Undefined Components Research Articles

Poly(norepinephrine)-Mediated Universal Surface Modification for Patterning Human Pluripotent Stem Cell Culture and Differentiation.

Maintaining undifferentiated states of human pluripotent stem cells (hPSCs) is key to accomplishing successful hPSC research. Specific culture conditions, including hPSC-compatible substrates, are required for the hPSC culture. Over the past two decades, substrates supporting hPSC self-renewal have evolved from undefined and xenogeneic protein components to chemically defined and xenogeneic-free materials. However, these synthetic substrates are often costly and complex to use, leading many laboratories to continue using simpler undefined extracellular matrix (ECM) protein mixtures. In this study, we present a method using poly(norepinephrine) (pNE) for surface modification to enhance the immobilization of ECM proteins on various substrates, including polydimethylsiloxane (PDMS) and ultralow attachment (ULA) hydrogels, thereby supporting hPSC culture and maintenance of pluripotency. The pNE-mediated surface modification enables spatial patterning of ECM proteins on nonadhesive ULA surfaces, facilitating tunable macroscopic cell patterning. This approach improves hPSC attachment and growth and allows for cell patterning to study the effects of anisotropic environments on the hPSC fate. Our findings demonstrate the versatility and simplicity of pNE-mediated surface modification for improving hPSC culture and spatially controlled differentiation into endothelial cells and cardiomyocytes on previously nonamenable substrates, providing a valuable tool for tissue engineering and regenerative medicine applications.

Assessing the Efficacy and Feasibility of Conditioned Medium as a Culture Reagent for Tumor Organoid Culture: A Comprehensive Evaluation

Tumor organoids are three-dimensional models of tumor cells grown in vitro which derived from primary tumors of patients. Tumor organoids highly mimic the characteristics of the primary tumors, retain tumor heterogeneity, and can be used for functional testing in vitro, including high-throughput drug screening and personalized therapeutic strategies. However, the establishment of tumor organoids facing such dilemma as low success rate and growth rate. The use of appropriate and effective culture reagent is pivotal to the success of tumor organoids culture. Nowadays, the most commonly and long-lasting used media in cell culture is conditioned medium, while its application in organoids culture has been controversial due to undefined components. In response, our study conducts a comprehensive analysis, offering compelling evidence supporting the efficacy of conditioned medium in promoting organoids growth and expansion. This research aims to address uncertainties surrounding its use and contribute valuable insights to enhance tumor organoids culture practices.

Induktyvinis metodas komponentinių programų sintezėje

The automatic programming and automatic software synthesis systems are relevant software engineering research objects. This article presents the prospects of induction as a component-based software synthesis method. The usage of induction in the component-based software synthesis system can solve main synthesis problems: problem of specification, problem of undefined components and problem of non-functional requirements. However using induction synthesis system can lose soundness provided by structural synhesis of programs method. To avoid this any inductive conclusion should be verified.

Robust Expansion Of Human Blood-derived γδ T Cells Using A Chemically-defined Medium

Abstract Gamma delta (γδ) T cells have recently emerged as an attractive candidate for cancer immunotherapy treatments due to their inherent cytotoxicity against cancer cells. Moreover, γδ T cells provide a platform for allogeneic cell therapies as they can recognize antigens independent of MHC recognition and do not cause graft-versus-host disease (GVHD). However, the lack of robust and GMP-compatible serum-free ex vivo expansion system for γδ T cells hinders the development of scalable expansion processes for advancing γδ T cell immunotherapies. In the current study, we characterize the expansion of human peripheral blood-derived γδ T cells from various donors using TheraPEAK ®T-VIVO™ Medium, a novel chemically-defined cell expansion medium which is free of undefined and animal-origin components. Peripheral blood mononuclear cells (PBMCs) stimulated with zoledronic acid resulted in more than 1,000 fold expansion of γδ T cells in only 14 days with TheraPEAK ®T-VIVO™ Medium containing 100 IU/mL human recombinant IL-2, without the need for human serum supplementation. Similarly, PBMCs stimulated with agonist antibodies targeting Vδ1 or Vδ2 T cell receptors (TCR) induced robust expansion of Vδ1 or Vδ2 T cells, respectively, in TheraPEAK ®T-VIVO™ Medium. Collectively, these data support the use of TheraPEAK ®T-VIVO™ Medium to advance the development of γδ T cell manufacturing processes.

Macromolecular crowding in animal component-free, xeno-free and foetal bovine serum media for human bone marrow mesenchymal stromal cell expansion and differentiation.

Background: Cell culture media containing undefined animal-derived components and prolonged in vitro culture periods in the absence of native extracellular matrix result in phenotypic drift of human bone marrow stromal cells (hBMSCs). Methods: Herein, we assessed whether animal component-free (ACF) or xeno-free (XF) media formulations maintain hBMSC phenotypic characteristics more effectively than foetal bovine serum (FBS)-based media. In addition, we assessed whether tissue-specific extracellular matrix, induced via macromolecular crowding (MMC) during expansion and/or differentiation, can more tightly control hBMSC fate. Results: Cells expanded in animal component-free media showed overall the highest phenotype maintenance, as judged by cluster of differentiation expression analysis. Contrary to FBS media, ACF and XF media increased cellularity over time in culture, as measured by total DNA concentration. While MMC with Ficoll™ increased collagen deposition of cells in FBS media, FBS media induced significantly lower collagen synthesis and/or deposition than the ACF and XF media. Cells expanded in FBS media showed higher adipogenic differentiation than ACF and XF media, which was augmented by MMC with Ficoll™ during expansion. Similarly, Ficoll™ crowding also increased chondrogenic differentiation. Of note, donor-to-donor variability was observed for collagen type I deposition and trilineage differentiation capacity of hBMSCs. Conclusion: Collectively, our data indicate that appropriate screening of donors, media and supplements, in this case MMC agent, should be conducted for the development of clinically relevant hBMSC medicines.

Cell Death Pathways: The Variable Mechanisms Underlying Fine Particulate Matter-Induced Cytotoxicity.

Recently, the advent of health risks due to the cytotoxicity of fine particulate matter (FPM) is concerning. Numerous studies have reported abundant data elucidating the FPM-induced cell death pathways. However, several challenges and knowledge gaps are still confronted nowadays. On one hand, the undefined components of FPM (such as heavy metals, polycyclic aromatic hydrocarbons, and pathogens) are all responsible for detrimental effects, thus rendering it difficult to delineate the specific roles of these copollutants. On the other hand, owing to the crosstalk and interplay among different cell death signaling pathways, precisely determining the threats and risks posed by FPM is difficult. Herein, we recapitulate the current knowledge gaps present in the recent studies regarding FPM-induced cell death, and propose future research directions for policy-making to prevent FPM-induced diseases and improve knowledge concerning the adverse outcome pathways and public health risks of FPM.

Learning Disentangled Representation Implicitly Via Transformer for Occluded Person Re-Identification

Person re-IDentification (re-ID) under various occlusions has been a long-standing challenge as person images with different types of occlusions often suffer from misalignment in image matching and ranking. Most existing methods tackle this challenge by aligning spatial features of body parts according to external semantic cues or feature similarities but this alignment approach is complicated and sensitive to noises. We design DRL-Net, a disentangled representation learning network that handles occluded re-ID without requiring strict person image alignment or any additional supervision. Leveraging transformer architectures, DRL-Net achieves alignment-free re-ID via global reasoning of local features of occluded person images. It measures image similarity by automatically disentangling the representation of undefined semantic components, e.g., human body parts or obstacles, under the guidance of semantic preference object queries in the transformer. In addition, we design a decorrelation constraint in the transformer decoder and impose it over object queries for better focus on different semantic components. To better eliminate interference from occlusions, we design a contrast feature learning technique (CFL) for better separation of occlusion features and discriminative ID features. Extensive experiments over occluded and holistic re-ID benchmarks show that the DRL-Net achieves superior re-ID performance consistently and outperforms the state-offi-the-art by large margins for occluded re-ID dataset.

Buffer Models for pH and Acid Changes Occurring in Cucumber Juice Fermented with Lactiplantibacillus pentosus and Leuconostoc mesenteroides

The pH changes that occur during the fermentation of vegetables by lactic acid bacteria depend on the production of weak acids and on the buffering of the fermentation medium. Undefined buffering components of fermentation media make estimates of pH from acid production difficult. The objective of this research was to develop buffer models for a model cucumber fermentation brine system linking pH changes to acid concentrations. A novel titration method was used to measure buffer capacity in cucumber juice medium made from three grades of pickling cucumbers based on diameter. Fermentation of juice made from cucumbers of different sizes resulted in differences in fermentation biochemistry. The results of modeling indicated that the pH of the medium after 24 and 48 h of fermentation by heterolactic Leuconostoc mesenteroides and homolactic Lactiplantibacillus pentosus could be predicted from acid concentrations based on the measured buffer capacity of the corresponding unfermented medium. The differences for all observed and predicted pH values of the fermentation samples, based on measured acid concentrations, had a root mean square error of 0.064 pH units. The buffer models included a quantitative measure of the effect on pH of the malolactic reaction caused by the lactic acid bacteria. These models may have application for assessing the influence of a variety of lactic acid bacteria buffering reactions on pH and fermentation ecology by linking pH to fermentation acid concentrations.

A worldwide survey on the use of animal-derived materials and reagents in scientific experimentation.

The use of cell and tissue‐based methods in basic, applied and regulatory science has been increasing exponentially. Animal‐derived components, including serum, coating materials, growth factors and antibodies are routinely used in cell/tissue cultures and in general laboratory practices. In addition to ethical issues, the use and production of animal‐derived materials and reagents raises many scientific concerns, generally associated with presence of undefined components and batch‐to‐batch variability, which may compromise experimental reproducibility. On the other hand, non‐animal materials and reagents, such as human cells, alternatives to animal sera or non‐animal recombinant antibodies, are becoming increasingly available, and their use is encouraged by the EU Directive 2010/63 and the Guidance Document on Good In vitro Method Practices (GIVIMP), published by the Organization for Economic Cooperation and Development (OECD). In an effort to map the current state of use of animal‐derived reagents across different sectors and to identify the obstacles possibly hampering the implementation of non‐animal derived alternatives, a global online survey addressed to scientists working on in vivo, in vitro, in silico methods, in academia as well as pharmaceutical or cosmetic companies, was conducted with the goal to understand: 1) the most commonly used animal‐derived materials and reagents, 2) the main issues associated with the production and use of animal‐derived materials and reagents, 3) the current level of knowledge on available non‐animal alternative materials and reagents, and 4) what educational and information sources could be most useful or impactful to disseminate knowledge on non‐animal alternatives. This paper provides an overview of the survey replies and discusses possible proposals to increase awareness, acceptance and use of non‐animal ingredients.

Evidence of Swim secretion and association with extracellular matrix in the Drosophila embryo.

Secreted wingless-interacting protein (Swim) is the Drosophila ortholog gene of the mammalian Tubulointerstitial Nephritis Antigen like 1 (TINAGL1), also known as lipocalin-7 (LCN7), or adrenocortical zonation factor 1 (AZ-1). Swim and TINAGL1 proteins share a significant homology, including the somatomedin B and the predictive inactive C1 cysteine peptidase domains. In mammals, both TINAGL1 and its closely related homolog TINAG have been identified in basement membranes, where they may function as modulators of integrin-mediated adhesion. In Drosophila, Swim was initially identified in the eggshell matrix and was subsequently detected in the culture medium of S2 cells. Further biochemical analysis indicated that Swim binds to wingless (wg) in a lipid-dependent manner. This observation, together with RNAi-knockdown studies, suggested that Swim is an essential cofactor of wg-signalling. However, recent elegant genetic studies ruled out the possibility that Swim is required alone to facilitate wg-signalling in Drosophila, because flies without Swim are viable and fertile. Here, we use the UAS/Gal4 expression system together with confocal imaging to analyze the in vivo localization of a chimeric Swim-GFP in the developing Drosophila embryo. Our data fully support the notion that Swim is an extracellular matrix component that is secreted upon ectopic expression and preferentially associates with the basement membranes of various organs and with the specialized tendon matrix at the muscle attachment sites (MAS). Interestingly, the accumulation of Swim at the MAS does not require integrins. In conclusion, Swim is an extracellular matrix component, and Swim may exhibit overlapping functions in concert with other undefined components.

Impact of Artificial Sputum Medium Formulation on Pseudomonas aeruginosa Secondary Metabolite Production

ABSTRACTIn vitro culture media are being developed to understand how host site-specific nutrient profiles influence microbial pathogenicity and ecology. To mimic the cystic fibrosis (CF) lung environment, a variety of artificial sputum media (ASM) have been created. However, the composition of these ASM vary in the concentration of key nutrients, including amino acids, lipids, DNA, and mucin. In this work, we used feature-based molecular networking (FBMN) to perform comparative metabolomics of Pseudomonas aeruginosa, the predominant opportunistic pathogen infecting the lungs of people with CF, cultured in nine different ASM. We found that the concentration of aromatic amino acids and iron from mucin added to the media contributes to differences in the production of P. aeruginosa virulence-associated secondary metabolites.IMPORTANCE Different media formulations aiming to replicate in vivo infection environments contain different nutrients, which affects interpretation of experimental results. Inclusion of undefined components, such as commercial porcine gastric mucin (PGM), in an otherwise chemically defined medium can alter the nutrient content of the medium in unexpected ways and influence experimental outcomes.

Mouse Primordial Germ Cells: In Vitro Culture and Conversion to Pluripotent Stem Cell Lines.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes. Despite decades of research, in vitro culture of PGCs remains a major challenge and has previously relied on undefined components such as serum and feeders. Notably, PGCs cultured for extended periods do not maintain their lineage identity but instead undergo conversion to form pluripotent stem cell lines called embryonic germ (EG) cells in response to LIF/STAT3 signaling. Here we report both established and new methodologies to derive EG cells, in a range of different conditions. We show that basic fibroblast growth factor is not required for EG cell conversion. We detail the steps taken in our laboratory to systematically remove complex components and establish a fully defined protocol that allows efficient conversion of isolated PGCs to pluripotent EG cells. In addition, we demonstrate that PGCs can adhere and proliferate in culture without the support of feeder cells or serum. This may well suggest novel approaches to establishing short-term culture of PGCs in defined conditions.

A Defined and Scalable Peptide-Based Platform for the Generation of Human Pluripotent Stem Cell-Derived Astrocytes.

Astrocytes comprise the most abundant cell type in the central nervous system (CNS) and play critical roles in maintaining neural tissue homeostasis. In addition, astrocyte dysfunction and death has been implicated in numerous neurological disorders such as multiple sclerosis, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD). As such, there is much interest in using human pluripotent stem cell (hPSC)-derived astrocytes for drug screening, disease modeling, and regenerative medicine applications. However, current protocols for generation of astrocytes from hPSCs are limited by the use of undefined xenogeneic components and two-dimensional (2D) culture surfaces, which limits their downstream applications where large-quantities of cells generated under defined conditions are required. Here, we report the use of a completely synthetic, peptide-based substrate that allows for the differentiation of highly pure populations of astrocytes from several independent hPSC lines, including those derived from patients with neurodegenerative disease. This substrate, which we demonstrate is compatible with both conventional 2D culture formats and scalable microcarrier (MC)-based technologies, leads to the generation of cells that express high levels of canonical astrocytic markers as well as display properties characteristic of functionally mature cells including production of apolipoprotein E (ApoE), responsiveness to inflammatory stimuli, ability to take up amyloid-β (Aβ), and appearance of robust calcium transients. Finally, we show that these astrocytes can be cryopreserved without any loss of functionality. In the future, we anticipate that these methods will enable the development of bioprocesses for the production of hPSC-derived astrocytes needed for biomedical research and clinical applications.

Benchtop to bioreactor: T-cell culture and expansion in chemically defined media

Background & Aim Cell therapy is a blossoming field that focuses on the application of cells as therapeutic products; accordingly, the associated manufacturing process is cell culture. The difficulty of generating sufficient cell numbers with consistent clinical quality, a major challenge in the field, can be mitigated by addressing two factors: cell source and media composition. The removal of undefined media components is essential for a dependable, reproducible manufacturing process and safe translation for clinical use. Therefore, the development of cGMP-manufactured T-cell culture media that can be used reliably to culture, differentiate, and expand T-cells under serum-free conditions is essential. Methods, Results & Conclusion Following FUJIFILM's rational design approach to media development, our extensive expertise has led to the creation of a chemically defined, animal-component free T-cell culture medium (CDM), with equal or better performance than other commercially available offers while being cGMP grade. Drawing from a vast registry of donors at HemaCare, we targeted peripheral blood mononuclear cells (PBMCs), positively selected CD3+ cells, and negatively isolated CD3+ cells, demonstrating the effectiveness of FUJIFILM Irvine Scientific's PRIME-XV T-cell CDM using multiple cell culture systems and activation methods. HemaCare's precisely isolated and purified cells cultured in PRIME-XV T-cell CDM maintained robust expansion, viability, and phenotype trends across donors and cell culture systems, from small-scale plates and flasks to scaled-up bioreactors. Cell therapy is a blossoming field that focuses on the application of cells as therapeutic products; accordingly, the associated manufacturing process is cell culture. The difficulty of generating sufficient cell numbers with consistent clinical quality, a major challenge in the field, can be mitigated by addressing two factors: cell source and media composition. The removal of undefined media components is essential for a dependable, reproducible manufacturing process and safe translation for clinical use. Therefore, the development of cGMP-manufactured T-cell culture media that can be used reliably to culture, differentiate, and expand T-cells under serum-free conditions is essential. Following FUJIFILM's rational design approach to media development, our extensive expertise has led to the creation of a chemically defined, animal-component free T-cell culture medium (CDM), with equal or better performance than other commercially available offers while being cGMP grade. Drawing from a vast registry of donors at HemaCare, we targeted peripheral blood mononuclear cells (PBMCs), positively selected CD3+ cells, and negatively isolated CD3+ cells, demonstrating the effectiveness of FUJIFILM Irvine Scientific's PRIME-XV T-cell CDM using multiple cell culture systems and activation methods. HemaCare's precisely isolated and purified cells cultured in PRIME-XV T-cell CDM maintained robust expansion, viability, and phenotype trends across donors and cell culture systems, from small-scale plates and flasks to scaled-up bioreactors.

Serum free medium for large scale microcarrier culture system of hmsc towards clinical cell-based therapy

Background & Aim Recent advances in human mesenchymal stem cell (hMSC)-based clinical therapies for stroke, graft vs. host disease and degenerative diseases has elevated the need for the production of large numbers of functional cells in scalable, homogeneous and cost-effective processes. Current 2D static culture method can not meet the anticipated demand for cells as it is labor intensive, not scalable and not a controlled system. Bioreactor system with microcarriers as a matrix in combination with optimized xeno-free (XF) medium represent a preferred culture structure towards clinical applications. The current xeno-free culture media for hMSC are specially designed for 2D static culture and are not necessarily suitable for 3D environment. The addition of undefined animal components such as serum to the media to enable the culture of hMSC on microcarriers in suspension pose safety concerns and it can be a major obstacle for obtaining regulatory approval when required. The present study evaluated a novel GMP compliant, serum-free (SF), XF hMSC expansion medium, with auxiliary solutions for attachment and dissociation suitable for microcarrier cultures in bioreactors. Methods, Results & Conclusion Results demonstrate the feasibility of expanding hMSC from various sources (one marrow, Wharton Jelly and adipose tissue) in a scalable microcarrier-based culture system using an optimized xeno-free medium while maintaining cell characteristics. Stability studies over 1 year, validated that the media was able to support robust hMSC growth and colony formation. The microcarrier suspension culture system combined with SF media represents a significant footstep for large-scale expansion of regulatory accepted hMSC for cellular therapeutics.

Principles of in vitro nutrition for human mesenchymal stromal cells

Background & Aim In the cultivation of stem cells in vitro, components of animal origin and/or chemically undefined extracts like human platelet lysate are still used for the formulation of culture media because the quality of the most common basal media is unsuitable for cell culture in chemically defined environments. Experimental evidence shows that the better the qualitative and quantitative composition of the basal medium, the fewer additives of animal origin or chemically undefined components are needed to achieve successful cultivation of stem cells. Methods, Results & Conclusion The contribution deals with essential aspects concerning identity and quality of culture media components for pre-clinical and clinical research. It describes the development of physico-chemically defined micro-environments for cell cultures that modulate functions of the metabolism of human stem cells from adipose tissue (hASCs). The development process consists of two parts. The first phase involves the development of fully defined cellular microenvironments capable of satisfying the essential physiological needs of the cell. For this purpose, a detailed analysis of the physical (pH, osmotic pressure) and chemical requirements (energy sources, amino acids, vitamins, precursors of fatty and nucleic acids, inorganic salts, trace elements and chemical impurities) of the cells is carried out, which are then selected for the second phase. The second phase involves the elimination of complex, chemically undefined variables such as fetal calf serum or human platelet lysate, which are to be considered as critical components in cell culture media. The selection of a suitable basal medium and relative preparation technique allows the achievement of the goal. The results show that good knowledge of the variables that influence cellular growth processes has numerous advantages. The two-step strategy described above allows the identification of the essential physiological needs of the cells, thus favouring control and modulation of the main metabolic functions. This perspective becomes even more interesting as the omission of complex and expensive medium additives in clinical culture processes leads to a reduction of contamination risk and production costs.

Identification of neurospheres generated from human dental pulp stem cells in xeno-/serum-free conditions

IntroductionCell-based therapies require an emerging alternative treatment using easily harvested cell sources. Neural stem cells derived from various tissues, including brain, bone marrow, skin and retina can give rise to both neurons and glial cells. Recently, human dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to have mesenchymal stem cell-like abilities such as self-renewal and multi-lineage differentiation, including neuron and glial cells. Moreover, DPSCs and SHED show a higher proliferation rate and a higher number of population doublings compared with adult bone marrow stromal stem cells. Therefore, DPSCs are a useful source that can be applied in cell replacement therapy for various neurological disorders. Generally, the conventional culture methods for DPSCs have used serum, therefore the undefined components in culture medium may complicate investigations of the molecular mechanisms that control the self-renewal and differentiation of DPSCs. However, neural stem cells proliferate to form ‘neurospheres’ in suspension in vitro in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). No study to date has obtained neurospheres from DPSCs in serum-free conditions in primary culture. Thus, the aim of this study was to establish a method for the proliferation and neural differentiation of DPSCs in xeno- and serum-free conditions in primary culture.MethodsDPSCs were obtained from the dental pulp of wisdom teeth from healthy individuals (18–41 years old) and cultured in conventional medium containing 15% fetal bovine serum and xeno-/serum-free medium. We evaluated the proliferation of DPSCs, neurosphere generation, and neural differentiation under xeno-/serum-free conditions by flow cytometry, immunohistochemistry, and real-time polymerase chain reaction.ResultsIn proliferation medium without xeno/serum, DPSCs can proliferate and generate neurospheres, however, the neurospheres had limited self-renewal ability. Under differentiation conditions, class III β-tubulin (TUBB3) and microtubule-associated protein (MAP2) were more significantly expressed in neurospheres derived from DPSCs in xeno-/serum-free culture conditions than in DPSCs in conventional culture conditions.ConclusionsOur result demonstrated that neurosphere generation from DPSCs in xeno-/serum-free culture may be an accessible source for clinical cell replacement therapies for neuronal degenerative diseases.

Amino acids are key substrates to Escherichia coli BW25113 for achieving high specific growth rate

Studying substrate consumption in nutrient-rich conditions is challenging because often the growth medium includes undefined components like yeast extract or peptone. For clear and consistent results, it is necessary to use defined medium, where substrate utilization can be followed. In the present work, Escherichia coli BW25113 batch growth in a medium supplemented with 20 proteinogenic amino acids and glucose was studied. Focus was on the quantitative differences in substrate consumption and proteome composition between minimal and nutrient-rich medium. In the latter, 72% of carbon used for biomass growth came from amino acids and 28% from glucose. Serine was identified as the most consumed substrate with 41% of total carbon consumption. Proteome comparison between nutrient-rich and minimal medium revealed changes in TCA cycle and acetate producing enzymes that together with extracellular metabolite data pointed to serine being consumed mainly for energy generation purposes. Serine removal from the growth medium decreased specific growth rate by 22%. In addition, proteome comparison between media revealed a large shift in amino acid synthesis and translation related proteins. Overall, this work describes in quantitative terms the batch growth carbon uptake profile and proteome allocation of E. coli BW25113 in minimal and nutrient-rich medium.

40: Noninvasive Monitoring of Transplanted Hepatocytes by Deep Red Imaging in Rats

Technologies for in vivo imaging gain significance to monitor distribution and integration of cells for the use in cell therapies during the regeneration of diseased organs. The aim of the study was to identify NIR dyes for visualization of transplanted hepatocytes in vivo in rats. Wild type and CD26-deficient hepatocytes and livers from wild type Fisher rats were used. A total of six dyes (DY676, 681, 776, 777, 782, 800 emission range 699-801nm) were analyzed using a multispectral imager. For quantification, the Regions-of-Interests (ROI) of defined mixtures of CD26-positive and negative cells were detected. Results were verified by flow cytometry. DY676, 776, 777, 782, 800 yielded non-specific conjugates with undefined cellular components. DY681 linked to the CD26-antibody specifically detected CD26-positive hepatocytes. DY681 displayed ROIs of 372, 375, 244, 102 and 9 [average signal] using defined mixtures of 100%, 85%, 50%, 15% and 0% CD26-positive cells. Flow cytometry confirmed 99%, 78%, 56%, 21% and 0.2% CD26-positive cells. Wildtype livers treated in situ with DY681-CD26 antibody displayed positive signals throughout the whole organ. The DY681-linked antibody is eligible for the non-invasive labeling of transplanted hepatocytes. Thus, the hepatic distribution of the cells and repopulation of the liver might be observed, which helps to reduce the amount of animal experiments. Wilhelm Roux Program 19/38.

Abstract 711: Therapeutic Effects of Engineered Human Pluripotent Stem Cell-derived Lymphatic Endothelial Cells on Experimental Lymphedema

Current systems generating lymphatic endothelial cell (LEC) from human pluripotent stem cells (hPSCs) have limited value due to low purity, the use of undefined components for differentiation, and poor cell survival in vivo . Here, we developed a fully defined system to differentiate hPSCs into LECs and evaluated their therapeutic and engraftment potential when encapsulated in a nanomatrix gel. hPSCs were cultured with GSK3-β inhibitor for 3 days to induce differentiation into the mesodermal lineage. The mesodermal cells were cultured for another 6 days and double-sorted by PDPN and FLT4. These hPSC-PDPN + FLT4 + cells showed highly purified and fully functional LEC characteristics in vitro. These hPSC-LECs express LEC markers such as PDPN, LYVE1, PROX1, and FLT4 at the mRNA level and the protein level, and formed tube-like structures in Matrigel. We next determined the lymphatic vascular reparative effects of engineered hPSC-LECs. After inducing lymphedema in the tail of mouse, hPSC-LECs, hPSC-LECs/PA-RGDS, human dermal lymphatic endothelial cells (hdLEC), PA-RGDS, or PBS were injected into the tail of mouse. Tail thickness significantly decreased in the groups injected with hPSC-LECs with or without PA-RGDS compared to the other groups at day 28. At day 45, mice injected with PA-RGDS encapsulated hPSC-LECs showed significant decrease in the tail diameter compared to all other groups including those injected with hPSC-LECs. This study demonstrated for the first time that PA-RGDS encapsulation can substantially improve lymphedema repair in mouse tail through enhancement of cell survival and lymphatic neovascularizationThis engineered hPSC-LEC therapy represents a novel option for treating lymphedema.