A Comparative Analysis of the Wound Healing-Related Heterogeneity of Adipose-Derived Stem Cells Donors.

Adipose-derived Stem cells (ASCs) are on the verge of being available for large clinical trials in wound healing. However, for developing advanced therapy medicinal products (ATMPs), potency assays mimicking the mode of action are required to control the product consistency of the cells. Thus, greater effort should go into the design of product assays. Therefore, we analyzed three ASC-based ATMPs from three different donors with respect to their surface markers, tri-lineage differentiation, proliferation, colony-forming unit capacity, and effect on fibroblast proliferation and migration, endothelial proliferation, migration, and angiogenesis. Furthermore, the transcriptome of all three cell products was analyzed through RNA-sequencing. Even though all products met the criteria by the International Society for Cell and Gene Therapy and the International Federation for Adipose Therapeutics and Science, we found one product to be consistently superior to others when exploring their potency in the wound healing specific assays. Our results indicate that certain regulatory genes associated with extracellular matrix and angiogenesis could be used as markers of a superior ASC donor from which to use ASCs to treat chronic wounds. Having a panel of assays capable of predicting the potency of the product would ensure the patient receives the most potent product for a specific indication, which is paramount for successful patient treatment and acceptance from the healthcare system.

Support for mobilising medical students to join the COVID-19 pandemic emergency healthcare workforce: a cross-sectional questionnaire survey.

OBJECTIVE: To identify what motivates medical students to join a pandemic emergency healthcare workforce. DESIGN: Cross-sectional study. SETTING: Aalborg University, Denmark. PARTICIPANTS: All medical students. MAIN OUTCOME MEASURES: Motivational points as perceived by the students to be important. Demographic characteristics and 11 motivational domains scored on a Visual Analog Scale from 0 (low) to 100 (high) responding to the question: 'To what degree are the following statements important for you to join a national emergency preparedness workforce?' The questionnaire was developed by an expert panel in a process of four iterations. RESULTS: A total of 486 students of 688 (70.6%) completed the survey within 7 days in March 2020. 80% had decided to join the pandemic emergency healthcare workforce. Ranked median scores for motivational statements in each domain were: care, 100; learn, 90; pride, 83; team, 77; needed, 75; safety, 75; supervision, 75; job, 73; duty, 66; salary, 62; historic, 50. Supervision (p<0.001), salary (p<0.001) and duty (p=0.001) were given increasing priority with advancing study years. Interestingly, students added that support by the university and clarification of study plans were priorities. CONCLUSIONS: Results guide decision-makers and colleagues on how to motivate or reinforce medical students in joining the pandemic emergency healthcare workforce. Importantly, students emphasised protection for themselves.

Regeneration of the oesophageal muscle layer from oesophagus acellular matrix scaffold using adipose-derived stem cells.

This study explored the feasibility of constructing a tissue engineered muscle layer in the oesophagus using oesophageal acellular matrix (OAM) scaffolds and human aortic smooth muscle cells (hASMCs) or human adipose-derived stem cells (hASCs). The second objective was to investigate the effect of hypoxic preconditioning of seeding cells on cell viability and migration depth. Our results demonstrated that hASMCs and hASCs could attach and adhere to the decellularized OAM scaffold and survive and proliferate for at least 7 days depending on the growth conditions. This indicates adipose-derived stem cells (ASCs) have the potential to substitute for smooth muscle cells (SMCs) in the construction of tissue engineered oesophageal muscle layers.

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells.

Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.

Hypoxia Enhances Differentiation of Adipose Tissue-Derived Stem Cells toward the Smooth Muscle Phenotype.

Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p < 0.01). Cells differentiated in 5% oxygen conditions showed greater contraction effect (p < 0.01). Hypoxia influences differentiation of smooth muscle cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

Maintaining RNA Integrity for Transcriptomic Profiling of Ex Vivo Cultured Limbal Epithelial Stem Cells after Fluorescence-Activated Cell Sorting (FACS).

BACKGROUND: Transcriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS). METHODS: To address this problem, we systematically analyzed the critical steps, and found that ethanol fixation together with optimized downstream procedures provided a pipeline that yielded high quality total RNA in amounts to readily support the RNA-seq procedure, while still preserving good discrimination between the individual hLESC immunophenotypes. RESULTS: The average RNA integrity number (RIN) was 7.7 ± 0.4, and the average yield was 4.6 ± 1.7 pg of RNA per cell. The sequencing analysis of the isolated RNA produced high quality data with more than 70% of read pairs mapping uniformly to the reference genome and 80% of bases with a Phred score of at least 30. CONCLUSION: In this study, we developed a reliable FACS-based procedure using ethanol as a fixative that would support accurate isolation of limbal epithelial progenitor subpopulations along with RNA yield and quality sufficient to enable deep transcriptomic profiling.

Mass spectrometry analysis of adipose-derived stem cells reveals a significant effect of hypoxia on pathways regulating extracellular matrix.

BACKGROUND: Adipose-derived stem cells (ASCs) are being increasingly recognized for their potential to promote tissue regeneration and wound healing. These effects appear to be partly mediated by paracrine signaling pathways, and are enhanced during hypoxia. Mass spectrometry (MS) is a valuable tool for proteomic profiling of cultured ASCs, which may help to reveal the identity of the factors secreted by the cells under different conditions. However, serum starvation which is essentially required to obtain samples compatible with secretome analysis by MS can have a significant influence on ASCs. Here, we present a novel and optimized culturing approach based on the use of a clinically relevant serum-free formulation, which was used to assess the effects of hypoxia on the ASC proteomic profile. METHODS: Human ASCs from three human donors were expanded in StemPro® MSC SFM XenoFree medium. Cells were cultured for 24 h in serum- and albumin-free supplements in either normoxic (20 %) or hypoxic (1 %) atmospheres, after which the cells and conditioned medium were collected, subfractionated, and analyzed using MS. Prior to analysis, the secreted proteins were further subdivided into a secretome (>30 kDa) and a peptidome (3-30 kDa) fraction. RESULTS: MS analysis revealed the presence of 342, 98, and 3228 proteins in the normoxic ASC secretome, peptidome, and proteome, respectively. A relatively small fraction of the proteome (9.6 %) was significantly affected by hypoxia, and the most regulated proteins were those involved in extracellular matrix (ECM) synthesis and cell metabolism. No proteins were found to be significantly modulated by hypoxic treatment across all cultures for the secretome and peptidome samples. CONCLUSIONS: This study highlights ECM remodeling as a significant mechanism contributing to the ASC regenerative effect after hypoxic preconditioning, and further underscores considerable inter-individual differences in ASC response to hypoxia. The novel culture paradigm provides a basis for future proteomic studies under conditions that do not induce a stress response, so that the best responders can be accurately identified for prospective therapeutic use. Data are available via ProteomeXchange with identifier PXD003550.

Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes.

Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.

Transcriptional dissection of human limbal niche compartments by massive parallel sequencing.

Corneal epithelium is maintained throughout life by well-orchestrated proliferation of limbal epithelial stem cells (LESCs), followed by migration and maturation centripetally towards the ocular surface. Disturbance of LESCs can potentially lead to a blinding condition, which can be reversed by reconstitution of a functional LESC pool. The current clinical procedures are effective to some degree, however, deeper knowledge of the molecular interplay within the limbal niche is necessary to achieve a fully satisfactory patient outcome. The present study was thus undertaken to carry out a comprehensive transcriptome analysis of four distinct human limbal compartments, including basal limbal crypts (BLCs), superficial limbal crypts (SLCs), cornea, and the supporting stroma, with the aid of laser capture microdissection and deep RNA sequencing. The tissue harvest pipeline was rigorously optimized so that the exposure to cold ischemia would be less than five minutes. The global gene ontology analysis confirmed existence of primitive cells in BLCs, migratory and activated cells in SLCs, and differentiated cells in cornea. Interestingly, many significantly upregulated genes in SLCs mapped to processes involved in regulation of vasculature, such as sFLT1. In contrast, BLCs exhibited many genes mapping to neurogenic processes and processes related to cell development. The primitive nature of BLCs was, furthermore, confirmed by the KEGG pathway analysis, and some potential regulators of LESCs were revealed, such as Lrig1 and SOX9. The analysis also yielded comprehensive lists of uniquely expressed genes in both BLCs and cornea, which may be useful to identify possible biomarkers. In conclusion, the current investigation provides new insight into the relationship between distinct cell populations within the limbal niche, identifies candidates to be verified for novel biological functions, and yields a wealth of information for prospective data mining.

Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation.

The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth medium supplemented with serum (3T3 system) or without feeder cells in a dedicated serum-free medium (EpiLife). During the culture, the cells were maintained either at ambient oxygen tension (20%) or at different levels of hypoxia (15, 10, 5, and 2% oxygen). The effect of oxygen on cell growth, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18 days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET).

Adipose-derived stem cells from the brown bear (Ursus arctos) spontaneously undergo chondrogenic and osteogenic differentiation in vitro.

In the den, hibernating brown bears do not develop tissue atrophy or organ damage, despite almost no physical activity. Mesenchymal stem cells could play an important role in tissue repair and regeneration in brown bears. Our objective was to determine if adipose tissue-derived stem cells (ASCs) can be recovered from wild Scandinavian brown bears and characterize their differentiation potential. Following immobilization of wild brown bears 7-10 days after leaving the den in mid-April, adipose tissue biopsies were obtained. ASCs were recovered from 6 bears, and shown to be able to undergo adipogenesis and osteogenesis in monolayer cultures and chondrogenesis in pellet cultures. Remarkably, when grown in standard cell culture medium in monolayer cultures, ASCs from yearlings spontaneously formed bone-like nodules surrounded by cartilaginous deposits, suggesting differentiation into osteogenic and chondrogenic lineages. This ability appears to be lost gradually with age. This is the first study to demonstrate stem cell recovery and growth from brown bears, and it is the first report of ASCs spontaneously forming extracellular matrix characteristic of bone and cartilage in the absence of specific inducers. These findings could have implications for the use of hibernating brown bears as a model to study disuse osteoporosis.

Hypoxia and adipose-derived stem cell-based tissue regeneration and engineering.

INTRODUCTION: Realization that oxygen is one of the key regulators of development and differentiation has a profound significance on how current cell-based and tissue engineering applications using adipose-derived stem cells (ASCs) can be further improved. AREAS COVERED: The article provides an overview of mechanisms of hypoxic responses during physiological adaptations and development. Furthermore, a synopsis of the hypoxic responses of ASCs is provided, and this information is presented in context of their utility as a major source of stem cells across the regenerative applications explored to date. EXPERT OPINION: The reader will obtain insight into a highly specific area of stem cell research focusing on ASCs and hypoxia. In order to enhance the level of comprehension, a broader context with other stem cell and experimental systems is provided. It is emphasized that the pericellular oxygen tension is a critical regulatory factor that should be taken into account when devising novel stem cell-based therapeutic applications along with other parameters, such as biochemical soluble factors and the growth substrates.

Powdery mildew fungal effector candidates share N-terminal Y/F/WxC-motif.

BACKGROUND: Powdery mildew and rust fungi are widespread, serious pathogens that depend on developing haustoria in the living plant cells. Haustoria are separated from the host cytoplasm by a plant cell-derived extrahaustorial membrane. They secrete effector proteins, some of which are subsequently transferred across this membrane to the plant cell to suppress defense. RESULTS: In a cDNA library from barley epidermis containing powdery mildew haustoria, two-thirds of the sequenced ESTs were fungal and represented approximately 3,000 genes. Many of the most highly expressed genes encoded small proteins with N-terminal signal peptides. While these proteins are novel and poorly related, they do share a three-amino acid motif, which we named "Y/F/WxC", in the N-terminal of the mature proteins. The first amino acid of this motif is aromatic: tyrosine, phenylalanine or tryptophan, and the last is always cysteine. In total, we identified 107 such proteins, for which the ESTs represent 19% of the fungal clones in our library, suggesting fundamental roles in haustoria function. While overall sequence similarity between the powdery mildew Y/F/WxC-proteins is low, they do have a highly similar exon-intron structure, suggesting they have a common origin. Interestingly, searches of public fungal genome and EST databases revealed that haustoria-producing rust fungi also encode large numbers of novel, short proteins with signal peptides and the Y/F/WxC-motif. No significant numbers of such proteins were identified from genome and EST sequences from either fungi which do not produce haustoria or from haustoria-producing Oomycetes. CONCLUSION: In total, we identified 107, 178 and 57 such Y/F/WxC-proteins from the barley powdery mildew, the wheat stem rust and the wheat leaf rust fungi, respectively. All together, our findings suggest the Y/F/WxC-proteins to be a new class of effectors from haustoria-producing pathogenic fungi.

Escherichia coli phylogenetic groups are associated with site of infection and level of antibiotic resistance in community-acquired bacteraemia: a 10 year population-based study in Denmark.

OBJECTIVES: The aim of this study was to assess whether Escherichia coli phylogenetic groups were associated with the site of infection and the level of antibiotic resistance in community-acquired bacteraemia (CAB). METHODS: The population-based cohort study included 1533 unique isolates of E. coli from Danish patients with CAB during a 10 year period. Triplex PCR was used to classify the phylogenetic groups, and susceptibility testing was performed by disc diffusion. Data were analysed using contingency tables and logistic regression. RESULTS: Overall, 65.9% of the 1533 E. coli isolates belonged to phylogroup B2, 16.6% to D, 13.1% to A and 4.4% to B1. B2 was the most prevalent group for all sites of infection, ranging from 69.9% in cases with a urinary tract site of infection to 54.8% in cases with a hepatobiliary tract site of infection. Antibiotic resistance to one and more than three antibiotics, respectively, was most frequent in group D (11.4%/33.9%), followed by A (5.5%/26.9%), B1 (5.9%/19.1%) and B2 (6.7%/7.5%). Regression analysis, with group B2 as reference, confirmed that groups A and B1 were associated with a site of infection other than the urinary tract and that groups A and D were associated with resistance to antibiotics including ampicillin, sulphonamide, trimethoprim, gentamicin and quinolones. CONCLUSIONS: Phylogenetic group B2 was predominant in E. coli CAB. This was the least resistant of the four groups. Phylogroups A and B1 were associated with sites of infection other than the urinary tract, and resistance to multiple antibiotics was most prevalent for groups A and D.

Transcript elongation factor TFIIS is involved in arabidopsis seed dormancy.

Transcript elongation factor TFIIS promotes efficient transcription by RNA polymerase II, since it assists in bypassing blocks during mRNA synthesis. While yeast cells lacking TFIIS are viable, inactivation of mouse TFIIS causes embryonic lethality. Here, we have identified a protein encoded in the Arabidopsis genome that displays a marked sequence similarity to TFIIS of other organisms, primarily within domains II and III in the C-terminal part of the protein. TFIIS is widely expressed in Arabidopsis, and a green fluorescent protein-TFIIS fusion protein localises specifically to the cell nucleus. When expressed in yeast cells lacking the endogenous TFIIS, Arabidopsis TFIIS partially complements the sensitivity of mutant cells to the nucleotide analog 6-azauridine, which is a typical characteristic of transcript elongation factors. We have characterised Arabidopsis lines harbouring T-DNA insertions in the coding sequence of TFIIS. Plants homozygous for T-DNA insertions are viable, and genomewide transcript profiling revealed that compared to control plants, a relatively small number of genes are differentially expressed in mutant plants. TFIIS(-/-) plants display essentially normal development, but they flower slightly earlier than control plants and show clearly reduced seed dormancy. Plants with RNAi-mediated knockdown of TFIIS expression also are affected in seed dormancy. Therefore, TFIIS plays a critical role in Arabidopsis seed development.

The expression level of the chromatin-associated HMGB1 protein influences growth, stress tolerance, and transcriptome in Arabidopsis.

High mobility group (HMG) proteins of the HMGB family are small and relatively abundant chromatin-associated proteins. As architectural factors, the HMGB proteins are involved in the regulation of transcription and other DNA-dependent processes. We have examined Arabidopsis mutant plants lacking the HMGB1 protein, which is a typical representative of the plant HMGB family. In addition, our analyses included transgenic plants overexpressing HMGB1 and mutant plants that were transformed with the HMGB1 genomic region (complementation plants), as well as control plants. Both the absence and overexpression of HMGB1 caused shorter primary roots and affected the sensitivity towards the genotoxic agent methyl methanesulfonate. The overexpression of HMGB1 decreased the seed germination rate in the presence of elevated concentrations of NaCl. The complementation plants that expressed HMGB1 at wild-type levels did not show phenotypic differences compared to the control plants. Transcript profiling by microarray hybridization revealed that a remarkably large number of genes were differentially expressed (up- and down-regulated) in plants lacking HMGB1 compared to control plants. Among the down-regulated genes, the gene ontology category of stress-responsive genes was overrepresented. Neither microscopic analyses nor micrococcal nuclease digestion experiments revealed notable differences in overall chromatin structure, when comparing chromatin from HMGB1-deficient and control plants. Collectively, our results show that despite the presence of several other HMGB proteins, the lack and overexpression of HMGB1 affect certain aspects of plant growth and stress tolerance and it has a marked impact on the transcriptome, suggesting that HMGB1 has (partially) specialized functions in Arabidopsis.

Extraction and annotation of SAGE tags using sequence quality values.

Data analysis of serial analysis of gene expression (SAGE) tag experiments begins with the extraction of tags from single-pass sequence files of ditag concatemers. When using DNA base quality values generated during base calling, it is possible to control the false-positive discovery rate of unique tags. This chapter describes how to set up a system for generating tag lists from quality associated sequence data.

Duplicate ditag analysis in LongSAGE.

The Long serial analysis of gene expression (SAGE) protocol generates ditags from tags with overlapping overhangs, thereby increasing the probability of duplicate ditag formation in LongSAGE. In this chapter, a tool is presented that facilitates the analysis of duplicate ditags in LongSAGE studies to determine whether they should be included or not.