scholarly journals High-density hiPSCs expansion supported by growth factors accumulation in a simple dialysis-culture platform

2020 ◽  
Author(s):  
Fuad Torizal ◽  
Qiao You Lau ◽  
Masato Ibuki ◽  
Yoshikazu Kawai ◽  
Masato Horikawa ◽  
...  
Keyword(s):  
Shear Stress ◽  
Growth Factors ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
High Density ◽  
Production Costs ◽  
High Density Culture ◽  
Differentiation Potential ◽  
Dialysis Culture ◽  
Factor Accumulation

Abstract Three-dimensional aggregate-suspension culture can produce large numbers of human induced pluripotent stem cells (hiPSCs); however, use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device allowed growth-factor accumulation and improved cell expansion up to ~ 32 × 106 cells/mL, and reduction of excessive shear stress and agglomeration following addition of the functional polymer FP003 supported high-density expansion. The results revealed accumulation of several growth factors, including fibroblast growth factor 2 and insulin, along with endogenous NODAL, which acts as a substitute for depleted transforming growth factor-β1 in maintaining pluripotency. Because we used the same growth-factor formulation per volume in the upper culture compartment, cost reduction increased significantly in proportional manner with cell density. We showed that growth-factor-accumulation dynamics in a low-shear-stress environment successfully improved hiPSC proliferation, pluripotency, and differentiation potential. This miniaturised dialysis-culture system demonstrated the feasibility and cost-effective mass production of hiPSCs in high-density culture.

scholarly journals A miniature dialysis-culture device allows high-density human-induced pluripotent stem cells expansion from growth factor accumulation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Fuad Gandhi Torizal ◽  
Qiao You Lau ◽  
Masato Ibuki ◽  
Yoshikazu Kawai ◽  
Masato Horikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Shear Stress ◽  
Growth Factors ◽  
Growth Factor ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
High Density ◽  
Induced Pluripotent ◽  
Dialysis Culture ◽  
Factor Accumulation

AbstractThree-dimensional aggregate-suspension culture is a potential biomanufacturing method to produce a large number of human induced pluripotent stem cells (hiPSCs); however, the use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency, respectively. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device improved cell expansion up to approximately ~3.2 to 4×107 cells/mL. The high-density expansion was supported by reduction of excessive shear stress and agglomeration mediated by the addition of the functional polymer FP003. The results revealed accumulation of several growth factors, including fibroblast growth factor 2 and insulin, along with endogenous Nodal, which acts as a substitute for depleted transforming growth factor-β1 in maintaining pluripotency. Because we used the same growth-factor formulation per volume in the upper culture compartment, the cost reduced in inverse proportional manner with the cell density. We showed that growth-factor-accumulation dynamics in a low-shear-stress environment successfully improved hiPSC proliferation, pluripotency, and differentiation potential. This miniaturised dialysis-culture system demonstrated the feasibility of cost-effective mass production of hiPSCs in high-density culture.

Interrupted Treatment With Growth Factors in Combination With Hydrodynamic Forces Enhances ECM Deposition in Tissue-Engineered Cartilage

2011 ◽  
Author(s):  
Yueh-Hsun Yang ◽  
Gilda A. Barabino
Keyword(s):  
Shear Stress ◽  
Growth Factors ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Hydrodynamic Forces ◽  
Tissue Growth ◽  
In Vitro Cultivation ◽  
Mechanical Stimuli ◽  
Engineered Cartilage

Achievement of viable engineered tissues through in-vitro cultivation in bioreactor systems requires a thorough understanding of the complex interplay between mechanical forces and biochemical cues. Briefly, bioreactors have been employed to impart mechanical stimuli to support tissue growth and development. Continuous fluid-induced shear stress, for example, has been shown to influence morphology and properties of engineered cartilage.1 Fluid flow enhances mass transfer mechanisms and simultaneously provides mechanical stimuli across or through the construct to emulate shear forces that occur in the knee or other joints. Critical biochemical factors, such as growth factors, are secreted by cells2,3 and involved in cell-to-cell signaling. Guided by these molecules, cells can communicate with each other and work synergistically to accomplish a specific task. It has also been demonstrated that the pathways of certain growth factors, such as transforming growth factor-β (TGF-β) family and insulin-like growth factor-1 (IGF-1), are responsive to shear stress, resulting in enhanced cell and tissue activities, and their expression is also up-regulated by fluid-induced shear stress.4,5 This evidence suggests their involvement in mechanotransduction mechanisms. However, a combination of mechanical and biochemical stimuli results in a complex culture environment which is not yet fully characterized. The present study was designed to obtain an understanding of the combined effects of hydrodynamic forces and growth factors on cartilage regeneration by employing a custom-designed wavy-walled bioreactor1 (WWB) and by selecting IGF-1 and TGF-β1 as two model molecules. We hypothesized that bioprocessing conditions which optimize mechanical, biochemical and compositional properties of tissue-engineered cartilage can be achieved under hydrodynamic stimuli in combination with an appropriate use of IGF-1 or TGF-β.

scholarly journals Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 679
Author(s):  
Benedict-Uy Fabia ◽  
Joshua Bingwa ◽  
Jiyeon Park ◽  
Nguyen-Mihn Hieu ◽  
Jung-Hoon Ahn
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Pseudomonas Fluorescens ◽  
Abc Transporter ◽  
Transforming Growth Factor Beta ◽  
Deletion Mutant ◽  
Transforming Growth Factor ◽  
Gene Knockout ◽  
Type I ◽  
Double Deletion

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

Mechanisms of renal tubular cell hypertrophy: mitogen-induced suppression of proteolysis

1997 ◽  
Vol 273 (3) ◽  
pp. C843-C851 ◽  
Author(s):  
H. A. Franch ◽  
P. V. Curtis ◽  
W. E. Mitch
Keyword(s):  
Protein Synthesis ◽  
Growth Factors ◽  
Growth Factor ◽  
Protein Degradation ◽  
Transforming Growth Factor ◽  
Primary Cultures ◽  
Renal Tubular Cell ◽  
Kidney Cells ◽  
Tgf Beta ◽  
Lysosomal Proteolysis

The combination of epidermal growth factor (EGF) plus transforming growth factor-beta 1 (TGF-beta 1) causes hypertrophy in renal epithelial cells. One mechanism contributing to hypertrophy is that EGF induces activation of the cell cycle and increases protein synthesis, whereas TGF-beta 1 prevents cell division, thereby converting hyperplasia to hypertrophy. To assess whether suppression of proteolysis is another mechanism causing hypertrophy induced by these growth factors, we measured protein degradation in primary cultures of proximal tubule cells and in cultured NRK-52E kidney cells. A concentration of 10(-8) M EGF alone or EGF plus 10(-10) M TGF-beta 1 decreased proteolysis by approximately 30%. TGF-beta 1 alone did not change protein degradation. Using inhibitors, we examined which proteolytic pathway is suppressed. Neither proteasome nor calpain inhibitors prevented the antiproteolytic response to EGF + TGF-beta 1. Inhibitors of lysosomal proteases eliminated the antiproteolytic response to EGF + TGF-beta 1, suggesting that these growth factors act to suppress lysosomal proteolysis. This antiproteolytic response was not caused by impaired EGF receptor signaling, since lysosomal inhibitors did not block EGF-induced protein synthesis. We conclude that suppression of lysosomal proteolysis contributes to growth factor-mediated hypertrophy of cultured kidney cells.

Regulation of mesenchymal extracellular matrix protein synthesis by transforming growth factor-beta and glucocorticoids in tumor stroma

1995 ◽  
Vol 108 (6) ◽  
pp. 2153-2162 ◽  
Author(s):  
J.F. Talts ◽  
A. Weller ◽  
R. Timpl ◽  
M. Ekblom ◽  
P. Ekblom
Keyword(s):  
Extracellular Matrix ◽  
Growth Factors ◽  
Growth Factor ◽  
Mrna Expression ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Tenascin C ◽  
Extracellular Matrix Components ◽  
Growth Factor Beta ◽  
Matrix Components

We have here studied the composition and regulation of stromal extracellular matrix components in an experimental tumor model. Nude mice were inoculated with WCCS-1 cells, a human Wilms' tumor cell line. In the formed tumors the stroma was found to contain mesenchymal extracellular matrix proteins such as tenascin-C, fibulins-1 and 2 and fibronectin, but no nidogen. Nidogen was confined to basement membranes of tumor blood vessels. Since glucocorticoids have been shown to downregulate tenascin-C expression in vitro, we tested whether dexamethasone can influence biosynthesis of extracellular matrix components during tumor formation in vivo. A downregulation of tenascin-C mRNA and an upregulation of fibronectin mRNA expression by dexamethasone was noted. Transforming growth factor-beta 1 mRNA levels were unaffected by the dexamethasone treatment. Glucocorticoids can thus downregulate tenascin-C synthesis although local stimulatory growth factors are present. The competition between a negative and a positive extrinsic factor on synthesis of stromal extracellular matrix components was studied in a fibroblast/preadipocyte cell line. Transforming growth factor-beta 1 stimulated tenascin-C synthesis but did not affect fibronectin or fibulin-2 synthesis. Dexamethasone at high concentrations could completely suppress the effect of transforming growth factor-beta 1 on tenascin-C mRNA expression. Transforming growth factor-beta 1 could in turn overcome the downregulation of tenascin-C mRNA expression caused by a lower concentration of dexamethasone. We therefore suggest that the limited expression of tenascin-C in part is due to a continuous suppression by physiological levels of glucocorticoids, which can be overcome by local stimulatory growth factors when present in sufficient amounts.

Metalloproteinase and growth factor interactions: do they play a role in pulmonary fibrosis?

2002 ◽  
Vol 283 (1) ◽  
pp. L1-L11 ◽  
Author(s):  
Margaret K. Winkler ◽  
John L. Fowlkes
Keyword(s):  
Acute Lung Injury ◽  
Growth Factors ◽  
Growth Factor ◽  
Pulmonary Fibrosis ◽  
Lung Injury ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Inflammatory Cells ◽  
Transforming Growth Factor Β ◽  
Target Cells

Chronic lung disease due to interstitial fibrosis can be a consequence of acute lung injury and inflammation. The inflammatory response is mediated through the migration of inflammatory cells, actions of proinflammatory cytokines, and the secretion of matrix-degrading proteinases. After the initial inflammatory insult, successful healing of the lung may occur, or alternatively, dysregulated tissue repair can result in scarring and fibrosis. On the basis of recent insights into the mechanisms underlying acute lung injury and its long-term consequences, data suggest that proteinases, such as the matrix metalloproteinases (MMPs), may not only be involved in the breakdown and remodeling that occurs during the injury but may also cause the release of growth factors and cytokines known to influence growth and differentiation of target cells within the lung. Through the release of and activation of fibrosis-promoting cytokines and growth factors such as transforming growth factor-β1, tumor necrosis factor-α, and insulin-like growth factors by MMPs, we propose that these metalloproteinases may be integral to the initiation and progression of pulmonary fibrosis.

Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2708-2715 ◽  
Author(s):  
Taku Kouro ◽  
Kay L. Medina ◽  
Kenji Oritani ◽  
Paul W. Kincade
Keyword(s):  
B Cells ◽  
Growth Factor ◽  
B Lymphocyte ◽  
Transforming Growth Factor ◽  
Early Stage ◽  
Transforming Growth Factor Β ◽  
Ki 67 ◽  
Differentiation Potential ◽  
Murine Bone Marrow ◽  
B Lineage

Abstract Recently, a collection of surface markers was exploited to isolate viable Lin− TdT+ cells from murine bone marrow. These early pro-B cells were enriched for B-lineage lymphocyte precursor activity measured by short-term culture and had little responsiveness to myeloid growth factors. Early precursors can be propagated with remarkably high cloning frequencies in stromal cell–free, serum-free cultures, permitting this analysis of direct regulatory factors. Expression of the interleukin-7 receptor (IL-7Rα) chain marks functional precursors and IL-7 is necessary for progression beyond the CD45RA+ CD19− stage. Efficient survival and differentiation were only observed when stem cell factor and Flt-3 ligand were also present. IL-7–responsive CD19+precursors are estrogen resistant. However, B-lineage differentiation was selectively abrogated when highly purified Lin− precursors were treated with hormone in the absence of stromal cells. In addition, early stages of B lymphopoiesis were arrested by limitin, a new interferon (IFN)–like cytokine as well as IFN-α, IFN-γ, or transforming growth factor β (TGF-β), but not by epidermal growth factor (EGF). Lin− TdT+early pro-B cells are shown here to be CD27+AA4.1+/−Ki-67+ Ly-6C−Ly-6A/Sca-1Lo/−Thy-1−CD43+CD4+/−CD16/32Lo/−CD44Hi and similar in some respects to the “common lymphoid progenitors” (CLP) identified by others. Although early pro-B cells have lost myeloid differentiation potential, transplantation experiments described here reveal that at least some can generate T lymphocytes. Of particular importance is the demonstration that a pivotal early stage of lymphopoiesis is directly sensitive to negative regulation by hormones and cytokines.

Induction of Growth Factor Expression is Reduced during Healing of Tympanic Membrane Perforations in Glucocorticoid-Treated Rats

2002 ◽  
Vol 111 (10) ◽  
pp. 947-953 ◽  
Author(s):  
Shin-Ichi Ishimoto ◽  
Toshio Ishibashi
Keyword(s):  
Wound Healing ◽  
Growth Factors ◽  
Growth Factor ◽  
Mrna Expression ◽  
Messenger Rna ◽  
Transforming Growth Factor ◽  
Keratinocyte Growth Factor ◽  
Treated Group ◽  
Transforming Growth Factor Α ◽  
Tympanic Membrane Perforations

The participation of growth factors in wound healing of tympanic membranes (TMs) is established. To determine the possible role of these growth factors in normal healing, we examined the regulation of keratinocyte growth factor (KGF), transforming growth factor–α (TGF-α), and basic fibroblast growth factor (bFGF) messenger RNA (mRNA) expression in wounded TMs of glucocorticoid-treated rats; these rats have severe wound healing abnormalities. Induction of KGF, TGF-α, and bFGF mRNA expression after TM injury was significantly reduced in these rats. Moreover, we found that the average number of bromodeoxyundine-positive cells in a glucocorticoid-treated group was significantly lower than that in controls. The data suggest that reduced expression of these genes might be partially responsible for the wound healing defects seen in these animals. These results provide a possible explanation for the beneficial effect of exogenous KGF, TGF-α, or bFGF in treatment of wound healing disorders of the TM.

Sign in / Sign up

Export Citation Format

Share Document