Organotypic Differentiation Research Articles

The liver organoid's past, present and future: a personalized medicine strategy

Organoids are three-dimensional (3D) cell culture systems derived from human pluripotent stem cells or organotypic differentiation, replicating the complex interactions and functionalities of actual organs. These systems offer significant advantages for studying human tissue and organ biology, addressing limitations of animal models related to sample accessibility and ethical concerns. Liver organoids, in particular, are advanced models developed to study hepatic phenotypes, encompassing various cell types and enabling detailed investigation of cellular, molecular, and genetic aspects of liver diseases, drug metabolism, and protein secretion. They hold promise for fundamental research, drug discovery, and regenerative medicine applications. Despite their potential, organoids face limitations such as simplicity, lack of high-fidelity cell types, flexibility, and atypical physiology. Enhancements in organotypic liver-like surrogates, incorporating in vivo-like cell interactions and architecture, along with advancements in microfluidic chip technology, are expected to improve models for disease, toxicity, and drug discovery, paving the way for new treatments. This review will provide an overview of the history and development of liver organoids, their current progress, challenges, applications, and future prospects in the field of personalized medicine.

Morphogenetic Promoters in the Development of Provisional and Definitive Urinary Organs in Humans and Vertebrates

The aim is to show the regulatory significance of promoter organs of morphogenesis in a stable state of the kidneys of vertebrates (fish, birds) and humans. Material and methods. 118 embryos at stages 12-23 of Carnegie (SC) and 28 human fetuses of 9-12 weeks of fetogenesis, 268 embryos of meat chickens (cross "Hibro PG +") from the stage of 48 hours to 20 days of broiler egg incubation, 50 fry of peled Coregonus peled were studied (Gmelin, 1788) at the stage of yolk sac atrophy (stage 36-37 according to the classification of Detlaf, 1975). To study the method of light microscopy, the material fixed in a 10% neutral form, embedded in paraffin. Sections stained with Mayer's hematoxylin and eosin, McManus CHIC method. Ki-67, CD31, CD34, positive cells are detected by immunohistochemistry. Primary kidney examination by low-vacuum electron microscopy. Embryos and human fetuses are observed in the Tyumen health care facility during medical abortion on demand in anamnestic healthy women with their warning about emergency work. Observation chicken embryos at the poultry farm “Tyumensky Broiler”, incubation workshop (Kaskara village, Tyumen region). Morphometric analysis and statistical processing of the actual material were carried out. Results. It was shown that the organotypic differentiation of the intermediate mesenchyme and the formation of nephrons of the provisional and definitive urinary organs control the state of the mesonephric duct and metanephritic diverticulum. The mesonephric duct initiates the organotypic antibiotic of the perifocal intermediate mesonephric mesenchyme and detects the proximodistal vector of saltatory mesonephronogenesis. The metanephritic diverticulum and its daughter branches initiate the organotypic differentiation of the intermediate mesenchyme and control the fan mechanism of metanephronogenesis. Activity of nephronogenesis Determination of the dynamics of convergence of immunocompetent cells. The absence of ventrodorsal generation of mesonephrons in humans is associated with a significant vital cycle of a normal kidney, atrophy of the mesonephric duct, and a decrease in the activity of immunocompetent cells. An increase in the expression of CD31, CD34, Bcl-2, Ki-67 positive cells is realized in combination with the formation of nephron rudiments as the promoter organs move in the developing organ of urinary formation. Conclusion. Prenatal organogenesis monitors and constantly monitors the formation of promoter organs of morphogenesis - the mesonephric duct and the metanephric disserticulum. Organotypic infection of the intermediate mesenchyme during the development of fish, birds and humans correlates with the chronovector of the convergence of immunocompetent cells.

Liver Organoids: Recent Developments, Limitations and Potential.

Liver cell types derived from induced pluripotent stem cells (iPSCs) share the potential to investigate development, toxicity, as well as genetic and infectious disease in ways currently limited by the availability of primary tissue. With the added advantage of patient specificity, which can play a role in all of these areas. Many iPSC differentiation protocols focus on 3 dimensional (3D) or organotypic differentiation, as these offer the advantage of more closely mimicking in vivo systems including; the formation of tissue like architecture and interactions/crosstalk between different cell types. Ultimately such models have the potential to be used clinically and either with or more aptly, in place of animal models. Along with the development of organotypic and micro-tissue models, there will be a need to co-develop imaging technologies to enable their visualization. A variety of liver models termed “organoids” have been reported in the literature ranging from simple spheres or cysts of a single cell type, usually hepatocytes, to those containing multiple cell types combined during the differentiation process such as hepatic stellate cells, endothelial cells, and mesenchymal cells, often leading to an improved hepatic phenotype. These allow specific functions or readouts to be examined such as drug metabolism, protein secretion or an improved phenotype, but because of their relative simplicity they lack the flexibility and general applicability of ex vivo tissue culture. In the liver field these are more often constructed rather than developed together organotypically as seen in other organoid models such as brain, kidney, lung and intestine. Having access to organotypic liver like surrogates containing multiple cell types with in vivo like interactions/architecture, would provide vastly improved models for disease, toxicity and drug development, combining disciplines such as microfluidic chip technology with organoids and ultimately paving the way to new therapies.

Retinoic Acid Benefits Glomerular Organotypic Differentiation from Adult Renal Progenitor Cells In Vitro.

When in certain culture conditions, organotypic cultures are able to mimic developmental stages of an organ, generating higher-order structures containing functional subunits and progenitor niches. Despite the major advances in the area, researchers have not been able to fully recapitulate the complexity of kidney tissue. Pluripotent stem cells are extensively used in the field, but very few studies make use of adult stem cells. Herein, we describe a simple and feasible method for achieving glomerular epithelial differentiation on an organotypic model comprising human renal progenitor cells from adult kidney (hRPCs). Their glomerular differentiative potential was studied using retinoic acid (RA), a fundamental molecule for intermediate mesoderm induction on early embryogenesis. Immunofluorescence, specific cell surface markers expression and gene expression analysis confirm the glomerular differentiative potential of RA in a short-term culture. We also compared the potential of RA with a potent WNT agonist, CHIR99021, on the differentiative capacity of hRPCs. Gene expression and immunofluorescence analysis confirmed that hRPCs are more sensitive to RA stimulation when compared to CHIR9901. Endothelial cells were also included on the spheroids, resulting in a higher organizational level. The assembly potential of these cells and their selective stimulation will give new insights on adult organotypic cell culture studies and will hopefully guide more works in this important area of research.

Modern dressing based on polymer microfibers with aluminum oxyhydroxide: properties and mechanism of wound healing

Objective: to investigate the structure of the new VitaVallis dressing and determine the mechanism of its wound healing effect. Materials and methods. To study the structure of the VitaVallis dressing, transmission and scanning electron microscopy methods were used. The course of the wound process was studied in 60 male mice of the Balb / c line. The healing of a wound defect under the influence of dressings was studied on the model of a “skin flap”. Morphometric evaluation of histological preparations was carried out by computer-aided graphical analysis of samples. Statistical processing of the results was carried out using the parametric (Student's test) and non-parametric (Wilcoxon's test) methods. For analysis, the program Statistica 6.0 was used. Results. The structure of the VitaVallis dressing is randomly spaced polymer fibers with a diameter of 1.0—5.0 microns, on the surface of which nanosheet structures of aluminum oxyhydroxide (AlOOH) are immobilized. In vivo experiments have shown that the use of VitaVallis dressing in the treatment of model wounds in mice promotes accelerated healing and leads to better epidermis organotypic differentiation and accelerates the maturation of granulation tissue. This effect is due to a combination of factors such as: reduction of inflammatory processes due to the removal and retention of wound exudate, including pathogenic microflora, stimulation of marginal epithelization, protection of the formed granulation tissue from drying out and acceleration of its maturation. Conclusion. Analysis of the structure and mechanism of the wound-healing action of the dressing VitaVallis suggests the effectiveness of its use in the local treatment of wounds.

Special review series on 3D organotypic culture models: Introduction and historical perspective.

Three dimensional (3D) organ-like (organotypic) culture models are a rapidly advancing area of in vitro biological science. In contrast to monolayer cell culture methods which were developed to achieve proliferation of animal cells in the beginning of in vitro biology, the advancements in 3D culture methods are designed to promote cellular differentiation, and to achieve in vivo–like 3D structure and organotypic functions. This project was conceived through the Society for In Vitro Biology to draw on the expertise of individual scientists with special expertise in organotypic cultures of selected tissues or associated interrogation methods to prepare individual-focused reviews in this series. This introductory manuscript will review the early achievements of animal cell culture in monolayer culture and the limitations of that approach to reproduce functioning organ systems. Among these are the nature and 3D architecture of the substrate on which or in which the cells are grown, physical and mechanical clues from the substrate, cell-cell interactions, and defined biochemical factors that trigger the induction of the 3D organotypic differentiation. The organoid culture requires a source of cells with proliferative capacity (ranging from tissue-derived stem or immortalized cells to the iPSC cultures), a suitable substrate or matrix with the mechanical and stimulatory properties appropriate for the organotypic construct and the necessary stimulation of the culture to drive differentiation of the cell population to form the functioning organotypic construct. Details for each type of organotypic construct will be provided in the following papers.

Organ-specific lymphatic vasculature: From development to pathophysiology.

Recent discoveries of novel functions and diverse origins of lymphatic vessels have drastically changed our view of lymphatic vasculature. Traditionally regarded as passive conduits for fluid and immune cells, lymphatic vessels now emerge as active, tissue-specific players in major physiological and pathophysiological processes. Lymphatic vessels show remarkable plasticity and heterogeneity, reflecting their functional specialization to control the tissue microenvironment. Moreover, alternative developmental origins of lymphatic endothelial cells in some organs may contribute to the diversity of their functions in adult tissues. This review aims to summarize the most recent findings of organotypic differentiation of lymphatic endothelial cells in terms of their distinct (patho)physiological functions in skin, lymph nodes, small intestine, brain, and eye. We discuss recent advances in our understanding of the heterogeneity of lymphatic vessels with respect to the organ-specific functional and molecular specialization of lymphatic endothelium, such as the hybrid blood-lymphatic identity of Schlemm's canal, functions of intestinal lymphatics in dietary fat uptake, and discovery of meningeal lymphatic vasculature and perivascular brain lymphatic endothelial cells.

Spindle cell oncocytoma of the pituitary gland with follicle-like component: organotypic differentiation to support its origin from folliculo-stellate cells

Spindle cell oncocytoma (SCO) is a rare, non-adenomatous tumor originating from the anterior pituitary gland. Composed of fusiform, mitochondrion-rich cells sharing several immunophenotypic and ultrastructural properties with folliculo-stellate cells (FSC), SCO has been proposed to represent a neoplastic counterpart of the latter. To date, however, SCO has failed to meet one criterion commonly used in histological-based taxonomy and diagnostics; that of recapitulating any of FSCs' morphologically defined developmental or physiological states. We describe a unique example of SCO wherein a conventional fascicular texture was seen coexisting with and organically merging into follicle-like arrangements. The sellar tumor of 2.7 × 2.6 × 2.5 cm was transphenoidally resected from a 55-year old female. Preoperative magnetic resonance imaging indicated an isointense, contrast enhancing mass with suprasellar extension. Histology showed multiple rudimentary to well-formed, follicle-like cavities on a classical spindle cell background; while all the participating cells exhibited an SCO immunophenotype, including positivity for S100 protein, vimentin, EMA, Bcl-2, and TTF-1, as well as staining with the antimitochondrial antibody 113-1. Conversely no expression of GFAP, follicular-epithelial cytokeratin, carcinoembryonic antigen, or anterior pituitary hormones was detected. Ultrastructurally, tumor cells facing follicular lumina displayed organelles of epithelial specialization, in particular surface microvilli and apical tight junctions. This constellation is felt to be reminiscent of FSCs' metaplastic transition to follicular epithelium, as observed during embryonic development and physiological renewal of the hormone-secreting parenchyma. Such finding is apt to being read as a supporting argument for SCO's descent from the FSC lineage.

Mesenchymale Stammzellen für das „tissue engineering“ des Knochens

Human mesenchymal stem cells (MSC) represent an attractive option for cell replacement strategies (tissue engineering, TE). TE applications require stability of a stem cell/biomaterial-hybrid via cell migration, matrix-remodelling and differentiation. We focus on these mechanisms in organotypic culture systems for bone TE using MSC from the umbilical cord (UC-MSC) and from bone marrow (BM-MSC). For the organotypic differentiation of MSC into functional osteoblasts, MSC were embedded in a collagenous matrix and subjected to osteogenic differentiation. Under these culture conditions, UC-MSC exceeded BM-MSC in the expression and synthesis of extracellular matrix (ECM) proteins, while BM-MSC show enhanced osteogenic gene upregulation. In both cell types the biosynthetic activity was accompanied by the ultrastructural appearance of hydroxyapatite/calcium crystals. Following secretion of matrix metalloproteinases, both MSC types migrated into and colonised the collagenous matrix causing matrix strengthening and contraction. In conclusion, MSC promise a broad therapeutical application for a variety of connective tissues requiring ECM synthesis and remodelling.

Tubes, Branches, and Pillars

The angiogenic cascade is getting increasingly complex. A few years ago, vasculogenesis and angiogenesis were considered as the primary mechanisms leading to the formation of new blood vessels. The original definition of vasculogenesis denotes the formation of a primary embryonic vascular network from in situ differentiating angioblastic cells.1 In contrast, angiogenesis primarily referred to the sprouting of blood vessels from preexisting vessels.1 Recent advances in the identification of molecules that regulate angiogenesis and vascular remodeling have shown that the simplistic model of an invading capillary sprout is not sufficient to appreciate the whole spectrum of morphogenic events that are required to form a neovascular network (Figure 1).1–3 Undoubtedly, vascular endothelial growth factor (VEGF) acts at an early point in the hierarchical order of morphogenic events and probably fulfills all criteria to be considered as a master switch of the angiogenic cascade. In contrast, the angiopoietins and their receptor Tie-2 as well as the ephrins and their corresponding Eph receptors appear to act at a somewhat later stage of neovessel formation. These molecules orchestrate a number of related, yet functionally and molecularly not well understood, processes such as vessel assembly (network formation and formation of anastomoses), vessel maturation (recruitment of mural cells [pericytes and smooth muscle cells], and extracellular matrix assembly, pruning of the primary vascular bed), and acquisition of vessel identity (formation of arteries, capillaries, and veins)3,4 (Figure 2). Lastly, the mechanisms of organotypic differentiation of the vascular tree (continuous endothelium, discontinuous endothelium, fenestrated endothelium) are not at all understood and the first molecules that govern subpopulation-specific vascular growth and differentiation are just being uncovered.5,6 Figure 1. Change of paradigm. From sprouting angiogenesis to vascular morphogenesis. Basement membrane degradation, directed endothelial cell migration, and proliferation (left) were considered as the primary mechanisms of angiogenesis. …

Ultrastructural and Immunohistochemical Characterization of an in Vitro Model of Human Epidermis: Its Potential for Sulfur Mustard Study

AbstractThe morphopathology of sulfur mustard dermal toxicity has been studied largely with in vivo models such as the hairless guinea pig and domestic weanling pig. Although these models have provided useful clues toward the understanding of mechanisms of sulfur mustard-induced dermal lesions, the correlation with human skin is not exact and the continuing availability of any animal model is problematic. In the interest of reducing research dependence on animal models, and to provide a laboratory model for sulfur mustard study with some level of correlation to human skin, we are evaluating an in vitro human epidermal model developed by Rhoads. The model was generated by seeding human keratinocytes onto Millipore Milli-Cm inserts precoated with type I collagen. The model, grown in culture for 7 days, presented a replicate display of epidermal structural components typically found in vivo. There was evidence of organotypic differentiation and stratification with characteristic cell types recognized to each...

Synaptic organization of an organotypic slice culture of the mammalian retina.

Vertical slices of postnatal day 6 (P6) rat retina were cut and cultured using the roller-tube technique. The organotypic differentiation during a culture period of up to 30 days has been described in a previous study (Feigenspan et al., 1993a). Here we concentrated on the synaptic organization in the retinal slice culture. Electron microscopy revealed the presence of ribbon synapses in the outer plexiform layer and conventional and ribbon synapses in the inner plexiform layer. Immunofluorescence with antibodies that recognize specific subunits of GABAA or glycine receptors revealed a punctate distribution of the receptors. They were aggregated in "hot spots" that correspond to a concentration of receptors at postsynaptic sites. Different isoforms of GABAA and glycine receptors occurred in the slice cultures. The experiments show that there is a differentiation of synapses and a diversity of transmitter receptors in the slice cultures that is comparable to the in vivo retina.

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture.

Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.

A new approach to the molecular basis of neoplastic transformation in the brain.

Gene transfer into living organisms has evolved as a powerful approach to study in vivo effects of specific genes and to devise animal models of hereditary disorders. We have been particularly interested in an approach to introducing transforming genes into the nervous system. Since specific promoter sequences for targeting the expression of a transgene to many cell types of the brain are not yet isolated, a suitable transgenic mouse model was not available for these experiments. This has prompted us to develop an alternative strategy for gene transfer into the brain. The rationale is to introduce foreign genes into fetal brain transplants using embryonic CNS as donor tissue and replication-defective retroviral vectors as genetic vehicles. This technique relies on the extraordinary organotypic differentiation capacity of neural grafts and the expression of retrovirally transmitted genes in different cell types of CNS transplants. In contrast to transgenic animals but analogous to sporadic tumour formation, target cells for the retroviral vector will develop in an environment of unmodified neural tissue. We have introduced a number of neurotropic oncogenes into fetal brain transplants to study potential effects of such genes on the brain. This review will summarize some of the findings which have emerged from this experimental study including the tropism of several genes for endothelial cells, attempts to identify cooperating combinations of transforming genes and an experimental model for primitive neuroectodermal tumours in neural grafts.

Intact organ culture of murine metanephros

A murine metanephric whole-organ culture system is described which maintains normal structural relationships of epithelial and mesenchymal elements and undergoes advanced organotypic differentiation in vitro. Manipulation of the culture conditions permits study of the developmental, immunologic, and biochemical aspects of normal and abnormal renal organogenesis.

Retrovirus‐Mediated Oncogene Transfer into Neural Transplants

A gene transfer model was developed which allows for the identification of transformation pathways in the developing nervous system. Transforming genes were introduced into fetal brain transplants using embryonic CNS as donor tissue and replication-defective retroviral vectors as genetic vehicles. This technique relies on the extraordinary organotypic differentiation capacity of neural grafts and the expression of retrovirally transmitted genes in various cell types of CNS transplants. In contrast to transgenic animals but analogous to sporadic tumor formation, target cells for the retroviral vector develop in an environment of unmodified neural tissue. We have introduced a number of neurotropic oncogenes into fetal brain transplants including genes with an associated tyrosine kinase activity (polyoma medium T, v-src), a novel member of the fibroblast growth factor (fgf) gene family and the SV40 large T antigen. These experiments have demonstrated a significant transformation potential of oncogenes in specific target cells of the brain, provided evidence for a dominant complementary transforming effect of simultaneously expressed ras and myc genes in neural precursor cells and have yielded intriguing model systems for human CNS neoplasms such as the cerebellar medulloblastoma. This review describes the transplantation model, demonstrates several striking phenotypes induced by oncogene expression in neural grafts and elaborates on future prospects of this experimental approach.

Serological H-Y antigen in the female chicken occurs during gonadal differentiation

Abstract In the chicken, serological H-Y antigen is specific for the female sex. Male gonad differentiation can be experimentally influenced by estrogens, resulting in the transient formation of an ovotestis. The sex-inverted gonad becomes positive for H-Y antigen. Therefore, the question arises whether, in normal gonadogenesis also, the female gonad at the indifferent stage, before estrogens are produced, is negative for H-Y antigen. Here we show that this is indeed the case. The female gonad becomes positive for H-Y antigen when the ovary starts its organotypic differentiation at about day 6 1/ 2 of embryonal development. It is assumed that estrogens are responsible for the occurrence of H-Y antigen. This finding supports the view that H-Y antigen plays a role in primary ovogenesis in the chicken.

Influence of Recombinant Deoxyribonucleic Acid-Derived Bovine Growth Hormone on α-Lactalbumin Production by Bovine Mammary Tissue Maintained in Athymic Nude Mice

Mammary tissue from five cows was cut into pieces approximately 3mm2×.2mm and placed subcutaneously in athymic mice (10 to 12 pieces/mouse). After 30 d, all mice were injected for 10 d with 17β-estradiol (1μg), progesterone (1mg), ovine prolactin (1mg), and growth hormone (recombinant DNA-derived bovine) (1mg). Four grafts were then removed from each mouse, mice were ovariectomized, and subsequently mice were injected daily for 7 d with hydrocortisone (.2mg, all mice), growth hormone (0, .25, .5, or 1.0mg), and 0 or .5mg ovine prolactin (three mice/treatment per udder). α-Lactalbumin content of grafts was 36±9μg/mg DNA after treatment with estradiol, progesterone, growth hormone, and prolactin but before hydrocortisone, prolactin, and growth hormone treatment. Hydrocortisone treatment increased α-lactalbumin to 147μg/mg DNA. Growth hormone plus hydrocortisone treatment increased α-lactalbumin to 391, 451, and 480μg/mg DNA for .25, .5, and 1.0 mg/d of growth hormone, respectively. Prolactin plus hydrocortisone treatment increased α-lactalbumin to 466μg/mg DNA. With exogenous prolactin, growth hormone increased α-lactalbumin content of grafts to 581, 647, and 689μg/mg DNA for .25, .5, and 1.0 mg/d of growth hormone, respectively. Histological examination of tissues indicated that the effect of growth hormone was not mediated through increased epithelial area. Data suggest that bovine mammary tissue is capable of organotypic differentiation in athymic mice.

Formation of organoid structures and extracellular matrix production in an intestinal epithelial cell line during long-term in vitro culture

During the long-term in vitro maintenance of an epithelial cell line established from rat duodenum (IEC-17) we have observed progressive morphological changes which, after approximately 4–5 months in culture, led to a loss of substrate adherence and to the formation of organoid structures characterized by organized layers of cells separated by continuous extracellular-like material and delimiting close lumina. The cells exhibited a defined polarity with deposition of extracellular matrix components on one side and development of microvilli on the opposite surface. The morphological changes observed did not appear to be the expression of spontaneous transformation since the cells retained a normal diploid rat karyotype and did not grow in soft agar. In this report we present the optical and electron microscopical characterization of the progressive organotypic differentiation of the cell line. Further studies are currently in progress to characterize the extracellular matrix during the process of differentiation.

Organotypic differentiation of trypsin-dissociated fetal rat intestine

These studies examined the potential for reorganization and differentiation of dissociated 18-day fetal rat intestine. Cultures of trypsin-dissociated fetal intestine were maintained in vitro for 1 week on a three-dimensional matrix, then transplanted into syngeneic hosts. When harvested after 4 weeks, these transplants consistently demonstrated organotypic differentiation. Spherical structures containing crypts with frequent mitotic figures and villi lined with columnar epithelium had formed. PAS staining demonstrated positive epithelial cell brush borders, goblet cells, and luminal contents. Significant levels of the microvillus membrane enzymes lactase, sucrase, maltase, and alkaline phosphatase were present in the luminal contents. Sucrase-isomaltase, an enzyme characteristic of postweaning small intestine, was demonstrated by immunoprecipitation and SDS-PAGE. Thus, both morphological and biochemical maturation occurred in the transplants.