Proteome of Personalized Tissue-Engineered Veins.

Vascular diseases are the largest cause of death globally and impose a major global burden on healthcare. The gold standard for treating vascular diseases is the transplantation of autologous veins, if applicable. Alternative treatments still suffer from shortcomings, including low patency, lack of growth potential, the need for repeated intervention, and a substantial risk of developing infections. The use of a vascular ECM scaffold reconditioned with the patient's own cells has shown successful results in preclinical and clinical studies. In this study, we have compared the proteomes of personalized tissue-engineered veins of humans and pigs. By applying tandem mass tag (TMT) labeling LC/MS-MS, we have investigated the proteome of decellularized (DC) veins from humans and pigs and reconditioned (RC) DC veins produced through perfusion with the patient's whole blood in STEEN solution, applying the same technology as used in the preclinical studies. The results revealed high similarity between the proteomes of human and pig DC and RC veins, including the ECM texture after decellularization and reconditioning. In addition, functional enrichment analysis showed similarities in signaling pathways and biological processes involved in the immune system response. Furthermore, the classification of proteins involved in immune response activity that were detected in human and pig RC veins revealed proteins that evoke immunogenic responses, which may lead to graft rejection, thrombosis, and inflammation. However, the results from this study imply the initiation of wound healing rather than an immunogenic response, as both systems share the same processes, and no immunogenic response was reported in the preclinical and clinical studies. Finally, our study assessed the application of STEEN solution in tissue engineering and identified proteins that may be useful for the prediction of successful transplantations.

Personalized tissue-engineered veins - long term safety, functionality and cellular transcriptome analysis in large animals.

Tissue engineering is a promising methodology to produce advanced therapy medicinal products (ATMPs). We have developed personalized tissue engineered veins (P-TEV) as an alternative to autologous or synthetic vascular grafts utilized in reconstructive vein surgery. Our hypothesis is that individualization through reconditioning of a decellularized allogenic graft with autologous blood will prime the tissue for efficient recellularization, protect the graft from thrombosis, and decrease the risk of rejection. In this study, P-TEVs were transplanted to vena cava in pig, and the analysis of three veins after six months, six veins after 12 months and one vein after 14 months showed that all P-TEVs were fully patent, and the tissue was well recellularized and revascularized. To confirm that the ATMP product had the expected characteristics one year after transplantation, gene expression profiling of cells from P-TEV and native vena cava were analyzed and compared by qPCR and sequencing. The qPCR and bioinformatics analysis confirmed that the cells from the P-TEV were highly similar to the native cells, and we therefore conclude that P-TEV is functional and safe in large animals and have high potential for use as a clinical transplant graft.

De- and recellularized urethral reconstruction with autologous buccal mucosal cells implanted in an ovine animal model.

OBJECTIVES: Patients with urethral stricture due to any type of trauma, hypospadias or gender dysphoria suffer immensely from impaired capacity to urinate and are in need of a new functional urethra. Tissue engineering with decellularization of a donated organ recellularized with cells from the recipient patient has emerged as a promising alternative of advanced therapy medicinal products. The aim of this pilot study was to develop an ovine model of urethral transplantation and to produce an individualized urethra graft to show proof of function in vivo. METHODS: Donated urethras from ram abattoir waste were decellularized and further recellularized with autologous buccal mucosa epithelial cells excised from the recipient ram and expanded in vitro. The individualized urethral grafts were implanted by reconstructive surgery in rams replacing 2.5 ± 0.5 cm of the native penile urethra. RESULTS: After surgery optimization, three ram had the tissue engineered urethra implanted for one month and two out of three showed a partially regenerated epithelium. CONCLUSIONS: Further adjustments of the model are needed to achieve a satisfactory proof-of-concept; however, we interpret these findings as a proof of principle and a possible path to develop a functional tissue engineered urethral graft with de- and recellularization and regeneration in vivo after transplantation.

Repair of Long Nerve Defects with a New Decellularized Nerve Graft in Rats and in Sheep.

Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The decellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep compared with AG. Evaluation of nerve regeneration and functional recovery was performed by clinical, electrophysiology and ultrasound tests. No significant differences were found in functional recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve.

Personalized tissue-engineered arteries as vascular graft transplants: A safety study in sheep.

Patients with cardiovascular disease often need replacement or bypass of a diseased blood vessel. With disadvantages of both autologous blood vessels and synthetic grafts, tissue engineering is emerging as a promising alternative of advanced therapy medicinal products for individualized blood vessels. By reconditioning of a decellularized blood vessel with the recipient's own peripheral blood, we have been able to prevent rejection without using immunosuppressants and prime grafts for efficient recellularization in vivo. Recently, decellularized veins reconditioned with autologous peripheral blood were shown to be safe and functional in a porcine in vivo study as a potential alternative for vein grafting. In this study, personalized tissue engineered arteries (P-TEA) were developed using the same methodology and evaluated for safety in a sheep in vivo model of carotid artery transplantation. Five personalized arteries were transplanted to carotid arteries and analyzed for safety and patency as well as with histology after four months in vivo. All grafts were fully patent without any occlusion or stenosis. The tissue was well cellularized with a continuous endothelial cell layer covering the luminal surface, revascularized adventitia with capillaries and no sign of rejection or infection. In summary, the results indicate that P-TEA is safe to use and has potential as clinical grafts.

Individualized tissue-engineered veins as vascular grafts: A proof of concept study in pig.

Personalized tissue engineered vascular grafts are a promising advanced therapy medicinal product alternative to autologous or synthetic vascular grafts utilized in blood vessel bypass or replacement surgery. We hypothesized that an individualized tissue engineered vein (P-TEV) would make the body recognize the transplanted blood vessel as autologous, decrease the risk of rejection and thereby avoid lifelong treatment with immune suppressant medication as is standard with allogenic organ transplantation. To individualize blood vessels, we decellularized vena cava from six deceased donor pigs and tested them for cellular removal and histological integrity. A solution with peripheral blood from the recipient pigs was used for individualized reconditioning in a perfusion bioreactor for seven days prior to transplantation. To evaluate safety and functionality of the individualized vascular graft in vivo, we transplanted reconditioned porcine vena cava into six pigs and analyzed histology and patency of the graft at different time points, with three pigs at the final endpoint 4-5 weeks after surgery. Our results showed that the P-TEV was fully patent in all animals, did not induce any occlusion or stenosis formation and we did not find any signs of rejection. The P-TEV showed rapid recellularization in vivo with the luminal surface covered with endothelial cells. In summary, the results indicate that P-TEV is functional and have potential for use as clinical transplant grafts.

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.

Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells.

Pluripotent human embryonic stem cell (hESC) lines are a promising model system in developmental and tissue regeneration research. Differentiation of hESCs towards the three germ layers and finally tissue specific cell types is often performed through the formation of embryoid bodies (EBs) in suspension or hanging droplet culture systems. However, these systems are inefficient regarding embryoid body (EB) formation, structural support to the EB and long term differentiation capacity. The present study investigates if agarose, as a semi solid matrix, can facilitate EB formation and support differentiation of hESC lines. The results showed that agarose culture is able to enhance EB formation efficiency with 10% and increase EB growth by 300%. The agarose culture system was able to maintain expression of the three germ layers over 8 weeks of culture. All of the four hESC lines tested developed EBs in the agarose system although with a histological heterogeneity between cell lines as well as within cell lines. In conclusion, a 3-D agarose culture of spherical hESC colonies improves EB formation and growth in a cost effective, stable and non-laborious technique.

High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules.

The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N6,N6-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.

Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control.

The successful transfer of human embryonic stem cell (hESC) technology and cellular products into clinical and industrial applications needs to address issues of automation, standardization and the generation of relevant cell numbers of high quality. In this study, we combined microcarrier technology and controlled stirred tank bioreactors, to develop an efficient and scalable system for expansion of pluripotent hESCs. We demonstrate the importance of controlling pO(2) at 30% air saturation to improve hESCs growth. This concentration allowed for a higher energetic cell metabolism, increased growth rate and maximum cell concentration in contrast to 5% pO(2) where a shift to anaerobic metabolism was observed, decreasing cell expansion 3-fold. Importantly, the incorporation of an automated perfusion system in the bioreactor enhanced culture performance and allowed the continuous addition of small molecules assuring higher cell concentrations for a longer time period. The expanded hESCs retained their undifferentiated phenotype and pluripotency. Our results show, for the first time, that the use of controlled bioreactors is critical to ensure the production of high quality hESCs. When compared to the standard colony culture, our strategy improves the final yield of hESCs by 12-fold, providing a potential bioprocess to be transferred to clinical and industrial applications.

Osteogenic potential of human mesenchymal stem cells and human embryonic stem cell-derived mesodermal progenitors: a tissue engineering perspective.

INTRODUCTION: Human mesenchymal stem cells (hMSCs) are promising candidates for bone engineering and regeneration with a considerable number of experimental successes reported over the last years. However, hMSCs show several limitations for tissue engineering applications, which can be overcome by using human embryonic stem cell-derived mesodermal progenitors (hES-MPs). The aim of this study was to investigate and compare the osteogenic differentiation potential of hMSCs and hES-MPs. MATERIALS AND METHODS: The osteogenic differentiation and mineralization behavior of both cell types were evaluated at passage 5, 10, 15, and 20. Expression of COL1A1, RUNX2, OPN, and OC was evaluated by reverse transcription (RT)-polymerase chain reaction, whereas mineralization was examined by photospectrometry, von Kossa staining, and time-of-flight secondary ion mass spectrometry. The immunoprofile of both cell types was investigated by flow cytometry. RESULTS: We demonstrated that, under proper stimulation, hES-MPs undergo osteogenic differentiation and exhibit significantly increased mineralization ability compared to hMSCs after protracted expansion. hES-MPs were also found to express lower amount of human leukocyte antigens class II proteins. CONCLUSIONS: The high osteogenic ability of hES-MPs, together with low expression of human leukocyte antigens class II, makes these cells an attractive alternative for bulk production of cells for bone engineering applications.

Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo.

OBJECTIVE: To develop an embryoid body-free directed differentiation protocol for the rapid generation of functional vascular endothelial cells derived from human embryonic stem cells (hESCs) and to assess the system for microRNA regulation and angiogenesis. METHODS AND RESULTS: The production of defined cell lineages from hESCs is a critical requirement for evaluating their potential in regenerative medicine. We developed a feeder- and serum-free protocol. Directed endothelial differentiation of hESCs revealed rapid loss of pluripotency markers and progressive induction of mRNA and protein expression of vascular markers (including CD31 and vascular endothelial [VE]-cadherin) and angiogenic growth factors (including vascular endothelial growth factor), increased expression of angiogenesis-associated microRNAs (including miR-126 and miR-210), and induction of endothelial cell morphological features. In vitro, differentiated cells produced nitric oxide, migrated across a wound, and formed tubular structures in both the absence and the presence of 3D matrices (Matrigel). In vivo, we showed that cells that differentiated for 10 days before implantation were efficient at the induction of therapeutic neovascularization and that hESC-derived cells were incorporated into the blood-perfused vasculature of recipient mice. CONCLUSIONS: The directed differentiation of hESCs is efficient and effective for the differentiation of functional endothelial cells from hESCs.

Human embryonic mesodermal progenitors highly resemble human mesenchymal stem cells and display high potential for tissue engineering applications.

Adult stem cells, such as human mesenchymal stem cells (hMSCs), show limited proliferative capacity and, after long-term culture, lose their differentiation capacity and are therefore not an optimal cell source for tissue engineering. Human embryonic stem cells (hESCs) constitute an important new resource in this field, but one major drawback is the risk of tumor formation in the recipients. One alternative is to use progenitor cells derived from hESCs that are more lineage restricted but do not form teratomas. We have recently derived a cell line from hESCs denoted hESC-derived mesodermal progenitors (hES-MPs), and here, using genome-wide microarray analysis, we report that the process of hES-MPs derivation results in a significantly altered expression of hESC characteristic genes to an expression level highly similar to that of hMSCs. However, hES-MPs displayed a significantly higher proliferative capacity and longer telomeres. The hES-MPs also displayed lower expression of HLA class II proteins before and after interferon-gamma treatment, indicating that these cells may somewhat be immunoprivileged and potentially used for HLA-incompatible transplantation. The hES-MPs are thus an appealing alternative to hMSCs in tissue engineering applications and stem-cell-based therapies for mesodermal tissues.

The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking.

This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.

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.

Human embryonic stem cell-derived mesenchymal progenitors--potential in regenerative medicine.

Tissue engineering and cell therapy require large-scale production of homogeneous populations of lineage-restricted progenitor cells that easily can be induced to differentiate into a specific tissue. We have developed straightforward protocols for the establishment of human embryonic stem (hES) cell-derived mesenchymal progenitor (hES-MP) cell lines. The reproducibility was proven by derivation of multiple hES-MP cell lines from 10 different hES cell lines. To illustrate clinical applicability, a xeno-free hES-MP cell line was also derived. None of the markers characteristic for undifferentiated hES cells were detected in the hES-MP cells. Instead, these cells were highly similar to mesenchymal stem cells with regard to morphology and expression of markers. The safety of hES-MP cells following transplantation was studied in severely combined immunodeficient (SCID) mice. The implanted hES-MP cells gave rise to homogeneous, well-differentiated tissues exclusively of mesenchymal origin and no teratoma formation was observed. These cells further have the potential to differentiate toward the osteogenic, adipogenic, and chondrogenic lineages in vitro. The possibility of easily and reproducibly generating highly expandable hES-MP cell lines from well-characterized hES cell lines with differentiation potential into several mesodermal tissues entails an enormous potential for the field of regenerative medicine.

Coculture of human embryonic stem cells and human articular chondrocytes results in significantly altered phenotype and improved chondrogenic differentiation.

Human embryonic stem (hES) cells have been suggested as a cell source for the repair of cartilage lesions. Here we studied how coculture with human articular chondrocytes affects the expansion potential, morphology, expression of surface markers, and differentiation abilities of hES cells, with special regard to chondrogenic differentiation. Undifferentiated hES cells were cocultured with irradiated neonatal or adult articular chondrocytes in high-density pellet mass cultures for 14 days. Cocultured hES cells were then expanded on plastic and their differentiation potential toward the adipogenic, osteogenic, and chondrogenic lineages was compared with that of undifferentiated hES cells. The expression of different surface markers was investigated using flow cytometry and teratoma formation was studied using injection of the cells under the kidney capsule. Our results demonstrate that although hES cells have to be grown on Matrigel, the cocultured hES cells could be massively expanded on plastic with a morphology and expression of surface markers similar to mesenchymal stem cells. Coculture further resulted in a more homogenous pellet and significantly increased cartilage matrix production, both in high-density pellet mass cultures and hyaluronan-based scaffolds. Moreover, cocultured cells formed colonies in agarose suspension culture, also demonstrating differentiation toward chondroprogenitor cells, whereas no colonies were detected in the hES cell cultures. Coculture further resulted in a significantly decreased osteogenic potential. No teratoma formation was detected. Our results confirm the potential of the culture microenvironment to influence hES cell morphology, expansion potential, and differentiation abilities over several population doublings.

Low molecular weight compounds for in vitro fate determination of human embryonic stem cells.

Over the last decade, human stem cells have received much attention in the scientific, as well as in the political arena. Based on their fundamental properties of self-renewal and pluripotency, these cells are anticipated to contribute to further our understanding of human development and the causes of congenital birth defects. In addition, human stem cells are beginning to find a practical use in new and improved models for drug discovery, and in the future, these cells may also potentially be used for the treatment of severe human diseases. In order to realize the great promise of human stem cells, there is a need for highly selective and efficient methods for maintenance and differentiation of the cells to produce large quantities of undifferentiated cells or populations of particular cell types. Currently, most protocols for stem cell expansion and differentiation involve poorly defined mixtures of protein growth factors and signaling molecules alone, or in combination with cellular genetic manipulations. In addition, the exploitation of specific low molecular weight compounds has proven successful for affecting basic stem cell properties such as proliferation, self-renewal, and differentiation. In the present review, we discuss some of the opportunities of modulating the state of human embryonic stem (hES) cells by exogenous synthetic substances. We also highlight some strategies for the identification of novel low molecular weight compounds capable of altering the hES cell fate. The access to inexpensive and specific tools for in vitro hES cell manipulations will be critical for realizing the industrial and medical applications of these cells.

Testing potential developmental toxicants with a cytotoxicity assay based on human embryonic stem cells.

Since the differentiation of embryonic stem cells mimics early development, these cells could potentially permit the detection of embryotoxicants which interfere with this process. Although reliable tests based on murine embryonic stem cells exist, no such methods are available for human embryonic stem (hES) cells. Nonetheless, to avoid the false classification of substances due to inter-species differences, human-relevant toxicity tests are needed. We therefore developed an assay based on three human cell types, representing different degrees of developmental maturation, namely, human foreskin fibroblasts, hES cell-derived progenitor cells, and pluripotent hES cells. A set of embryotoxicants for which existing in vivo data were available, namely, all-trans retinoic acid (ATRA), 13-cis retinoic acid (13CRA), valproic acid (VPA) and dimethyl sulphoxide (DMSO), were tested. 5-fluorouracil (5-FU) was used as a positive control, and saccharin as a negative control. Two methods were compared for the assessment of cell viability -- the determination of intracellular ATP content and of resazurin reduction. In addition, the protective capacity of basic fibroblast growth factor (bFGF) against retinoid-induced toxicity was investigated. This novel assay system reliably detected the embryotoxic potentials of the test substances, 5-FU, ATRA, 13-CRA (a substance that displays inter-species differences in its effects) and VPA. This was possible due to the apparent differences in the sensitivities of the human cell types used in the assay system. Thus, our results clearly indicate the advantages and relevance of using hES cells in in vitro developmental toxicity testing.

The formation of pores in the basal lamina of regenerated renal tubules.

Little information is available concerning the generation of renal tubules, but this information is urgently needed in regenerative medicine for the future treatment of acute and chronic renal failures. Of major interests are the integration of stem/progenitor cells, the cellular development and the tubular growth in a spatial environment. In this regard, we investigated the basal aspect of renal tubules generated at the interphase of an artificial interstitium. Stem/progenitor cells derived from neonatal rabbit kidney were mounted inside a specific tissue holder and covered by layers of polyester fleece. The tissue was then kept in a perfusion culture container for 13 days in chemically defined IMDM containing aldosterone (1 x 10(-7)m) as a tubulogenic factor. The spatial development of tubules was registered on whole-mount specimens and on cryo-sections labeled with soybean agglutinin (SBA) and tissue-specific antibodies indicating that collecting duct tubules were developed. Scanning electron microscopy (SEM) revealed that the generated tubules were completely covered by a basal lamina. Most interestingly, the matrix was not consistently composed, but exhibited three categories of pores. The most frequently found pore type had an apparent diameter of 133+/-26 nm followed by a medium-sized pore type of 317+/-35 nm. Another category of pores with a diameter of 605+/-101 nm was rather rarely found. All of the pores were evenly distributed and not restricted to particular sites. The newly detected pores are not related to culture artifacts, since they were also detected in collecting duct tubules of the neonatal rabbit kidney. It remains to be evaluated whether these pores support physiological transport functions or if they indicate the site where extracellular matrix proteins are inserted into newly synthesized basal lamina.