Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.     

Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes

Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron‐scale control of cell position can be achieved over centimeter‐length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro‐stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel?) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH‐3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches. Biotechnol. Bioeng. 2012; 109: 2630–2641. © 2012 Wiley Periodicals, Inc.     

Variations in the epigenetic regulation of lineage-specific genes among human pluripotent stem cell lines

Monocyte exposure to tumor cells induces a transient state in which these cells are refractory to further exposure to cancer. This phenomenon, termed "tumor tolerance", is characterized by a decreased production of proinflammatory cytokines in response to tumors. In the past, we found that this effect comprises IRAK-M up regulation and TLR4 and CD44 activation. Herein we have established a human model of tumor tolerance and have observed a marked down-regulation of MHCII molecules as well as the MHCII master regulator, CIITA, in monocytes/macrophages. These cells combine an impaired capability for antigen presentation with potent phagocytic activity and exhibit an M2-like phenotype. In addition circulating monocytes isolated from Chronic Lymphocytic Leukemia patients exhibited the same profile as tumor tolerant cells after tumor ex vivo exposition.     

Self-renewing endodermal progenitor lines generated from human pluripotent stem cells

The use of human pluripotent stem cells for laboratory studies and cell-based therapies is hampered by their tumor-forming potential and limited ability to generate pure populations of differentiated cell types in vitro. To address these issues, we established endodermal progenitor (EP) cell lines from human embryonic and induced pluripotent stem cells. Optimized growth conditions were established that allow near unlimited (>10(16)) EP cell self-renewal in which they display a morphology and gene expression pattern characteristic of definitive endoderm. Upon manipulation of their culture conditions in vitro or transplantation into mice, clonally derived EP cells differentiate into numerous endodermal lineages, including monohormonal glucose-responsive pancreatic β-cells, hepatocytes, and intestinal epithelia. Importantly, EP cells are nontumorigenic in vivo. Thus, EP cells represent a powerful tool to study endoderm specification and offer a potentially safe source of endodermal-derived tissues for transplantation therapies.     

Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue

AIM To investigate genotype variation among induced pluripotent stem cell(iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. METHODS Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer(OCT3/4, SOX2, and KLF4). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using nextgeneration sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants(SNVs)(missense, nonsense,and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbS NP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer. RESULTS In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2, TTN, ULK4, TSSK1 B, FLT4, STK19, STK31, TRRAP, WNK1, PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer(stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the noncancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer(stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells.CONCLUSION The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.     

Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.     

Single cell heterogeneity in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.     

Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property

Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal tendon cell lines (TT-E4, TT-G11, and TT-D6) were established using transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen. Proliferation of these cells was significantly enhanced by treatment with bFGF and TGF-beta but not BMP2. Tendon phenotype-related genes such as those encoding scleraxis, Six1, EphA4, COMP, and type I collagen were expressed in these tendon cell clones. In addition to tendon phenotype-related genes, expression of osteopontin and Cbfal was observed. These clonal cell lines formed hard fibrous connective tissue when implanted onto chorioallantoic membrane in ovo. Furthermore, these cells also formed tendon-like tissues when they were implanted into defects made in patella tendon in mice. As these tendon cell lines also produced fibrocartilaginous tissues in tendon defect implantation experiments, mesenchymal stem cell properties were examined. Interestingly, these cells expressed genes related to osteogenic, chondrogenic, and adipogenic lineages at low levels when examined by RT-PCR. TT-G11 and TT-E4 cells differentiated into either osteoblasts or adipocytes, respectively, when they were cultured in cognate differentiation medium. These observations indicated that the established tendon cell line possesses mesenchymal stem cell-like properties, suggesting the existence of mesenchymal stem cell in tendon tissue.     

Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines

There are two critical stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. One is the production of high titer virus required to reprogram somatic cells; the other is identification of true hiPSC colonies from heterogeneous cell populations, and their isolation and expansion to generate a sustainable, pluripotent stem cell line. Here we describe simple, time-saving methods to address the current difficulties at these two critical junctures. First, we have developed a method to increase the number of infectious viral units 600-fold. Second, we have developed a TRA-1-81-based positive selection column method for isolating “true” hiPSCs from the heterogeneous cell populations, which overcomes the labor-intensive and highly subjective method of manual selection of hiPSC colonies. We have used these techniques to produce 8 hiPSC lines from human fibroblasts and we believe that they are of considerable utility to researchers in the hiPSC field.     

Applications of genetically engineered human pluripotent stem cell reporters in cardiac stem cell biology

1. Download : Download high-res image (129KB)
2. Download : Download full-size image

     

Engineering the human pluripotent stem cell microenvironment to direct cell fate

Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, offer a potential cell source for research, drug screening, and regenerative medicine applications due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Cell fate decisions are based on components of the surrounding microenvironment, including soluble factors, substrate or extracellular matrix, cell–cell interactions, mechanical forces, and 2D or 3D architecture. Depending on their spatio-temporal context, these components can signal hPSCs to either self-renew or differentiate to cell types of the ectoderm, mesoderm, or endoderm. Researchers working at the interface of engineering and biology have identified various factors which can affect hPSC fate, often based on lessons from embryonic development, and they have utilized this information to design in vitro niches which can reproducibly direct hPSC fate. This review highlights culture systems that have been engineered to promote self-renewal or differentiation of hPSCs, with a focus on studies that have elucidated the contributions of specific microenvironmental cues in the context of those culture systems. We propose the use of microsystem technologies for high-throughput screening of spatial–temporal presentation of cues, as this has been demonstrated to be a powerful approach for differentiating hPSCs to desired cell types.     

Calcium handling in human induced pluripotent stem cell derived cardiomyocytes

Background

The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca²⁺-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).

Methodology/Principal Findings

RT-PCR and immunocytochemistry experiments identified the expression of key Ca²⁺-handling proteins. Detailed laser confocal Ca²⁺ imaging demonstrated spontaneous whole-cell [Ca²⁺]_i transients. These transients required Ca²⁺ influx via L-type Ca²⁺ channels, as demonstrated by their elimination in the absence of extracellular Ca²⁺ or by administration of the L-type Ca²⁺ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca²⁺ store, contributing to [Ca²⁺]_i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca²⁺) and ryanodine (decreasing [Ca²⁺]_i). Similarly, the importance of Ca²⁺ reuptake into the SR via the SR Ca²⁺ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca²⁺]_i transients elimination. Finally, the presence of an IP3-releasable Ca²⁺ pool in hiPSC-CMs and its contribution to whole-cell [Ca²⁺]_i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.

Conclusions/Significance

Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca²⁺ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca²⁺]_i transients in hiPSC-CMs on both sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and intracellular store Ca²⁺ release.     

Laser-assisted selection and passaging of human pluripotent stem cell colonies

The derivation of somatic cell products from human embryonic stem cells (hESCs) requires a highly standardized production process with sufficient throughput. To date, the most common technique for hESC passaging is the manual dissection of colonies, which is a gentle, but laborious and time-consuming process and is consequently inappropriate for standardized maintenance of hESC. Here, we present a laser-based technique for the contact-free dissection and isolation of living hESCs (laser microdissection and pressure catapulting, LMPC). Following LMPC treatment, 80.6 ± 8.7% of the cells remained viable as compared to 88.6 ± 1.7% of manually dissected hESCs. Furthermore, there was no significant difference in the expression of pluripotency-associated markers when compared to the control. Flow cytometry revealed that 83.8 ± 4.1% of hESCs isolated by LMPC expressed the surface marker Tra-1-60 (control: 83.9 ± 3.6%). In vitro differentiation potential of LMPC treated hESCs as determined by embryoid body formation and multi-germlayer formation was not impaired. Moreover, we could not detect any overt karyotype alterations as a result of the LMPC process. Our data demonstrate the feasibility of standardized laser-based passaging of hESC cultures. This technology should facilitate both colony selection and maintenance culture of pluripotent stem cells.