scholarly journals Expansion of Submucosal Bladder Wall TissueIn VitroandIn Vivo

2016 ◽  
Vol 2016 ◽  
pp. 1-9
Author(s):  
Gisela Reinfeldt Engberg ◽  
Clara Ibel Chamorro ◽  
Agneta Nordenskjöld ◽  
Magdalena Fossum
Keyword(s):  
Smooth Muscle ◽  
Bladder Wall ◽  
Subcutaneous Fat ◽  
Culture Conditions ◽  
Detrusor Muscle ◽  
Step Procedure ◽  
Standard Culture ◽  
One Step

In order to develop autologous tissue engineering of the whole wall in the urinary excretory system, we studied the regenerative capacity of the muscular bladder wall. Smooth muscle cell expansion on minced detrusor musclein vitroandin vivowith or without urothelial tissue was studied. Porcine minced detrusor muscle and urothelium were culturedin vitrounder standard culture conditions for evaluation of the explant technique and in collagen for tissue sectioning and histology. Autografts of minced detrusor muscle with or without minced urothelium were expanded on 3D cylinder moulds by grafting into the subcutaneous fat of the pig abdominal wall. Moulds without autografts were used as controls. Tissue harvesting, mincing, and transplantation were performed as a one-step procedure. Cells from minced detrusor muscle specimens migrated and expandedin vitroon culture plastic and in collagen.In vivostudies with minced detrusor autografts demonstrated expansion and regeneration in all specimens. Minced urothelium autografts showed multilayered transitional urothelium when transplanted alone but not in cotransplantation with detrusor muscle; thus, minced bladder mucosa was not favored by cografting with minced detrusor. No regeneration of smooth muscle or epithelium was seen in controls.

Glomerular laminin isoform transitions: errors in metanephric culture are corrected by grafting

2001 ◽  
Vol 280 (4) ◽  
pp. F695-F705 ◽  
Author(s):  
Patricia L. St. John ◽  
Ruixue Wang ◽  
Yong Yin ◽  
Jeffrey H. Miner ◽  
Barry Robert ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Organ Culture ◽  
Growth Factor Receptor ◽  
Culture Conditions ◽  
Type Iv ◽  
Standard Culture ◽  
Metanephric Culture ◽  
Endothelial Growth Factor

Glomerular basement membrane (GBM) assembly and maturation are marked by the replacement of laminin-1 (containing α1-, β1-, and γ1-chains) with laminin-11 (consisting of α5-, β2-, and γ1-chains). Similarly, the α1- and α2-chains of type IV collagen are replaced by collagen α3-, α4-, and α5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed metanephric organ cultures. Standard culture conditions do not support endothelial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cultures, including the formation of foot processes and assembly of a GBM-like matrix. Here, we show that the podocyte-specific markers, glomerular epithelial protein 1 and nephrin, which are normally expressed in capillary loop stage glomeruli in vivo, are also expressed by glomerular figures that form in organ culture. However, the GBM-like segments that form in vitro do not undergo normal laminin isoform switching. Instead, both laminin α1- and α5-chains are present, as is the β1-chain, but not β2. When avascular organ-cultured kidneys are grafted into anterior eye chambers, however, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 ( E12) vascular endothelial growth factor receptor (Flk1) -lacZ kidneys that had first been grown in organ culture for 6–7 days and then grafted into wild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings indicate that endothelial cells, and/or factors present in the circulation, mediate normal GBM laminin isoform transitions in vivo.

scholarly journals Exofucosylation of Adipose Mesenchymal Stromal Cells Alters Their Secretome Profile

2020 ◽  
Vol 8 ◽  
Author(s):  
David García-Bernal ◽  
Mariano García-Arranz ◽  
Ana I. García-Guillén ◽  
Ana M. García-Hernández ◽  
Miguel Blanquer ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Culture Conditions ◽  
Peripheral Tissues ◽  
Standard Culture ◽  
Anti Inflammatory ◽  
Selectin Binding ◽  
The Impact

Mesenchymal stromal cells (MSCs) constitute the cell type more frequently used in many regenerative medicine approaches due to their exclusive immunomodulatory properties, and they have been reported to mediate profound immunomodulatory effects in vivo. Nevertheless, MSCs do not express essential adhesion molecules actively involved in cell migration, a phenotypic feature that hampers their ability to home inflamed tissues following intravenous administration. In this study, we investigated whether modification by fucosylation of murine AdMSCs (mAdMSCs) creates Hematopoietic Cell E-/L-selectin Ligand, the E-selectin-binding CD44 glycoform. This cell surface glycan modification of CD44 has previously shown in preclinical studies to favor trafficking of mAdMSCs to inflamed or injured peripheral tissues. We analyzed the impact that exofucosylation could have in other innate phenotypic and functional properties of MSCs. Compared to unmodified counterparts, fucosylated mAdMSCs demonstrated higher in vitro migration, an altered secretome pattern, including increased expression and secretion of anti-inflammatory molecules, and a higher capacity to inhibit mitogen-stimulated splenocyte proliferation under standard culture conditions. Together, these findings indicate that exofucosylation could represent a suitable cell engineering strategy, not only to facilitate the in vivo MSC colonization of damaged tissues after systemic administration, but also to convert MSCs in a more potent immunomodulatory/anti-inflammatory cell therapy-based product for the treatment of a variety of autoimmune, inflammatory, and degenerative diseases.

scholarly journals Transplantation of Autologous Minced Bladder Mucosa for a One-Step Reconstruction of a Tissue Engineered Bladder Conduit

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Gisela Reinfeldt Engberg ◽  
Johan Lundberg ◽  
Clara Ibel Chamorro ◽  
Agneta Nordenskjöld ◽  
Magdalena Fossum
Keyword(s):  
Abdominal Wall ◽  
Surgical Intervention ◽  
Bladder Wall ◽  
Bladder Mucosa ◽  
Small Particles ◽  
Microscopic Appearance ◽  
Step Procedure ◽  
One Step ◽  
Gross Examination

Surgical intervention is sometimes needed to create a conduit from the abdominal wall to the bladder for self-catheterization. We developed a method for tissue engineering a conduit for bladder emptying withoutin vitrocell culturing as a one-step procedure. In a porcine animal model bladder, wall tissue was excised and the mucosa was minced to small particles. The particles were attached to a tube in a 1 : 3 expansion rate with fibrin glue and transplanted back by attaching the tube to the bladder and through the abdominal wall. Sham served as controls. After 4-5 weeks, conduits were assessed in respect to macroscopic and microscopic appearance in 6 pigs. Two pigs underwent radiology before termination. Gross examination revealed a patent conduit with an opening to the bladder. Histology and immunostaining showed a multilayered transitional uroepithelium in all cases. Up to 89% of the luminal surface area was neoepithelialized but with a loose attachment to the submucosa. No epithelium was found in control animals. CT imaging revealed a patent channel that could be used for filling and emptying the bladder. Animals that experienced surgical complications did not form conduits. Minced autologous bladder mucosa can be transplanted around a tubular mold to create a conduit to the urinary bladder withoutin vitroculturing.

Neo-Vasculogenesis In Vivo Is Facilitated by Oxygen Sensing Mesenchymal Stem and Pogenitor Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 699-699
Author(s):  
Nicole A Hofmann ◽  
Anna Ortner ◽  
Rodrigo O Jacamo ◽  
Andreas Reinisch ◽  
Katharina Schallmoser ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Progenitor Cells ◽  
Wound Repair ◽  
Culture Conditions ◽  
Angiogenic Therapy ◽  
Ischemic Tissue ◽  
Oxygen Conditions ◽  
Standard Culture

Abstract Abstract 699 Rationale: Vascular repair after hypoxic tissue damage requires a stringent interaction between somatic endothelial colony-forming progenitor cells (ECFCs) and mesenchymal stem and progenitor cells (MSPCs). Stem cell therapy to re-vascularize ischemic tissue has been a promising tool for various therapeutic targets including stroke, myocardial infarction and peripheral artery disease. Despite promising experimental data, therapeutic approaches employing endothelial progenitor cells have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. Hypoxia in ischemic tissue is an extensively studied key factor that influences pro- and anti-angiogenic treatment by driving the revascularization machinery. We and others have shown that despite hypoxic stimulation, ECFCs in vivo only form patent vessels in the presence of MSPCs. Here we show that MSPCs but not ECFCs are the oxygen sensors enabling vasculogenesis in vivo. Methods: Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. In in vitro studies progenitor cell phenotype, long-term proliferation, molecular cellular response, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen conditions (5% O2) and standard culture conditions (20% O2). ECFC and MSPC interaction in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelete lysate). To investigate the respective roles of MSPCs and ECFCs during vasculogenesis under hypoxia in vivo chemical and genetic inhibitors against protein synthesis (cycloheximide) and HIF-1α (YC-1, shRNA) were employed. Immune histochemistry, immune fluorescence and TUNEL assays were performed on plugs in the time course after transplantation. Results: In vitro studies showed that compared to 20% O2, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced at 5% O2, and even more at 1% O2. Standard culture conditions resulted in a shift in the progenitor hierarchy with an augmented number of high proliferative potential (HPP)-ECFC colonies (60±18% of total colonies) as compared to venous oxygen conditions (9±6%) and a complete loss of HPP-ECFC colonies under severe hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair function in scratch assays and the ability to form vascular-like networks in matrigel assays in vitro were diminished with declining oxygen supply. The re-oxygenation to 20% O2 of ECFCs which where precultured at 1% or 5% O2 led to enhanced proliferation, colony size and function. Single cell analysis revealed that ECFCs stabilized hypoxia-inducing factor-1α (HIF-1α) only at 1% O2 while MSPCs stabilize HIF-1α at 1% O2 as well as 5% O2 conditions. In a mouse model, subcutaneously injected ECFCs underwent apoptosis after 24h and attracted mouse leucocytes. In contrast, ECFCs co-implanted in vivo with MSPCs were rescued from apoptotic death and formed perfused human vessels 7 days after transplantation independent of matrix. Perivascular cells, but not ECFCs, were positive for HIF-1α in vivo. Inhibition of MSPCs but not ECFCs protein synthesis and HIF-1α prior to co-implantation blocked vessel formation. Conclusion: These data demonstrates that hypoxic ECFCs alone show reduced functuionality in vitro and form patent vessels in vivo. In contrast, MSPCs react to the low oxygen environment more sensitively than ECFCs and promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis. Surprisingly, this study shows that therapeutic vasculogenesis can occur independent of endothelial HIF stabilization and protein synthesis. This data indicate that in addition to their established role regulating hematopoiesis, MSPCs oxygen sensing is crucial during vascular regeneration. This suggests a shift of focus from endothelial cells to perivascular cells as a therapeutic target in regenerative medicine and anti-angiogenic therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modelling fibroid pathology: development and manipulation of a myometrial smooth muscle cell macromolecular crowding model to alter extracellular matrix deposition

2020 ◽  
Vol 26 (7) ◽  
pp. 498-509 ◽  
Author(s):  
Ann Winter ◽  
Lois A Salamonsen ◽  
Jemma Evans
Keyword(s):  
Ascorbic Acid ◽  
Smooth Muscle ◽  
In Vitro Model ◽  
Uterine Fibroids ◽  
Macromolecular Crowding ◽  
Culture Conditions ◽  
Myometrial Smooth Muscle Cell ◽  
Vitro Model

ABSTRACT Current treatment options for uterine fibroids are limited to hormonal manipulation or surgical intervention. We aimed to develop an in vitro model to mirror collagen deposition and extracellular matrix (ECM) formation, the principal features of uterine fibroids, to enable testing of novel therapeutics. Macromolecular crowding with Ficoll 400 and Ficoll 70 in cultures of human uterine myometrial smooth muscle cells containing ascorbic acid, provided the basis for this model. These culture conditions mimic the ‘crowded’ nature of the in vivo extracellular environment by incorporating neutral, space-filling macromolecules into conventional cell cultures. This method of culture facilitates appropriate ECM deposition, thus closely representing the in vivo fibrotic phenotype of uterine fibroids. Macromolecular crowding in Ficoll cultures containing ascorbic acid reduced myometrial smooth muscle cell proliferation and promoted collagen production. Under these conditions, collagen was processed for extracellular deposition as demonstrated by C-propeptide cleavage from secreted procollagen. The fibrosis marker activin was increased relative to its natural inhibitor, follistatin, in crowded culture conditions while addition of exogenous follistatin reduced collagen (Col1A1) gene expression. This in vitro model represents a promising development for the testing of therapeutic interventions for uterine fibroids. However, it does not recapitulate the full in vivo pathology which can include specific genetic and epigenetic alterations that have not been identified in the myometrial smooth muscle (hTERT-HM) cell line. Following screening of potential therapeutics using the model, the most promising compounds will require further assessment in the context of individual subjects including those with genetic changes implicated in fibroid pathogenesis.

scholarly journals Adipose Tissue: Understanding the Heterogeneity of Stem Cells for Regenerative Medicine

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 918
Author(s):  
Wee Kiat Ong ◽  
Smarajit Chakraborty ◽  
Shigeki Sugii
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Regenerative Medicine ◽  
Species Difference ◽  
Subcutaneous Fat ◽  
Cell Types ◽  
Culture Conditions ◽  
Cellular Heterogeneity

Adipose-derived stem cells (ASCs) have been increasingly used as a versatile source of mesenchymal stem cells (MSCs) for diverse clinical investigations. However, their applications often become complicated due to heterogeneity arising from various factors. Cellular heterogeneity can occur due to: (i) nomenclature and criteria for definition; (ii) adipose tissue depots (e.g., subcutaneous fat, visceral fat) from which ASCs are isolated; (iii) donor and inter-subject variation (age, body mass index, gender, and disease state); (iv) species difference; and (v) study design (in vivo versus in vitro) and tools used (e.g., antibody isolation and culture conditions). There are also actual differences in resident cell types that exhibit ASC/MSC characteristics. Multilineage-differentiating stress-enduring (Muse) cells and dedifferentiated fat (DFAT) cells have been reported as an alternative or derivative source of ASCs for application in regenerative medicine. In this review, we discuss these factors that contribute to the heterogeneity of human ASCs in detail, and what should be taken into consideration for overcoming challenges associated with such heterogeneity in the clinical use of ASCs. Attempts to understand, define, and standardize cellular heterogeneity are important in supporting therapeutic strategies and regulatory considerations for the use of ASCs.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Targeting Matrix Metalloproteinases in Atherosclerosis and Cardiovascular Dysfunction

2019 ◽  
Vol 70 (2) ◽  
pp. 718-720
Author(s):  
Lucia Corina Dima-Cozma ◽  
Sebastian Cozma ◽  
Delia Hinganu ◽  
Cristina Mihaela Ghiciuc ◽  
Florin Mitu
Keyword(s):  
Smooth Muscle ◽  
Matrix Metalloproteinases ◽  
Cholesterol Synthesis ◽  
Balloon Dilation ◽  
Aortic Aneurysms ◽  
Arterial Lesions ◽  
Smooth Muscle Cell Migration ◽  
And Migration

Matrix metalloproteinases (MMPs) are the primary mediators of extracellular remodeling and their properties are useful in diagnostic evaluation and treatment. They are zinc-dependent proteases. MMPs have been involved in the mechanisms of atherosclerosis in various arterial areas, ischemic heart disease and myocardial infarction, atrial fibrillation and aortic aneurysms. Recently, MMP9 has been implicated in dyslipidemia and cholesterol synthesis by the liver. Increased MMP expression and activity has been associated with neointimal arterial lesions and migration of smooth muscle cells after arterial balloon dilation, while MMP inhibition decreases smooth muscle cell migration in vivo and in vitro.

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