Effects of glucose, lactate and basic FGF as limiting factors on the expansion of human induced pluripotent stem cells.

Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses showed that reducing medium change frequency increased differentiation marker expressions but high glucose concentration downregulated these expressions. In contrast, heparan sulfate did not prevent differentiation marker expressions. According to analyses of growth rate, cell growth with extended medium change interval was decreased in later stage of log growth phase despite the existence of high glucose concentration and heparan sulfate. This result and culturing iPSCs with lactate showed that the accumulation of excreted lactate decreased the growth rate regardless of pH control. Conclusively, these experiments show that adding glucose and removing lactate are important to expand iPSCs with reduced culture medium usage. This knowledge should be useful to design economical iPSC mass production and differentiation system.

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

MAPs/bFGF-PLGA microsphere composite-coated titanium surfaces promote increased adhesion and proliferation of fibroblasts.

Infection and epithelial downgrowth are two major problems with maxillofacial transcutaneous implants, and both are mainly due to lack of stable closure of soft tissues at transcutaneous sites. Fibroblasts have been shown to play a key role in the formation of biological seals. In this work, titanium (Ti) model surfaces were coated with mussel adhesive proteins (MAPs) utilizing its unique adhesion ability on diverse inorganic and organic surfaces in wet environments. Prepared basic fibroblast growth factor (bFGF)-poly(lactic-co-glycolic acid) (PLGA) microspheres can be easily synthesized and combined onto MAPs-coated Ti surfaces, due to the negative surface charges of microspheres in aqueous solution, which is in contrast to the positive charges of MAPs. Titanium model surfaces were divided into three groups. Group A: MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces. Group B: MAPs-coated Ti surfaces. Group C: uncoated Ti surfaces. The effects of coated Ti surfaces on adhesion of fibroblasts, cytoskeletal organization, proliferation, and extracellular matrix (ECM)-related gene expressions were examined. The results revealed increased adhesion (P < 0.05), enhanced actin cytoskeletal organization, and up-regulated ECM-related gene expressions in groups A and B compared with group C. Increased proliferation of fibroblasts during five days of incubation was observed in group A compared with groups B and C (P < 0.05). Collectively, the results from this in vitro study demonstrated that MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces had the ability to increase fibroblast functionality. In addition, MAPs/bFGF-PLGA microsphere composite-coated Ti surfaces should be studied further as a method of promoting formation of stable biological seals around transcutaneous sites.

Noninvasive and quantitative assessment of in vivo angiogenesis using RGD-based fluorescence imaging of subcutaneous sponges.

PURPOSE: There is a real need to adapt simple and reproducible imaging methodologies to evaluate noninvasively pro- and antiangiogenic activities of new treatments in a physiological context in mice. PROCEDURE: The angiogenic response to fibroblast growth factor 2 (FGF-2) in a model of subcutaneously implanted cellulose sponges was measured in parallel after an intravenous injection of a fluorescent αvß3 integrin-targeting molecule (Angiolone(TM)) and an fluorescence diffuse optical tomography optical imaging system and by measuring the hemoglobin content in the sponges. RESULTS: Optical measurements of angiogenesis correlated perfectly with the values obtained using hemoglobin quantification. This assay can be used to follow the activity of a pro- or antiangiogenic treatment like demonstrated after FGF-2 or angiostatin, respectively. CONCLUSION: The perfectly controlled quality of cellulose sponges combined to this noninvasive optical method allow rapid, accurate, and reproducible measurements of angiogenic activities in vivo at the preclinical level.

Growth and growth factor production by human nasal septal chondrocytes in serum-free media.

BACKGROUND: Tissue-engineered human cartilage offers vast possibilities as a source of graft implant material for reconstructive surgery. Serum-supplemented growth media is successful in supporting chondrocyte proliferation in vitro. Serum, however, contains exogenous growth factors that hamper the identification and quantification of growth factors autogenously produced by chondrocytes. We explore the possibility of using a commercially available serum-free medium UltraCULTURE as an alternative to modified Webber's medium (MWM), the standard media used in chondrocyte cell culture. METHODS: Human nasal septal chondrocytes were grown in UltraCULTURE containing various concentrations of basic fibroblast growth factor (bFGF; 0, 1, 10, and 100 ng/mL) with or without insulin-like growth factor and compared with chondrocytes grown in MWM. Growth curves and transforming growth factor (TGF) beta 1 production were analyzed. RESULTS: We found no differences in the ability to sustain cell viability in culture between the two base media types. We also found no statistically significant differences in TGF-beta 1 production by chondrocytes grown in either system. Finally, there were no statistically significant differences in chondrocyte proliferation between cultures supplemented with bFGF at 10 and 100 ng/mL. CONCLUSION: UltraCULTURE media is a cost-effective, serum-free alternative to standard media with compatible growth characteristics. It offers specific advantages over standard serum-containing media for the precise measurement of autogenous growth factor production by cultured chondrocytes. Furthermore, UltraCULTURE's serum-free environment would be ideal for safely producing tissue-engineered cartilage grafts.

Ultrasound assessment of angiogenesis in a matrigel model in rats.

Matrigel, a basement membrane extract, has been extensively used in in vivo angiogenesis. Contrast ultrasound imaging (CUI) of implanted Matrigel plugs with (+bFGF) and without basic fibroblast growth factor (-bFGF) was performed 7 and 14 d after implantation, followed by histologic analysis. Statistically significant differences between +bFGF and -bFGF plugs were apparent at d 7 in both plug size and contrast enhancement (both p < 0.05). Histopathology revealed differences in microvessel density (MVD) between +bFGF and -bFGF at d 7 and d 14. A significant correlation between MVD and both power Doppler contrast-enhanced area (r = 0.65, p < 0.05) and fraction of plug enhanced (r = 0.59, p < 0.05) was present. CUI of Matrigel plugs was shown to be a robust method for distinguishing between two different angiogenic states. Ultrasound measurements of blood flow in the plugs correlated with MVD, a histologic technique used to quantify tumor angiogenesis.

Angiogenesis: noninvasive quantitative assessment with contrast-enhanced functional US in murine model.

PURPOSE: To evaluate quantitative functional ultrasonography (US) in a murine gel model by using microbubble destruction kinetics to determine whether parametric indices provided with US could help assess angiogenesis. MATERIALS AND METHODS: Institutional Animal Subjects Committee approved experiments and procedures. In 36 normal mice, two 0.4-mL gel implants were placed subcutaneously on either side of spine. One implant contained 0.5, 1.0, or 1.5 microg human basic fibroblast growth factor (bFGF) per milliliter of gel. Functional US quantitative analysis of angiogenesis with microbubble contrast agent was performed on days 3, 6, 9, and 12; histologic data were collected. Time-intensity curve of implant was fitted to mathematic decay model to calculate fractional blood volume and fraction of blood replaced per unit of time. Microvascular density (MVD) and percentage of microvascular area (MVA) were measured after anti-CD31 staining. Spearman rank order correlation was used in analyses. RESULTS: bFGF-containing implants induced MVD of eight, 35, 42, and 42 vessels per square millimeter on days 3, 6, 9, and 12, respectively; in controls, MVD was four vessels/mm2 (P<.05 on days 6, 9, and 12). bFGF-containing implants induced percentage MVA of 2%, 5%, 20%, and 27%, respectively; in controls, it was 0.5% (P<.05). Maximum enhancement was significantly increased in bFGF implants (23.3 gray level+/-14.1 [standard deviation]) compared with controls (11.0+/-5.5, P<.001). Implants containing bFGF showed poor correlations between fractional blood volume and MVD (r2=0.42) or percentage MVA (r2=0.51) at US. There was no correlation between microbubble velocity and MVD (r2<0.05) or percentage MVA (r2<0.13). CONCLUSION: Functional US perfusion parameters do not correlate with current histologic indices for quantifying angiogenesis. MVD, as a histologic quantitative measurement of angiogenesis, may not be an appropriate standard for contrast-enhanced imaging that relies on perfused neovessels.

Assessment of endogenous and therapeutic arteriogenesis by contrast ultrasound molecular imaging of integrin expression.

BACKGROUND: We hypothesized that molecular imaging with contrast-enhanced ultrasound (CEU) and microbubbles targeted to endothelial integrins could be used to noninvasively assess early angiogenic responses to ischemia and growth factor therapy. METHODS AND RESULTS: Hindlimb ischemia was produced in 48 rats by ligation of an iliac artery. Half of the animals received intramuscular sustained-release fibroblast growth factor-2 (FGF-2). Immediately after ligation and at subsequent intervals from 4 to 28 days, blood flow and oxygen tension in the proximal adductor muscles were measured by CEU perfusion imaging and phosphor quenching, respectively. Targeted CEU imaging of alpha(v)- and alpha5beta1-integrin expression was performed with microbubbles bearing the disintegrin echistatin. Iliac artery ligation produced a 65% to 70% reduction in blood flow and oxygen tension. In untreated ischemic muscle, muscle flow and oxygen tension partially recovered by days 14 to 28. In these animals, signal from integrin-targeted microbubbles was intense and peaked before flow increase (days 4 to 7). In comparison to untreated animals, FGF-2-treated muscle had a greater rate and extent of blood flow recovery and greater signal intensity from integrin-targeted microbubbles, which peaked before maximal recovery of flow. On immunohistology, arteriolar but not capillary density increased in the ischemic limb after ligation, the rate and degree of which were greater in FGF-2-treated rats. Immunofluorescence demonstrated intense staining for alpha(v) in arterioles, the temporal course of which correlated with targeted imaging. CONCLUSIONS: Targeted CEU can be used to assess endogenous and therapeutic arteriogenesis before recovery of tissue perfusion. These results suggest that molecular imaging of integrin expression may be useful for evaluating proangiogenic therapies.

In vivo chamber angiogenesis assay: an optimized Matrigel plug assay for fast assessment of anti-angiogenic activity.

Efficient in vitro and in vivo angiogenesis assays, to assess and compare anti-angiogenic activity are a prerequisite for the discovery and characterization of anti-angiogenic targets. Here we describe an optimized Matrigel plug assay based on subcutaneously implanted chambers and two fast and reproducible measuring techniques. Plexiglas ring/nylon net filter-chambers (0.2 ml) containing growth factor-reduced Matrigel and 300 ng basic fibroblast growth factor (bFGF) were subcutaneously implanted into the right flank of rats. Chamber angiogenesis was scored on day 5 and day 10 post-implantation by computer image analysis of the chamber, and by optical density reading at 415 nm of a PBS solution of the chamber content. bFGF significantly induced chamber angiogenesis and histological examination confirmed that numerous blood vessels were present in the bFGF-induced chambers. The anti-angiogenic control compound TNP-470 (10 mg/kg/d s.c.) completely inhibited the bFGF-induced angiogenesis. In contrast, the anti-inflammatory or immuno-suppressive compounds cyclosporin A (15 mg/kg/d p.o.), indomethacin (1 mg/kg/d p.o.), and prednisolone (5 mg/kg/d p.o.) showed no anti-angiogenic activity, indicating that the bFGF-induced angiogenesis was not driven by an inflammatory response or by a foreign body reaction. Finally, two candidate anti-angiogenic compounds were tested in the assay. Continuous low-dose therapy with cyclophosphamide (25 mg/kg/d p.o.) significantly inhibited bFGF-induced angiogenesis, whereas 1alpha,25-dihydroxyvitamin D3 (0.5 micro g/kg/d p.o.) showed no significant anti-angiogenic activity. In conclusion, this in vivo chamber angiogenesis assay is a useful new tool for drug evaluation as well as research into anti-angiogenesis.

Quantitative assessment of cerebral neocapillary network and its remodeling in mice using intravital fluorescence videomicroscopy.

To assess the responses of different growth factors on cerebral neocapillary density (NCD), cerebral angiogenesis was induced in mice using growth factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at a concentration of 6 ng/ml each. Intravital fluorescence videomicroscopy was used to quantitatively evaluate microhemodynamic parameters such as diameter and red cell velocity. The gel-nylon mesh-sandwich system was implanted over the exposed cortex. After incubation for different periods of time (days 7, 14 or 28), fluorescein isothiocyanate (FITC)-labeled red cells were injected through a carotid artery and the neocapillaries on the upper surface of the nylon mesh were observed under a fluorescence videomicroscope. Based on the recorded videoimages, we evaluated the density, diameter and red cell velocity of the neocapillaries. The NCD in the bFGF group on day 7 was significantly higher than that in the PDGF group on day 7 (P < 0.01). The NCD (index) reached 100% on day 14, while it reduced significantly in both the groups on day 28. The neocapillary diameter was greater than that of the pre-existing capillaries on day 7. On day 14, a clear difference appeared in the capillary density between large and small vessels. The red cell velocity increased with the number of days after incubation. The response of cerebral neocapillaries to acetylcholine was measured after 28 days of incubation with growth factor bFGF and with PDGF. The red cell velocity increased significantly from its basal value in the PDGF group. These results suggest that the neocapillaries in the PDGF group matured earlier than those in the bFGF group.

Isolation of thecal cells: an assessment of purity and steroidogenic potential.

In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E(2)) and progesterone (P(4)) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E(2) could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P(4) in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E(2) was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P(4) (P<0.05) in the serum-free cell culture media. The stimulation of P(4) production was greater at higher LH concentration (50 ng/ml) (P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P(4) by LH-stimulated thecal cell cultures (P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC(50)<1 ng/ml at LH-20 ng/ml vs. EC(50)>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E(2) production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.

Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells.

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.

A cost-effective method for the automatic quantitative analysis of fibroblastic colony-forming units.

A great deal of the work characterizing stromal cell precursors in the bone marrow has been performed using the fibroblastic colony-forming unit (CFU-f) assay. However, the assay is limited in its usefulness by the necessity for manual colony counting which means that assay quantitation is highly subjective, time consuming, and much information regarding the colony size is lost. To rectify this, we have developed a computer-automated method for the analysis of CFU-f. Bone marrow cells were cultured at low density and treated with either prostaglandin E(2) (PGE(2)), basic fibroblast growth factor (bFGF), or dexamethasone, and colony formation was assessed by staining with methylene blue. After staining, the dishes were photographed over a light box using a digital camera and the image was then analyzed using Bioimage "Intelligent Quantifier" image analysis software which automatically locates and quantifies each individual colony. The data can then be imported to a spreadsheet program and processed. We have shown that this system can accurately identify, assign coordinates, and quantitate each individual colony. Colony numbers obtained with this method and manually counting showed a linear relationship with a correlation coefficient of 0.99. In addition, using the colony intensity and surface area data, the colony size can be calculated. With this methodology, we have shown that dexamethasone, PGE(2), and bFGF can all modulate total cell numbers in bone marrow stromal cells (BMSC) cultures but modulating both colony number and colony size.

In vitro assessment of the biological activity of basic fibroblast growth factor released from various polymers and biomatrices.

The kinetics of controlled release of basic fibroblast growth factor (bFGF) from polymers (sutures, polycarbonate, Hydron, and Elvax), biopolymers (alginate), and biomatrices (lens capsules), and conditions for storage of bFGF (temperature, plastic type, heparin) were evaluated in vitro. Tissue culture proliferation bioassays with 3T3 fibroblasts, showed that only lens capsules with bFGF had a sustained release of bFGF for up to three weeks. The other materials released all of the 'bound' bFGF with two hours or produced an inflammatory response in vivo. Therefore, the lens tissue had the most potential for controlled long-term delivery of bFGF in vivo. These studies emphasise the importance of in vitro analysis of release kinetics of growth factors from a range of materials as a basis for potential in vivo applications.