological SCA13R420H mutation, resulting in cell-autonomous progressive PC degeneration linked to cerebellum-driven eye-movement deficits as observed in SCA patients. This underscores that investigating PC-specific cerebellar neuropathologies in zebrafish allows for interconnecting bioimaging of disease mechanisms with behavioral analysis suitable for therapeutic compound testing.SIGNIFICANCE STATEMENT SCA13 patients carrying a KCNC3R420H allele have been shown to display mid-onset progressive cerebellar atrophy, but genetic modeling of SCA13 by expressing this pathogenic mutant in different animal models has not resulted in neuronal degeneration so far; likely because the transgene was expressed in heterologous cell types. We developed a genetic system for tunable PC-specific coexpression of several transgenes to manipulate and simultaneously monitor cerebellar PCs. We modeled a SCA13 zebrafish accessible for bioimaging to investigate disease progression, revealing robust PC degeneration, resulting in impaired eye movement. Our transgenic zebrafish mimicking both neuropathological and behavioral changes manifested in SCA-affected patients will be suitable for investigating causes of cerebellar diseases in vivo from the molecular to the behavioral level.

Partial reprogramming of heterologous cells by defined factors to generate megakaryocyte lineage-restricted biomolecules

The ability of transcriptional regulators to drive lineage conversion of somatic cells offers great potential for the treatment of human disease. To explore the concept of switching on specific target genes in heterologous cells, we developed a model system to screen candidate factors for their ability to activate the archetypal megakaryocyte-specific chemokine platelet factor 4 (PF4) in fibroblasts. We found that co-expression of the transcriptional regulators GATA1 and FLI1 resulted in a significant increase in levels of PF4, which became magnified over time. This finding demonstrates that such combinations can be used to produce potentially beneficial chemokines in readily available heterologous cell types.

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription

ABSTRACTDNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development.

Isolation, culture, characterization, and osteogenic differentiation of canine endometrial mesenchymal stem cell.

Aim:In this study, the canine endometrium tissue is characterized for its stem cell properties such as adherence to tissue culture plate (plasticity), short population doubling time, serial clonal passaging, long-term culturing properties, stem cell marker expression, and multilineage differentiation potential.Materials and Methods:The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the uterine endometrium and is compared with cells derived from umbilical cord matrix as a positive control. These cells are clonogenic, can undergo several population doublings in vitro, and can be differentiated to the osteocytes in mature mesenchymal tissues when grown in osteogenic differentiation media as detected by Alizarin Red-S staining.Results:It is reported for the first time that the cells derived from the canine endometrium (e-multipotent stem cells [MSCs]) were able to differentiate into a heterologous cell type: Osteocytes, thus demonstrating the presence of MSCs. Thus, the endometrium may be told as a potential source of MSCs which can be used for various therapeutic purposes.Conclusion:The endometrium can be used as a potential source of MSCs, which can be used for various therapeutic purposes.

Maintaining hair follicle stem cell identity in a dish.

Identifying and mimicking the signals that regulate stem cell self‐renewal, differentiation and maintenance in a petri dish is crucial to faithfully recapitulate stem cell behaviour in vitro . In this issue, Chacon‐Martinez et al (2017) describe novel culture conditions that allow the long‐term expansion and maintenance of functional murine hair follicle stem cells. This exciting discovery provides a faithful platform to study hair follicle stem cells in vitro and potentially perform drug screening for skin and hair follicle disorders.

Hair follicle stem cell cultures reveal self-organizing plasticity of stem cells and theirprogeny.

Understanding how complex tissues are formed, maintained, and regenerated through local growth, differentiation, and remodeling requires knowledge on how single-cell behaviors are coordinated on the population level. The self-renewing hair follicle, maintained by a distinct stem cell population, represents an excellent paradigm to address this question. A major obstacle in mechanistic understanding of hair follicle stem cell (HFSC) regulation has been the lack of a culture system that recapitulates HFSC behavior while allowing their precise monitoring and manipulation. Here, we establish an invitro culture system based on a 3D extracellular matrix environment and defined soluble factors, which for the first time allows expansion and long-term maintenance of murine multipotent HFSCs in the absence of heterologous cell types. Strikingly, this scheme promotes de novo generation of HFSCs from non-HFSCs and vice versa in a dynamic self-organizing process. This bidirectional interconversion of HFSCs and their progeny drives the system into a population equilibrium state. Our study uncovers regulatory dynamics by which phenotypic plasticity of cells drives population-level homeostasis within a niche, and provides a discovery tool for studies on adult stem cell fate.

Abstract LB-031: Generation of inducible oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing

Abstract Specific, reciprocal chromosomal translocations are found in numerous cancers, typically leading to the formation and expression of fusion genes with oncogenic properties. It has been demonstrated that by inducing double-stranded breaks (DSBs) in genomic DNA, using either zinc fingers nucleases (ZF), transcription activator-like effector nucleases (TALENs), or the CRISPR-Cas9 system, it is possible to faithfully recapitulate such chromosomal translocations in human cell lines. A potential pitfall is that the induction of the chromosomal translocation generates a constitutively active oncogenic fusion product, which can be detrimental to cell growth or viability in certain heterologous cell types. To overcome these obstacles, we have combined homology directed repair (HDR) with CRISPR-Cas9 genome editing to generate conditionally inducible chromosomal translocations. As proof of principle, we have used this approach to engineer human cell lines to carry the molecular hallmark present in Ewing Sarcoma, the t(11;22)(q24;q12) translocation. Our approach allows for the de novo generation and capture of the desired chromosomal translocation in the context of a conditional allele. Whole genome sequencing (WGS) and fluorescence in situ hybridization (FISH) confirmed the accuracy of our genomic editing approach, whilst analysis of mRNA and protein levels confirmed the selective inducibility and generation of biologically functional EWSR1-FLI1 fusion oncogene. Therefore, our approach establishes an innovative platform for constructing isogenic and conditionally inducible biologically relevant models for a variety of cancers driven by chromosomal translocations. Citation Format: Lee Spraggon, Luciano Martelotto, Julija Hmeljak, Emily Slotkin, Lu Wang, Jorge Reis-Filho, Marc Ladanyi. Generation of inducible oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-031.

Cell Cycle-dependent Changes in Localization and Phosphorylation of the Plasma Membrane Kv2.1 K+ Channel Impact Endoplasmic Reticulum Membrane Contact Sites in COS-1 Cells

The plasma membrane (PM) comprises distinct subcellular domains with diverse functions that need to be dynamically coordinated with intracellular events, one of the most impactful being mitosis. The Kv2.1 voltage-gated potassium channel is conditionally localized to large PM clusters that represent specialized PM:endoplasmic reticulum membrane contact sites (PM:ER MCS), and overexpression of Kv2.1 induces more exuberant PM:ER MCS in neurons and in certain heterologous cell types. Localization of Kv2.1 at these contact sites is dynamically regulated by changes in phosphorylation at one or more sites located on its large cytoplasmic C terminus. Here, we show that Kv2.1 expressed in COS-1 cells undergoes dramatic cell cycle-dependent changes in its PM localization, having diffuse localization in interphase cells, and robust clustering during M phase. The mitosis-specific clusters of Kv2.1 are localized to PM:ER MCS, and M phase clustering of Kv2.1 induces more extensive PM:ER MCS. These cell cycle-dependent changes in Kv2.1 localization and the induction of PM:ER MCS are accompanied by increased mitotic Kv2.1 phosphorylation at several C-terminal phosphorylation sites. Phosphorylation of exogenously expressed Kv2.1 is significantly increased upon metaphase arrest in COS-1 and CHO cells, and in a pancreatic β cell line that express endogenous Kv2.1. The M phase clustering of Kv2.1 at PM:ER MCS in COS-1 cells requires the same C-terminal targeting motif needed for conditional Kv2.1 clustering in neurons. The cell cycle-dependent changes in localization and phosphorylation of Kv2.1 were not accompanied by changes in the electrophysiological properties of Kv2.1 expressed in CHO cells. Together, these results provide novel insights into the cell cycle-dependent changes in PM protein localization and phosphorylation.

Alternative Super-Enhancer States Determine MYC Sensitivity to Notch and Brd4 Inhibitors in T Lymphoblastic Leukemia/Lymphoma

Gain-of-function NOTCH1 mutations are oncogenic drivers in a high fraction of T-cell lymphoblastic leukemia/lymphoma (T-LL). These mutations variously cause increased production or stabilization of the free intracellular domain of NOTCH1, which regulates gene expression by forming a transcription complex with the DNA-binding factor RBPJ and coactivators of the MAML family. Using expression profiling and ChIP-seq, we have shown that NOTCH1/RBPJ complexes activate most target genes by binding to super-enhancers, large regulatory elements that switch on transcription through long-range interactions with gene promoters. MYC is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates MYC is unknown. To understand which regulatory element(s) regulate MYC expression, we used chromatin conformation capture (3C) assays to test the interaction between putative enhancer(s) and the MYC promoter in T-LL cell lines, and reporter gene assays to confirm enhancer function of candidate sites. We identified a distal site located >1 Mb 3’ of human and murine MYC termed the Notch-dependent MYC enhancer (NDME) that binds Notch transcription complexes and physically interacts with the MYC proximal promoter. An ~1 kb DNA fragment containing this site activates a luciferase reporter gene in a Notch-dependent fashion in T-LL cells but not in heterologous cell types. The Notch binding site lies within a large enhancer region (>600 kb in breadth) containing multiple discrete H3K27ac peaks. Remarkably, acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer region and the MYC promoter that correlate with NOTCH1/RBPJ complex binding and MYC expression. T-LL cells selected for resistance to gamma-secretase inhibitors (GSIs) exhibit epigenetic silencing of the NDME and loss of NDME looping interactions with the MYC promoter, yet maintain MYC expression. 3C analysis of GSI resistant cells shows preferential interaction between the MYC promoter and a more 3’ enhancer element recently described as a BRD4-dependent regulator of MYC expression in acute myeloid leukemia cells. In line with this observation, BRD4 antagonists are potent inhibitors of MYC expression in GSI resistant T-LL cells but not GSI-sensitive cells. We also studied a case of Notch-mutated early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL). ChIP-Seq analysis of the leukemic blasts revealed an “AML-like” MYC enhancer chromatin state, and as predicted from our analysis of cell lines, the blasts rapidly down-regulated MYC in response to BRD4 inhibitor but not in response to GSI. These findings suggest that specific MYC chromatin states predict responsiveness to Notch and BRD4 inhibitors, and provide a rationale for use of Notch and BRD4 inhibitor combinations in Notch-mutated leukemias. DisclosuresNo relevant conflicts of interest to declare.

Rhythmic ganglion cell activity in bleached and blind adult mouse retinas.

In retinitis pigmentosa – a degenerative disease which often leads to incurable blindness- the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor’s dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance <200 µm) reveals synchrony among homologous RGC types and a constant phase shift (∼70 msec) among heterologous cell types (ON versus OFF). The rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the understanding of the degeneration process and may guide future rescue strategies.

Syncytial communication in descending vasa recta includes myoendothelial coupling.

Using dual cell patch-clamp recording, we examined pericyte, endothelial, and myoendothelial cell-to-cell communication in descending vasa recta. Graded current injections into pericytes or endothelia yielded input resistances of 220 ± 21 and 128 ± 20 MΩ, respectively (P < 0.05). Injection of positive or negative current into an endothelial cell depolarized and hyperpolarized adjacent endothelial cells, respectively. Similarly, current injection into a pericyte depolarized and hyperpolarized adjacent pericytes. During myoendothelial studies, current injection into a pericyte or an endothelial cell yielded small, variable, but significant change of membrane potential in heterologous cells. Membrane potentials of paired pericytes or paired endothelia were highly correlated and identical. Paired measurements of resting potentials in heterologous cells were also correlated, but with slight hyperpolarization of the endothelium relative to the pericyte, -55.2 ± 1.8 vs. -52.9 ± 2.2 mV (P < 0.05). During dual recordings, angiotensin II or bradykinin stimulated temporally identical variations of pericyte and endothelial membrane potential. Similarly, voltage clamp depolarization of pericytes or endothelial cells induced parallel changes of membrane potential in the heterologous cell type. We conclude that the descending vasa recta endothelial syncytium is of lower resistance than the pericyte syncytium and that high-resistance myoendothelial coupling also exists. The myoendothelial communication between pericytes and endothelium maintains near identity of membrane potentials at rest and during agonist stimulation. Finally, endothelia membrane potential lies slightly below pericyte membrane potential, suggesting a tonic role for the former to hyperpolarize the latter and provide a brake on vasoconstriction.

Influence of tumour micro-environment heterogeneity on therapeutic response

Tumour formation involves the co-evolution of neoplastic cells together with extracellular matrix, tumour vasculature and immune cells. Successful outgrowth of tumours and eventual metastasis is not determined solely by genetic alterations in tumour cells, but also by the fitness advantage such mutations confer in a given environment. As fitness is context dependent, evaluating tumours as complete organs, and not simply as masses of transformed epithelial cells, becomes paramount. The dynamic tumour topography varies drastically even throughout the same lesion. Heterologous cell types within tumours can actively influence therapeutic response and shape resistance.

Transcription factors interfering with dedifferentiation induce cell type-specific transcriptional profiles

Transcription factors (TFs) are able to regulate differentiation-related processes, including dedifferentiation and direct conversion, through the regulation of cell type-specific transcriptional profiles. However, the functional interactions between the TFs regulating different transcriptional profiles are not well understood. Here, we show that the TFs capable of inducing cell type-specific transcriptional profiles prevent the dedifferentiation induced by TFs for pluripotency. Of the large number of TFs expressed in a neural-lineage cell line, we identified a subset of TFs that, when overexpressed, strongly interfered with the dedifferentiation triggered by the procedure to generate induced pluripotent stem cells. This interference occurred through a maintenance mechanism of the cell type-specific transcriptional profile. Strikingly, the maintenance activity of the interfering TF set was strong enough to induce the cell line-specific transcriptional profile when overexpressed in a heterologous cell type. In addition, the TFs that interfered with dedifferentiation in hepatic-lineage cells involved TFs with known induction activity for hepatic-lineage cells. Our results suggest that dedifferentiation suppresses a cell type-specific transcriptional profile, which is primarily maintained by a small subset of TFs capable of inducing direct conversion. We anticipate that this functional correlation might be applicable in various cell types and might facilitate the identification of TFs with induction activity in efforts to understand differentiation.

Isolation, culture and characterization of caprine mesenchymal stem cells derived from amniotic fluid

Amniotic fluid (AF) represents heterologous cell types and a specific group of these cells show high growth rate and multipotent characteristics. The aim of the present study was to culture and fully characterize the putative stem cell population isolated from caprine mesenchymal stem cells. Plastic adherent fibroblastoid cell population could be successfully isolated from the caprine amniotic fluid. In vitro expanded caprine amniotic fluid derived mesenchymal stem cells (cAF-MSCs) showed high proliferation ratio with a doubling time of 33.1h and stained positive for alkaline phosphatase. Relative transcript abundance of CD-73, CD-90 and CD-105 surface markers were analyzed by SYBR green based real time PCR and their respective proteins were localized through immunocytochemistry, however cAF-MSCs were found negative for haematopoietic marker CD-34. When exposed to corresponding induction condition, cAF-MSCs differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through von Kossa, Oil Red O and Alcian blue staining respectively. Furthermore, these cells were found positive for undifferentiated embryonic stem cell markers like Oct-4, Nanog, Sox-2, SSEA-1 and SSEA-4 which accentuate their pluripotent property. In conclusion, caprine amniotic fluid represents a promising source of mesenchymal stem cells with high proliferative and differentiation potential and these cells offer their scope for multiple regenerative therapies.

Deconstructing the Complexity of a Microenvironmental Niche

Cells acquire their fate invivo in the context of a complex microenvironmental "niche" comprised ofheterologous cell types, signaling molecules, extracellular matrix, biophysical forces, and metabolic substrates. Now Calderón and Boehm report the "refunctionalization" of a defective thymic epithelial niche, offering insights into how signaling molecules may be hierarchically organized to direct differentiation outcomes.