Laminin receptor; Laminin A-chain fragment

The development of biomaterials for regenerating neurons from induced pluripotent stem (iPS) cells is crucial to the potential therapy for traumatic injury to nervous system. This study aims to guide differentiation of iPS cells into neuron-lineage cells in inverted colloidal crystal (ICC) scaffolds containing alginate, poly(γ-glutamic acid), and surface PA22-2/CSRARKQAASIKVAVSADR (peptide). The differentiation of iPS cells in ICC constructs was characterized by staining of embryonic and neuronal markers. The results indicated that hexagonal crystals of polystyrene microspheres shaped hydrogels into ICC scaffolds with interconnected pores. PA22-2/CSRARKQAASIKVAVSADR slightly enhanced the adhesion of iPS cells in ICC constructs and yielded no variation in the viability of iPS cells. Cultured ICC constructs with CSRARKQAASIKVAVSADR reduced the expression of stage-specific embryonic surface antigen-1 and raised the expression of β III tubulin of differentiating iPS cells. The induction with CSRARKQAASIKVAVSADR in ICC topography can improve the differentiation of iPS cells toward neurons for nerve tissue engineering[1].

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Adhesion and viability of iPS cells in peptide-grafted ICC constructs [1]
Fig. 6 shows the adhesion efficiency and viability of iPS cells in alginate/γ-PGA ICC constructs with surface PA22-2/CSRARKQAASIKVAVSADR. As indicated in this figure, the adhesion efficiency of iPS cells in ICC constructs enhanced slightly with an increasing concentration of CSRARKQAASIKVAVSADR. This was mainly because the positively charged amino groups of CSRARKQAASIKVAVSADR could attract iPS cells to the pore surface of alginate/γ-PGA ICC constructs via electrostatic interaction. However, the peptide sequence of CSRARKQAASIKVAVSADR could scarcely be recognized by cellular adhesion molecules on the membrane of iPS cells. This recognition ability might explain that at ralginate = 0.33, η was almost an invariant when npeptide increased from 10 μg/mL to 15 μg/mL. As displayed in Fig. 6, the adhesion efficiency of iPS cells in ICC construct with ralginate = 0.33 was slightly higher than that with ralginate = 0.67. This result deduced the following explanation. The interconnection in ICC construct with ralginate = 0.33 was higher than that with ralginate = 0.67 (images shown in Fig. 3). As a result, the infiltration of cell suspension into the former was easier than that into the latter. Thus, the utilization rate at space and surface for cell migration and attachment in ICC construct with ralginate = 0.33 was than higher than that with ralginate = 0.67. As revealed in Fig. 6, an increasing concentration of CSRARKQAASIKVAVSADR yielded insignificant effect on the viability of iPS cells. Typical example was that at ralginate = 0.33, PCV was almost an invariant when npeptide increased from 10 μg/mL to 15 μg/mL. In addition, the viability of iPS cells in ICC construct with ralginate = 0.33 was slightly higher than that with ralginate = 0.67. This was because the affinity of iPS cells to ICC construct with ralginate = 0.33 was stronger than that with ralginate = 0.67, rendering a higher capability of iPS cells to colonize the surface in the former. Moreover, an ICC construct with a high composition of γ-PGA might benefit the adaption and accommodation of iPS cells.

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Fluorescent images of embryonic and neuronal markers in ICC constructs [1]
Fig. 7 shows the micrographs of cultured ICC constructs stained against SSEA-1 and β III tubulin. The expression of SSEA-1 represented the embryonic or phenotypic characteristics of iPS cells in constructs. In addition, anti-β III tubulin identified the differentiation of iPS cells into neuron-lineage cells. As indicated in Fig. 7(a) and (b), the stain of SSEA-1 expressed by differentiating iPS cells in PA22-2/CSRARKQAASIKVAVSADR-grafted ICC construct (Fig. 7(b)) was smaller than that in peptide-free ICC construct (Fig. 7(a)). This suggested that the surface CSRARKQAASIKVAVSADR induced the differentiation of iPS cells and reduced the quantity of phenotypic iPS cells. As indicated in Fig. 7(c) and (d), the cultured iPS cells in both ICC constructs with and without CSRARKQAASIKVAVSADR expressed green stains. Moreover, the surface CSRARKQAASIKVAVSADR enlarged the region identified by anti-β III tubulin in ICC construct. This suggested that alginate/γ-PGA ICC constructs with surface CSRARKQAASIKVAVSADR accelerated neuronal differentiation of iPS cells.

Adhesion of iPS cells in peptide-grafted alginate/γ-PGA ICC constructs [1]
Cryopreserved iPS cells were unfrozen at 37 °C for 1 min. After dilution and centrifugation, iPS cells were cultured with ESGRO medium containing 1% (v/v) penicillin–streptomycin–glutamate solution in the humidified CO2 incubator. The concentration of iPS cells in ESGRO medium was evaluated by trypan blue exclusion with a hemocytometer under a phase-contrast biological microscope. The suspension of iPS cells at a density of 3 × 105 cells/construct was permeated into PA22-2CSRARKQAASIKVAVSADR-grafted alginate/γ-PGA ICC scaffolds, placed in 24-well microtiter plate, and cultured in the humidified CO2 incubator for 4 h. After seeding, the quantity of released iPC cells in 0.2 mL of ESGRO medium was determined by the hemocytometer and phase-contrast biological microscope. The adhesion efficiency of iPS cells in ICC constructs (η (%)) was defined as η (%) = [(3 × 105 − quantity of released iPS cells)/(3 × 105)] × 100%.

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Viability of iPS cells in peptide-grafted alginate/γ-PGA ICC constructs [1]
The suspension of iPS cells in ESGRO medium was injected into disinfected PA22-2/CSRARKQAASIKVAVSADR-grafted alginate/γ-PGA ICC scaffolds by a syringe. The ICC constructs with a cell density of 5 × 104 cells/construct in a 24-well plate (Corning, Horseheads, NY) were incubated in the humidified CO2 incubator at 37 °C for 4 h and cultured with 0.15 mL of ESGRO medium in the humidified CO2 incubator for 8 h. After removal of ESGRO medium and wash with DPBS, CSRARKQAASIKVAVSADR-grafted alginate/γ-PGA ICC constructs were centrifuged at 130 × g for 5 min and reacted with 50 μL of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide containing 2% (v/v) activation solution in the humidified CO2 incubator for 4 h. 200 μL of the liquid sample in a 96-well MicroWell™ polystyrene plate was analyzed by the ELISA spectrophotometer at 450 nm. The viability of iPS cells (PCV (%)) in CSRARKQAASIKVAVSADR-grafted alginate/γ-PGA ICC constructs is defined as (OD12 − ODXTT)/(OD4 − ODXTT) × 100%, where OD4, OD12, and ODXTT are, respectively, the optical density of XTT-treated sample after seeding for 4 h, that after incubation for 8 h, and the optical density of XTT solution.

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Immunochemical staining of antigenic markers in differentiating iPS cells [1]
After cultivation of iPS cells in PA22-2/CSRARKQAASIKVAVSADR-grafted alginate/γ-PGA ICC scaffolds for 5 days, the constructs with differentiating iPS cells were submerged in liquid nitrogen, enclosed in tissue-tek optimal cutting temperature compound, placed in an ultralow temperature freezer at −80 °C for 2 h, sliced by a cryostat microtome into samples of 10 μm. After wash with DPBS, the samples were reacted with 0.1% (v/v) methanol in hydrogen peroxide at room temperature for 10 min, treated with 0.5% (v/v) Triton X-100 at room temperature for 10 min, and immersed in serum blocking solution for 1 h. To identify the phenotype of iPS cells, the samples were stained with diluted anti-stage-specific embryonic surface antigen (SSEA)-1 cloning MC-480 phycoerythrin (PE) conjugate at 4 °C for 12 h in darkness. To identify the neuronal differentiation of iPS cells, the samples were stained with diluted rabbit monoclonal [EP1331Y] anti-β III tubulin at 4 °C for 12 h in darkness and treated with diluted goat polyclonal secondary antibody to rabbit IgG-H&L FITC conjugate at room temperature for 15 min in darkness. The stained fluorescence in the samples was maintained with ProLong gold antifade reagent. A confocal laser scanning microscope with an argon laser through a light filter was applied to visualize the images of markers expressed by differentiating iPS cells. The red stains of anti-SSEA-1 and green stains of anti-β III tubulin used 555 nm (excitation) and 578 nm (emission) and 495 nm (excitation) and 528 nm (emission), respectively.

[1]. Inverted colloidal crystal scaffolds with induced pluripotent stem cells for nerve tissue engineering. Colloids Surf B Biointerfaces. 2013 Feb 1:102:789-94.

[2]. Supported phospholipid bilayers as a platform for neural progenitor cell culture.J Biomed Mater Res A. 2008 Mar 15;84(4):940-53.

[3]. Cell-free synthesis of cytochrome bo(3) ubiquinol oxidase in artificial membranes. Anal Biochem. 2012 Apr 1;423(1):39-45.

The PA22-2/CSRARKQAASIKVAVSADR-grafted alginate/γ-PGA ICC scaffolds were prepared for differentiating iPS cells into neuron-lineage cells. The grafted PA22-2/CSRARKQAASIKVAVSADR enhanced the quantity of surface nitrogen in alginate/γ-PGA ICC scaffolds. In addition, CSRARKQAASIKVAVSADR on the pore surface of ICC constructs promoted the adhesion of iPS cells due to the electrostatic attraction. However, the viability of iPS cells in ICC constructs was irrelevant to the incorporated CSRARKQAASIKVAVSADR. Moreover, a high weight ratio of γ-PGA in ICC scaffolds improved the adhesion efficiency and viability of iPS cells. The surface CSRARKQAASIKVAVSADR and ICC geometry favored the differentiation of iPS cells toward neurons.[1]

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Supported phospholipid bilayers constitute a biomimetic platform for cell behavior studies and a new approach to the design of cell culture substrates. Phosphocholine bilayers are resistant to cell attachment, but can be functionalized with bioactive molecules to promote specific cell interactions. Here, we explore phosphocholine bilayers, functionalized with the laminin-derived IKVAV pentamer, as substrates for attachment, growth, and differentiation of neural progenitor cells (AHPs). By varying peptide concentration (0-10%), we discovered a strongly nonlinear relationship between cell attachment and IKVAV concentration, with a threshold of 1% IKVAV required for attachment, and saturation in cell binding at 3% IKVAV. This behavior, together with the 10-fold reduction in cell attachment when using a jumbled peptide sequence, gives evidence for a specific interaction between IKVAV and its AHP cell-surface receptor. After 8 days in culture, the peptide-functionalized bilayers promoted a high degree of cell cluster formation. This is in contrast to the predominant monolayer growth, observed for these cells on the standard laminin coated growth substrates. The peptide-functionalized bilayer did not induce differentiation levels over those observed for the laminin coated substrates. These results are promising in that peptide-functionalized bilayers can allow attachment and growth of stem cells without induction of differentiation.[2]

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The analysis of membrane proteins is notoriously difficult because isolation and detergent-mediated reconstitution often results in compromising the protein structure and function. We introduce a novel strategy of combining a cell-free expression method for synthesis of a protein species coping with one of the most important obstacles in membrane protein research-preserving the structural-functional integrity of a membrane protein species and providing a stable matrix for application of analytical tools to characterize the membrane protein of interest. We address integration and subsequent characterization of the cytochrome bo(3) ubiquinol oxidase (Cyt-bo(3)) from de novo synthesis without the effort of conventional cell culture, isolation, and purification procedures. The experimental output supports our idea of a suitable platform for in vitro protein synthesis and functional integration into a membrane-mimicking structure. We show the compatibility of different concepts of in vitro synthesis toward biosensor applicability by the example of Cyt-bo(3) protein expression. Our results obtained from in vitro synthesized proteins displayed similar behavior to proteins isolated from the cellular context. Overall, our approach is suitable for the in vitro expression of "complex" protein species such as Cyt-bo(3), which can be reproducible and stably synthesized and preserved in robust, synthetic planar membrane architecture. [3]

C82H150N32O25S

2016.33

2015.11705

131435-36-4

168510659

H-Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp-Arg-NH2; Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp-Arg-NH2

CSRARKQAASIKVAVSADR-NH2; CSRARKQAASIKVAVSADR

Typically exists as White to off-white solid at room temperature

-13.2

37

32

73

140

4200

20

[C@H](CCCCN)(C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@H](C(=O)N)CCCNC(N)=N)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CS

VWUQXESDDMVECY-YJTOHMMESA-N

InChI=1S/C82H150N32O25S/c1-12-39(6)60(79(139)107-48(21-14-16-28-84)72(132)113-58(37(2)3)77(137)101-44(11)66(126)112-59(38(4)5)78(138)111-53(33-115)74(134)100-43(10)64(124)108-52(32-57(119)120)73(133)102-46(61(87)121)22-17-29-94-80(88)89)114-76(136)55(35-117)109-65(125)41(8)97-62(122)40(7)98-69(129)51(25-26-56(86)118)106-70(130)47(20-13-15-27-83)104-71(131)50(24-19-31-96-82(92)93)103-63(123)42(9)99-68(128)49(23-18-30-95-81(90)91)105-75(135)54(34-116)110-67(127)45(85)36-140/h37-55,58-60,115-117,140H,12-36,83-85H2,1-11H3,(H2,86,118)(H2,87,121)(H,97,122)(H,98,129)(H,99,128)(H,100,134)(H,101,137)(H,102,133)(H,103,123)(H,104,131)(H,105,135)(H,106,130)(H,107,139)(H,108,124)(H,109,125)(H,110,127)(H,111,138)(H,112,126)(H,113,132)(H,114,136)(H,119,120)(H4,88,89,94)(H4,90,91,95)(H4,92,93,96)/t39-,40-,41-,42-,43-,44-,45-,46-,47-,48-,49-,50-,51-,52-,53-,54-,55-,58-,59-,60-/m0/s1

(3S)-3-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-amino-3-sulfanylpropanoyl]amino]-3-hydroxypropanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoyl]amino]-5-oxopentanoyl]amino]propanoyl]amino]propanoyl]amino]-3-hydroxypropanoyl]amino]-3-methylpentanoyl]amino]hexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]propanoyl]amino]-4-[[(2S)-1-amino-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-oxobutanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)