Footprint-free human induced pluripotent stem cells from articular cartilage with redifferentiation capacity: a first step toward a clinical-grade cell source.

Human induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine; however, clinical applications of iPSCs are restricted because of undesired genomic modifications associated with most reprogramming protocols. We show, for the first time, that chondrocytes from autologous chondrocyte implantation (ACI) donors can be efficiently reprogrammed into iPSCs using a nonintegrating method based on mRNA delivery, resulting in footprint-free iPSCs (no genome-sequence modifications), devoid of viral factors or remaining reprogramming molecules. The search for universal allogeneic cell sources for the ACI regenerative treatment has been difficult because making chondrocytes with high matrix-forming capacity from pluripotent human embryonic stem cells has proven challenging and human mesenchymal stem cells have a predisposition to form hypertrophic cartilage and bone. We show that chondrocyte-derived iPSCs can be redifferentiated in vitro into cartilage matrix-producing cells better than fibroblast-derived iPSCs and on par with the donor chondrocytes, suggesting the existence of a differentiation bias toward the somatic cell origin and making chondrocyte-derived iPSCs a promising candidate universal cell source for ACI. Whole-genome single nucleotide polymorphism array and karyotyping were used to verify the genomic integrity and stability of the established iPSC lines. Our results suggest that RNA-based technology eliminates the risk of genomic integrations or aberrations, an important step toward a clinical-grade cell source for regenerative medicine such as treatment of cartilage defects and osteoarthritis.

Labeled stem cells as disease models and in drug discovery.

Human pluripotent stem cells provide unique possibilities for in vitro studies of human cells in basic research, disease modeling as well as in industrial applications. By introducing relevant genome engineering technology, and thereby creating, for example, reporter cell lines, one will facilitate and improve safety pharmacology, toxicity testing, and can help the scientists to better understand pathological processes in humans. This review discusses how the merger of these two fields, human pluripotent stem cells and genome engineering, form extremely powerful tools and how they have been implemented already within the scientific community. In sharp contrast to immortalized human cell lines, which are both easy to expand and very simple to transfect, the genetically modified pluripotent stem cell line can be directed to a specific cell lineage and provide the user with highly relevant information. We highlight some of the challenges the field had to solve and how new technology advancements has removed the early bottlenecks.

Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro.

PURPOSE: The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial-like cells. METHODS: Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman's membrane for up to 9 days, and the culture dynamics documented in a time-lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial-like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6. RESULTS: The transplanted cells established and expanded on Bowman's membrane, forming a 1-4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3. CONCLUSION: This shows that it is possible to transplant cells originating from hESCs onto Bowman's membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial-like cells in vitro.

Principles for derivation of human embryonic stem cells.

This chapter describes the principles for derivation and maintenance of human embryonic stem cells. Detailed protocols are outlined and researchers who are generally skilled in mammalian cell culture should be able to repeat the processes successfully. Further, the protocols are intended for scientists who do not have access to advanced IVF equipment and therefore cannot perform, e.g. assisted hatching. In addition to derivation, we also discuss characterisation and banking of hES cells.

Single cell enzymatic dissociation of human embryonic stem cells: a straightforward, robust, and standardized culture method.

The routine culture and expansion of human embryonic stem (hES) cells has been and is still posing a challenge to researchers wishing to take advantage of the cells' unique potential. In contrast to mouse embryonic stem cells, hES cells usually have to be expanded by tedious mechanical microdissection or by enzymatic dissociation to cell clusters of a very narrow size range.It is essential to use a culture system that allows the robust and reproducible enzymatic dissociation of viable hES cell cultures to single cells to allow the scale-up of hES cell cultures as well as the application of hES cells in various experiments, such as FACS, electroporation, and clonal selection.By the development of enzyme-based protocols, which are less labor intensive and less time consuming, much progress has been made over the recent years with regard to improved culture systems for hES cell. We have developed a culture system that is based on single cell enzymatic dissociation (SCED) in combination with a highly supportive feeder cell layer of human foreskin fibroblasts (hFFs). The culture system allows defined enzymatic propagation while maintaining the hES cell lines in an undifferentiated, pluripotent, and normal state.In this chapter, we will show how hES cells, which have been derived and passaged by traditional mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. The protocols we describe are widely applicable and should therefore be of general use for the reliable mass cultivation of hES cells for various experiments.

Creation of engineered human embryonic stem cell lines using phiC31 integrase.

It has previously been shown that the phage-derived phiC31 integrase can efficiently target native pseudo-attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC-derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or the EF1alpha promoter. Stable clones were selected by antibiotic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo-attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression driven by the EF1alpha promoter was seen. Our results demonstrate the utility of phiC31 integrase to target pseudo-attP sites in hESC and show that integrase-mediated site-specific integration can efficiently create stably expressing engineered human embryonic stem cell clones.

Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation.

Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations. Disclosure of potential conflicts of interest is found at the end of this article.

Derivation of a xeno-free human embryonic stem cell line.

Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [Stem Cells 2006; 24:221-229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611.

The VEGF receptor flt-1 (VEGFR-1) is a positive modulator of vascular sprout formation and branching morphogenesis.

Sprouting angiogenesis is critical to blood vessel formation, but the cellular and molecular controls of this process are poorly understood. We used time-lapse imaging of green fluorescent protein (GFP)-expressing vessels derived from stem cells to analyze dynamic aspects of vascular sprout formation and to determine how the vascular endothelial growth factor (VEGF) receptor flt-1 affects sprouting. Surprisingly, loss of flt-1 led to decreased sprout formation and migration, which resulted in reduced vascular branching. This phenotype was also seen in vivo, as flt-1(-/-) embryos had defective sprouting from the dorsal aorta. We previously showed that loss of flt-1 increases the rate of endothelial cell division. However, the timing of division versus morphogenetic effects suggested that these phenotypes were not causally linked, and in fact mitoses were prevalent in the sprout field of both wild-type and flt-1(-/-) mutant vessels. Rather, rescue of the branching defect by a soluble flt-1 (sflt-1) transgene supports a model whereby flt-1 normally positively regulates sprout formation by production of sflt-1, a soluble form of the receptor that antagonizes VEGF signaling. Thus precise levels of bioactive VEGF-A and perhaps spatial localization of the VEGF signal are likely modulated by flt-1 to ensure proper sprout formation during blood vessel formation.

RP59, a marker for osteoblast recruitment, is also detected in primitive mesenchymal cells, erythroid cells, and megakaryocytes.

We recently described a novel protein in bone marrow of rats, RP59, as a marker for cells with the capacity to differentiate into osteoblasts. In this work, its expression pattern was further investigated to learn about the origin and biological relevance of RP59 expressing marrow cells. As revealed by in situ hybridization and by immunohistochemistry of yolk sac embryos, RP59 was found in the cells of the primitive ectoderm and primitive streak as well as in blood islands and extraembryonal mesoderm. Later, RP59 occurred in fetal liver cells and in circulating blood. From the time around birth, it was found in bone marrow and spleen cells. In addition, in vitro-formed blood vessels contained RP59-positive cells in the lumen. Endothelial cells and the vast majority of cells outside the blood vessels were not labeled. Concerning more mature hematopoietic cell types, RP59 was observed in megakaryocytes and nucleated erythroblasts, but absent from lymphoid cells. In conclusion, RP59 was induced in early mesoderm. It was maintained in the erythroid and megakaryotic lineages and, as earlier described, in young osteoblasts.