Antisense-oligonucleotide-mediated perturbation of long non-coding RNA reveals functional features in stem cells and across cell types.

Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell-type enrichment analysis identifies lncRNAs associated with pluripotency, while the knockdown of LINC02595, CATG00000090305.1, and RP11-148B6.2 modulates colony formation of iPSCs. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit a consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights that molecular phenotyping is more comprehensive in revealing affected pathways.

Recombination of repeat elements generates somatic complexity in human genomes.

Non-allelic recombination between homologous repetitive elements contributes to evolution and human genetic disorders. Here, we combine short- and long-DNA read sequencing of repeat elements with a new bioinformatics pipeline to show that somatic recombination of Alu and L1 elements is widespread in the human genome. Our analysis uncovers tissue-specific non-allelic homologous recombination hallmarks; moreover, we find that centromeres and cancer-associated genes are enriched for retroelements that may act as recombination hotspots. We compare recombination profiles in human-induced pluripotent stem cells and differentiated neurons and find that the neuron-specific recombination of repeat elements accompanies chromatin changes during cell-fate determination. Finally, we report that somatic recombination profiles are altered in Parkinson's and Alzheimer's disease, suggesting a link between retroelement recombination and genomic instability in neurodegeneration. This work highlights a significant contribution of the somatic recombination of repeat elements to genomic diversity in health and disease.

Decoding Neuronal Diversification by Multiplexed Single-cell RNA-Seq.

Cellular reprogramming is driven by a defined set of transcription factors; however, the regulatory logic that underlies cell-type specification and diversification remains elusive. Single-cell RNA-seq provides unprecedented coverage to measure dynamic molecular changes at the single-cell resolution. Here, we multiplex and ectopically express 20 pro-neuronal transcription factors in human dermal fibroblasts and demonstrate a widespread diversification of neurons based on cell morphology and canonical neuronal marker expressions. Single-cell RNA-seq analysis reveals diverse and distinct neuronal subtypes, including reprogramming processes that strongly correlate with the developing brain. Gene mapping of 20 exogenous pro-neuronal transcription factors further unveiled key determinants responsible for neuronal lineage specification and a regulatory logic dictating neuronal diversification, including glutamatergic and cholinergic neurons. The multiplex scRNA-seq approach is a robust and scalable approach to elucidate lineage and cellular specification across various biological systems.

Single-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids.

Human stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organization and function of a respective organ or part of an organ. Human midbrain organoids (hMOs) have been described to contain midbrain-specific dopaminergic neurons that release the neurotransmitter dopamine. However, the human midbrain contains also additional neuronal cell types, which are functionally interacting with each other. Here, we analysed hMOs at high-resolution by means of single-cell RNA sequencing (scRNA-seq), imaging and electrophysiology to unravel cell heterogeneity. Our findings demonstrate that hMOs show essential neuronal functional properties as spontaneous electrophysiological activity of different neuronal subtypes, including dopaminergic, GABAergic, glutamatergic and serotonergic neurons. Recapitulating these in vivo features makes hMOs an excellent tool for in vitro disease phenotyping and drug discovery.

Brainstem Organoids From Human Pluripotent Stem Cells.

The brainstem is a posterior region of the brain, composed of three parts, midbrain, pons, and medulla oblongata. It is critical in controlling heartbeat, blood pressure, and respiration, all of which are life-sustaining functions, and therefore, damages to or disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells (hPSCs) recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models are limited in the extent hPSCs recapitulate human brain development and hence are not able to fully elucidate the diseases affecting various components of the brain such as brainstem. Here, we developed a method to generate human brainstem organoids (hBSOs), containing midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, dopaminergic neurons, and neural crest lineage cells. Single-cell RNA sequence (scRNA-seq) analysis, together with evidence from proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem, which raises the possibility of making use of hBSOs in investigating central nervous system disorders affecting brainstem and in efficient drug screenings.

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions.

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.

C1 CAGE detects transcription start sites and enhancer activity at single-cell resolution.

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3'-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5'-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-ß of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization.

The essentiality of non-coding RNAs in cell reprogramming.

In mammals, short (mi-) and long non-coding (lnc) RNAs are immensely abundant and they are proving to be more functional than ever before. Particularly in cell reprogramming, non-coding RNAs are essential to establish the pluripotent network and are indispensable to reprogram somatic cells to pluripotency. Through systematic screening and mechanistic studies, diverse functional features of both miRNA and lncRNAs have emerged as either scaffolds, inhibitors, or co-activators, necessary to orchestrate the intricacy of gene regulation. Furthermore, the collective characterizations of both miRNA and lncRNA reveal their interdependency (e.g. sequestering the function of the other) to modulate cell reprogramming. This review broadly explores the regulatory processes of cell reprogramming - with key functional examples in neuronal and cardiac differentiations - in the context of both short and long non-coding RNAs.

Collagen hydrogels strengthened by biodegradable meshes are a basis for dermo-epidermal skin grafts intended to reconstitute human skin in a one-step surgical intervention.

Extensive full-thickness skin loss, associated with deep burns or other traumata, represents a major clinical problem that is far from being solved. A promising approach to treat large skin defects is the use of tissue-engineered full-thickness skin analogues with nearly normal anatomy and function. In addition to excellent biological properties, such skin substitutes should exhibit optimal structural and mechanical features. This study aimed to test novel dermo-epidermal skin substitutes based on collagen type I hydrogels, physically strengthened by two types of polymeric net-like meshes. One mesh has already been used in clinical trials for treating inguinal hernia; the second one is new but consists of a FDA-approved polymer. Both meshes were integrated into collagen type I hydrogels and dermo-epidermal skin substitutes were generated. Skin substitutes were transplanted onto immuno-incompetent rats and analyzed after distinct time periods. The skin substitutes homogeneously developed into a well-stratified epidermis over the entire surface of the grafts. The epidermis deposited a continuous basement membrane and dermo-epidermal junction, displayed a well-defined basal cell layer, about 10 suprabasal strata and a stratum corneum. Additionally, the dermal component of the grafts was well vascularized.

Tissue-engineered dermo-epidermal skin grafts prevascularized with adipose-derived cells.

The major problem in skin grafting is that tissue-engineered skin grafts after their transplantation are initially entirely dependent on diffusion. Since this process is slow and inefficient, nutrients, growth factors, and oxygen will insufficiently be supplied and the regenerating graft will undergo a physiological crisis, resulting in scar-like dermal structures and shrinkage. The tissue-engineering of a vascular network in human dermo-epidermal skin substitutes (DESS) is a promising approach to overcome this limitation. Here we report, for the first time, on the use of the adipose stromal vascular fraction (SVF)-derived endothelial cell population to tissue-engineer DESS containing a highly efficient capillary plexus. To develop vascular networks in vitro, we employed optimized 3D fibrin or collagen type I hydrogel systems. Upon transplantation onto immune-deficient rats, these pre-formed vascular networks anastomosed to the recipient's vasculature within only four days. As a consequence, the neo-epidermis efficiently established tissue homeostasis, the dermis underwent almost no contraction, and showed sustained epidermal coverage in vivo. Overall, the here described rapid and efficient perfusion of SVF-based skin grafts opens new perspectives for the treatment of hitherto unmet clinical needs in burn/plastic surgery and dermatology.

Bioengineering dermo-epidermal skin grafts with blood and lymphatic capillaries.

The first bioengineered, autologous, dermo-epidermal skin grafts are presently undergoing clinical trials; hence, it is reasonable to envisage the next clinical step at the forefront of plastic and burn surgery, which is the generation of autologous skin grafts that contain vascular plexuses, preformed in vitro. As the importance of the blood, and particularly the lymphatic vascular system, is increasingly recognized, it is attractive to engineer both human blood and lymphatic vessels in one tissue or organ graft. We show here that functional lymphatic capillaries can be generated using three-dimensional hydrogels. Like normal lymphatics, these capillaries branch, form lumen, and take up fluid in vitro and in vivo after transplantation onto immunocompromised rodents. Formation of lymphatic capillaries could be modulated by both lymphangiogenic and anti-lymphangiogenic stimuli, demonstrating the potential usefulness of this system for in vitro testing. Blood and lymphatic endothelial cells never intermixed during vessel development, nor did blood and lymphatic capillaries anastomose under the described circumstances. After transplantation of the engineered grafts, the human lymphatic capillaries anastomosed to the nude rat's lymphatic plexus and supported fluid drainage. Successful preclinical results suggest that these skin grafts could be applied on patients suffering from severe skin defects.

Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog.

PURPOSE: Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formation and stratification in a humanized animal model. METHODS: Dermo-epidermal skin grafts with either amniocytes or with fibroblasts in the dermis were compared in a rat model. Full-thickness skin wounds on the back of immuno-incompetent rats were covered with skin grafts with (1) amniocytes in the dermis, (2) fibroblasts in the dermis, or, (3) acellular dermis. Grafts were excised 7 and 21 days post transplantation. Histology and immunofluorescence were performed to investigate epidermis formation, stratification, and expression of established skin markers. RESULTS: The epidermis of skin grafts engineered with amniocytes showed near-normal anatomy, a continuous basal lamina, and a stratum corneum. Expression patterns for keratin 15, keratin 16, and Ki67 were similar to grafts with fibroblasts; keratin 1 expression was not yet fully established in all suprabasal cell layers, expression of keratin 19 was increased and not only restricted to the basal cell layer as seen in grafts with fibroblasts. In grafts with acellular dermis, keratinocytes did not survive. CONCLUSION: Dermo-epidermal skin grafts with amniocytes show near-normal physiological behavior suggesting that amniocytes substitute fibroblast function to support the essential cross-talk between mesenchyme and epithelia needed for epidermal stratification. This novel finding has considerable implications regarding tissue engineering.

Glucose sensing in human epidermis using mid-infrared photoacoustic detection.

No reliable non-invasive glucose monitoring devices are currently available. We implemented a mid-infrared (MIR) photoacoustic (PA) setup to track glucose in vitro in deep epidermal layers, which represents a significant step towards non-invasive in vivo glucose measurements using MIR light. An external-cavity quantum-cascade laser (1010-1095 cm(-1)) and a PA cell of only 78 mm(3) volume were employed to monitor glucose in epidermal skin. Skin samples are characterized by a high water content. Such samples investigated with an open-ended PA cell lead to varying conditions in the PA chamber (i.e., change of light absorption or relative humidity) and cause unstable signals. To circumvent variations in relative humidity and possible water condensation, the PA chamber was constantly ventilated by a 10 sccm N(2) flow. By bringing the epidermal skin samples in contact with aqueous glucose solutions with different concentrations (i.e., 0.1-10 g/dl), the glucose concentration in the skin sample was varied through passive diffusion. The achieved detection limit for glucose in epidermal skin is 100 mg/dl (SNR=1). Although this lies within the human physiological range (30-500 mg/dl) further improvements are necessary to non-invasively monitor glucose levels of diabetes patients. Furthermore spectra of epidermal tissue with and without glucose content have been recorded with the tunable quantum-cascade laser, indicating that epidermal constituents do not impair glucose detection.

Modified plastic compression of collagen hydrogels provides an ideal matrix for clinically applicable skin substitutes.

Tissue engineering of clinically applicable dermo-epidermal skin substitutes is crucially dependent on the three-dimensional extracellular matrix, supporting the biological function of epidermal and dermal cells. This matrix essentially determines the mechanical stability of these substitutes to allow for safe and convenient surgical handling. Collagen type I hydrogels yield excellent biological functionality, but their mechanical weakness and their tendency to contract and degrade does not allow producing clinically applicable transplants of larger sizes. We show here that plastically compressed collagen type I hydrogels can be produced in clinically relevant sizes (7×7 cm), and can be safely and conveniently handled by the surgeon. Most importantly, these dermo-epidermal skin substitutes mature into a near normal skin that can successfully reconstitute full-thickness skin defects in an animal model.

Formation of human capillaries in vitro: the engineering of prevascularized matrices.

Initial take, development, and function of transplanted engineered tissue substitutes are crucially dependent on rapid and adequate blood perfusion. Therefore, the development of rapidly and efficiently vascularized tissue grafts is vital for tissue engineering and regenerative medicine. Here we report on the construction of a network of highly organotypic capillaries in engineered tissue substitutes. We employed a three-dimensional culture system consisting of human microvascular endothelial cells. These were reproducibly expanded at high purity and subsequently seeded into biodegradable, fibrin-based hydrogels. The process of capillary formation in vitro followed the principles of both angiogenesis and postnatal vasculogenesis and a distinct sequence of other developmental steps that closely resemble embryonic neovascularization. Capillary lumen formation in vitro was initiated by the deposition of a basement membrane and intensive pinocytosis, followed by the generation of intracellular vacuoles, successive fusion of these vacuoles, and finally the formation of a long, continuous lumen. After transplantation the vascular structures were stabilized by mural cells of the recipient animal. Our findings suggest that the in vitro engineering of prevascularized matrices is within reach.