Bioengineering of Animal/Human Cells PDF

Handout Bioengineering of Animal/Human Cells SS2024 Prof. Dr. Julia von Maltzahn Stem Cell Biology of Aging Introduction to myogenesis and rhabdomyosarcoma (adapted from Schmidt et al., 2019) 1. Myogenesis Skeletal muscle fulfils multip...

Handout Bioengineering of Animal/Human Cells SS2024 Prof. Dr. Julia von Maltzahn Stem Cell Biology of Aging Introduction to myogenesis and rhabdomyosarcoma (adapted from Schmidt et al., 2019) 1. Myogenesis Skeletal muscle fulfils multiple functions in the body including voluntary locomotion, breathing and postural behaviour. Skeletal muscle possesses a remarkable ability to regenerate and to adapt to physiological demands such as growth or training (von Maltzahn J, 2013). Muscle stem cells - also termed satellite cells (SCs) - are a prerequisite for its regeneration (Lepper et al., 2011; Murphy et al., 2011; Sambasivan et al., 2011). Under resting conditions, muscle stem cells are quiescent and reside under the basal lamina of the myofiber (Chang and Rudnicki, 2014). While quiescent under resting conditions, muscle stem cells become activated, expand and differentiate during skeletal muscle regeneration, a process controlled by sequential expression of transcription factors, resembling the differentiation program of embryonic myogenesis (Hernandez-Hernandez et al., 2017; von Maltzahn J, 2013) (Fig.1). The paired box transcription factor Pax7 is expressed in all muscle stem cells (Lepper et al., 2009; Seale et al., 2000; von Maltzahn et al., 2013). Upon activation, muscle stem cells co-express Pax7 and MyoD – an early marker for myogenic commitment - leave the quiescent state and become myoblasts. Myoblasts then further differentiate into myocytes, before maturing to myofibers. Fig.1: Myogenic lineage progression and expression profile of key myogenic regulators. (a) Schematic illustration of the myogenic lineage progression. Satellite cells are activated, e.g. due to injury, start to proliferate, thereby generating myogenic progenitor cells. Upon differentiation myogenic progenitor cells differentiate into myocytes, which fuse to form myotubes and mature to become myofibers, the contractile unit of skeletal muscle. (b) Expression profile of key modulators of myogenic lineage progression. (Schmidt et al., 2019). 2. Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is a relatively rare neoplasm of skeletal muscle, yet it represents the most common soft tissue cancer in children. RMS is thought to originate from myogenic precursor cells at any site of the body (Skapek et al., 2019). RMS can be subdivided in two major subtypes, which were 2 historically classified by their appearance in light microscopy upon histological examination. While embryonal rhabdomyosarcoma (ERMS) with 67 % is the most frequent subtype of RMS in children and shares features of developing skeletal muscle tissue (Patton and Horn, 1962), alveolar rhabdomyosarcoma (ARMS) subtype accounts for 30 % of RMS cases and appears histologically as an accumulation of cells surrounding an open central space (Enterline and Horn, 1958). Since the expression of many muscle-specific proteins in the tumor cells, they are also termed rhabdomyoblasts. Once of hallmarks of rhabdomyoblasts is their deregulated myogenic differentiation program leading to continuous proliferation and impaired terminal differentiation. For ARMS the majority of tumors are characterized by a chromosomal translocation resulting in the expression of a chimeric transcription factor composed of PAX3 or PAX7and the forkhead transcription factor FOXO1 (Barr et al., 1993; Davis et al., 1994). In contrast to ARMS, ERMS tumors display a greater variety of tumor-promoting alterations. The ERMS subtype is associated with recurrent mutations in components of the RAS-RAF-MAPK and PI3K-AKT-mTOR pathways (Shern et al., 2014). Furthermore, alterations in insulin-like growth factor (IGF)-signaling are frequently observed in ERMS tumors, such as high expression of the ligand insulin-like growth factor 2 (IGF2) as a result of a loss of heterozygosity and loss of imprinting at the 11p15.5 locus (Makawita et al., 2009; Martins et al., 2011; Zhan et al., 1994). 3 Cell lines and Media:  MYOG-Reporter:  RD-HPRM44335  CT10-HPRM44335  SMS-CTR-HPRM44335  MYH3-Reporter:  RD-HPRM44328  CT10-HPRM44328  SMS-CTR-HPRM44328 Growth Medium: DMEM GlutaMax + 10%FCS + 1%PenStrep Differentiation Medium: DMEM GlutaMax + 2%Horse Serum + 1%PenStrep The promoter reporter cell lines were created by the transduction of eRMS cell lines with lentiviral particles. The reporter construct consists of two gene sections. The first gene segment contains the coding sequence of gaussia luciferase (GLuc) under the control of the promoter regions (P-Region) ~1300 bp upstream of the transcription start site of the human genes MYOG (HPRM44335) or MYH3 (HPRM44328). The second gene segment on the constructs consists of the constitutive promoter SV40 and the coding sequences for the secreted alkaline phosphatase (SeAP) and a puromycin-N-acetyltransferase (Puro), separated by a IRES sequence.  C2C12 cell line Growth Medium: DMEM low glucose + 10%FCS + 1%PenStrep Differentiation Medium: DMEM high glucose + 2%Horse Serum + 1%PenStrep C2C12 cells are an immortalized mouse myoblast cell line. 4 Reporter Experiment Reporter Cell seeding (d-1):  Cells are cultured in a T75 flask with 15ml growth medium  Wash cells 1x with 5ml PBS  2ml trypsin per T75 flask  Incubate at 37°C until cells detach  Add 8ml growth medium and transfer to 15ml tube  Centrifuge 5min at 900rpm  Solve pellet in 10ml growth medium and count:  Take 10µl cell suspension for the counting chamber  Seed 1*104 cells per well (96 well plate, 6 wells, 200µl total volume per well)  Incubate at 37°C Differentiation and siRNA transfection (d0):  Take off growth medium and add 200µl differentiation medium per well  Prepare siRNA:  25 µl OptiMEM + 1.5 µl Lipofectamine RNAiMAX  25 µl OptiMEM + 0.5 µl scrambled siRNA (10µM stock) (make sure, there is liquid in the tip)  25 µl OptiMEM + 1.5 µl Lipofectamine RNAiMAX  25 µl OptiMEM + 0.5 µl TRPS1 siRNA (10µM stock)  25 µl siRNA-OptiMEM mixed into Lipofectamine-OptiMEM mix  incubated 5 min at RT  Add 10µl of siRNA-Lipofectamine to cells 5 sampling (d3):  collect 100 µl supernatant from each well in separate tubes and freeze at -20°C Luciferase assay: This experiment will be performed with all the collected samples on one plate.  Collected samples are thawed at RT  Buffer GL-S (10x) is thawed at RT, vortex 3-5s  GL-S (10x) is diluted to 1x with distilled water (100 µl per sample) (calculate the amount needed for all samples and positive control (PC)!)  Prepare GLuc Working Solution:  add 1 µl Substrate GL per 100 µl 1x GL-S Buffer (calculate the amount needed for all samples and positive control (PC)!), invert to mix  Incubate GLuc Working Solution 25 min at RT capped and protected from light  10 µl of sample are added to a white 96 well plate  10µl EF1a-PG04 is added as positive control (PC) in a separate well  100 µl of GLuc Working Solution is added to samples with multichannel pipette  Plate is placed in plate reader and AG-Maltzahn_SecretedPair_Part1 is started  GLuc plate is heat treated at 65 °C for 15 min, then placed on ice  Buffer AP (10x) is thawed at RT, vortex 3-5s (start 5 min after heating)  Buffer AP (10x) is diluted to 1x with distilled water (100 µl per sample) (calculate the amount needed for all samples and positive control (PC)!)  Prepare SEAP Working Solution:  add 1 µl Substrate AP per 100µl 1x AP Buffer (calculate the amount needed for all samples and positive control (PC)!), invert to mix  Incubate SEAP Working Solution 5-10 min at RT capped and protected from light  10 µl of sample from the GLuc plate are added to a new white 96 well plate  100 µl of SEAP Working Solution is added to samples with multichannel pipette  Plate is placed in plate reader and AG-Maltzahn_SecretedPair_Part2 is started 6 Calculation of results:  To normalise the data the values of GLuc will be divided by the SeAP values (e.g. A1 of GLuc/A1 of SeAP).  Then the mean and standard deviation of each condition will be calculated from the triplicates.  Calculate the ratio of the siTRSP1 and the siSCR mean and show in a graph with statistical analysis 7 Immunofluorescence Experiment Reporter cells Reporter Cell seeding (d-1):  Cells are cultured in a T75 flask with 15ml growth medium  Wash cells 1x with 5ml PBS  2ml trypsin per T75 flask  Incubate at 37°C until cells detach  Add 8ml growth medium and transfer to 15ml tube  Centrifuge 5min at 900rpm  Solve pellet in 10ml growth medium and count:  Take 10µl cell suspension for the counting chamber  Seed 5*104 cells per well (24 well plate, 2 wells, 500µl total volume per well)  Incubate at 37°C Differentiation and siRNA transfection (d0):  Take off growth medium and add 500µl differentiation medium  Prepare siRNA:  25 µl OptiMEM + 1.5 µl Lipofectamine RNAiMAX  25 µl OptiMEM + 0.5 µl scrambled siRNA (10µM stock)  25 µl OptiMEM + 1.5 µl Lipofectamine RNAiMAX  25 µl OptiMEM + 0.5 µl TRPS1 siRNA (10µM stock)  50 µl siRNA-OptiMEM mixed into Lipofectamine-OptiMEM mix  incubated 5 min at RT  Add 50µl of siRNA-Lipofectamine to cells sampling (d3):  Remove medium and wash 1x with PBS  Fix cells with 2% PFA for 10min (500µl/well)  Remove PFA (collect in separate tube, do not discard in sink) and wash 2x 5min with PBS  Store in PBS at 4°C 8 Immunofluorescence staining:  Remove PBS and incubate 5min with 500µl permeabilization solution (PBS + 0.1%Triton)  wash 2x 5min with PBS  incubate 1h with 500µl blocking solution (PBS with 5% horse serum)  dilute primary antibody in blocking solution (500µl/well)  MF20 mouse IgG2b, 1:1  MYOG mouse IgG1, 1:1  Incubate over night at 4°C  wash 3x 5min with PBS  dilute secondary antibody in blocking solution (500µl/well)  α-mIgG2b-546 (red), 1:1000  α-mIgG1-488 (green), 1:1000  Incubate 1h at room temperature, in the dark!  wash 3x 5min with PBS  dilute nuclear stain in PBS (500µl/well)  DAPI, 1:5000  Incubate 10min at room temperature, in the dark!  wash 3x 5min with PBS  store in PBS at 4°C covered with foil documentation:  5 pictures (20x) per well will be taken by us at the Keyence microscope (14C.423)  Use Fiji software for counting: https://imagej.net/software/fiji/downloads how to count is explained in the provided protocol: see attachment  count all cells (DAPI)  count all MYOG positive cells  count all nuclei within MF20 positive cells  calculate % MYOG positive  TRPS1 vs scrambled siRNA  calculate % MF20 positive nuclei / all nuclei  TRPS1 vs scrambled siRNA  create a graph with statistical analysis 9 Immunofluorescence Experiment C2C12 cells C2C12 Cell seeding (d-1):  Cells are cultured in a T75 flask with 15ml growth medium  Wash cells 1x with 5ml PBS  2ml trypsin per T75 flask  Incubate at 37°C until cells detach  Add 8ml growth medium and transfer to 15ml tube  Centrifuge 5min at 900rpm  Solve pellet in 10ml growth medium and count:  Take 10µl cell suspension for the counting chamber  Seed 2*104 cells per well (24 well plate, 2 wells, 500µl total volume per well)  Incubate at 37°C Differentiation and siRNA transfection (d0):  Take off growth medium and add 500µl differentiation medium  Prepare siRNA:  25 µl OptiMEM + 1.5 µl Lipofectamine RNAiMAX  25 µl OptiMEM + 0.5 µl scrambled siRNA (10µM stock)  25 µl OptiMEM + 1.5 µl Lipofectamine RNAiMAX  25 µl OptiMEM + 0.5 µl Pank4 siRNA (10µM stock)  50 µl siRNA-OptiMEM mixed into Lipofectamine-OptiMEM mix  incubated 5 min at RT  Add 50µl of siRNA-Lipofectamine to cells sampling (d3):  Remove medium and wash 1x with PBS  Fix cells with 2% PFA for 10min (500µl/well)  Remove PFA (collect in separate tube, do not discard in sink) and wash 2x 5min with PBS  Store in PBS at 4°C 10 Immunofluorescence staining:  Remove PBS and incubate 5min with 500µl permeabilization solution (PBS + 0.1%Triton)  wash 2x 5min with PBS  incubate 1h with 500µl blocking solution (PBS with 5% horse serum)  dilute primary antibody in blocking solution (500µl/well)  MF20 mouse IgG2b, 1:1  MYOG mouse IgG1, 1:1  Incubate over night at 4°C  wash 3x 5min with PBS  dilute secondary antibody in blocking solution (500µl/well)  α-mIgG2b-546 (red), 1:1000  α-mIgG1-488 (green), 1:1000  Incubate 1h at room temperature, in the dark!  wash 3x 5min with PBS  dilute nuclear stain in PBS (500µl/well)  DAPI, 1:5000  Incubate 10min at room temperature, in the dark!  wash 3x 5min with PBS  store in PBS at 4°C covered with foil documentation:  5 pictures per well will be taken by us at the Keyence microscope (14C.423)  Use Fiji software for analysis: https://imagej.net/software/fiji/downloads  measure the Myotube diameter at the thickest part of each myotube  how to messure is explained in the provided protocol: see attachment  create a graph including the datapoints from everybody of your course with statistical analysis 11 Western Blot C2C12 Cell seeding (d-1):  Cells are cultured in a T75 flask with 15ml growth medium  Wash cells 1x with 5ml PBS  2ml trypsin per T75 flask  Incubate at 37°C until cells detach  Add 8ml growth medium and transfer to 15ml tube  Centrifuge 5min at 900rpm  Solve pellet in 10ml growth medium and count:  Take 10µl cell suspension for the counting chamber  Seed 6*105 cells per well (6 well plate, 2 wells, 2000µl total volume per well)  Incubate at 37°C Differentiation and siRNA transfection (d0):  Take off growth medium and add 2000µl differentiation medium to the wells  Prepare siRNA:  150 µl OptiMEM + 9 µl Lipofectamine RNAiMAX  150 µl OptiMEM + 3 µl scrambled siRNA (10µM stock)  150 µl OptiMEM + 9 µl Lipofectamine RNAiMAX  150 µl OptiMEM + 3 µl Pank4 siRNA (10µM stock)  150 µl siRNA-OptiMEM mixed into Lipofectamine-OptiMEM mix  incubated 5 min at RT  Add 250µl of siRNA-Lipofectamine to cells sampling (d3):  Wash cells 1x with cold PBS  Add ice cold 100µl (6well) RIPA buffer (with protease inhibitors freshly added: 40µl complete/ml, 50µl phosstop/ml)  Scrape cells and transfer to eppi (on ice)  Incubate 20min on ice, vortex every 5min  Centrifuge 30min at 10.000rpm, 4°C  Transfer supernatant to new eppi, freeze at -80°C or proceed 12 BCA-Assay (Pierce™ BCA Protein Assay Kit, Catalog Number 23225) Preparation of diluted albumin (BSA) standards (one standard for all of you)  Use RIPA-Buffer as diluent (the same diluent as used for the samples) Preparation of the BCA working reagent (WR) (prepare one mix for all of you)  Determine the total volume of WR required using the following formula: (# standards + # samples) × volume of WR per sample (200 µL) = total volume WR required  Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B)  Dilute samples 1:5 in RIPA buffer  Pipette 25 μL of each standard or unknown sample duplicate into a microplate well  Loding template:  Add 200 μL of the WR to each well  Place plate in plate reader with BCA assay program  Calculate amount of protein needed for 40µg total protein to be loaded and amount of lämmli buffer mean Conc. µl 5x Sample [µg/ml] dilution 1:5 µl/40µg lämmli = concentration * 5 = (40*1000) / concentration = µl / 5 13 Gel-Preparation (2 gels for the whole group)  Ammonium persulfate (APS) solidifies the solution. If it is not added until the end, both gels can be prepared at the same time. Prepare gels under the safety cabinet.  Clean glass plates with ethanol, assemble the rack for gel solidification, check with water if everything is tight  add about 5mL of the resolving gel solution  Carefully pipette 500µL of Isopropanol on top of the solution to remove the air bubbles  Wait 30min  Remove the Isopropanol  Overlay the running gel with the stacking gel and carefully insert the comb without any air bubbles  Wait until the gel is solidified (about 30min) 2 gels: 8% Stacking 3.5x Bis‐Tris buffer, mL 2.84 1 30% acrylamide (toxic), mL 2.68 0.46 H2O, mL 4.48 2.04 10% APS, µL 50 40 TEMED, µL 14 20 SDS-PAGE  mix sample with lämmli sample buffer according to calculated amounts after the protein assay  denature samples for 5min at 95°C, spin down briefly  Remove the comb carefully and flush pockets with 1x MOPS running buffer  Arrange gel chambers inside the electrophorator and fill the tank with 1 x MOPS running buffer (fill the inner chamber formed by the gels with new Buffer, the tank can be filled with used running buffer).  Load 5µL of prestained standard  add 40µg sample/lane Gelloading 1: nitrocellulose membrane, tank blot 1 2 3 4 5 6 7 8 9 10 Std 5µl scr siRNA Pank4 SiRNA Std 5µl scr siRNA Pank4 SiRNA Std 5µl scr siRNA Pank4 SiRNA - Gel: 8% Run: 120V Blot: tank, 100V, Glycine buffer Gelloading 2: nitrocellulose membrane, tank blot 1 2 3 4 5 6 7 8 9 10 Std 5µl scr siRNA Pank4 SiRNA Std 5µl scr siRNA Pank4 SiRNA - - - - Gel: 8% Run: 120V Blot: tank, 100V, Glycine buffer  Run at 100V through the resolving gel and at 120V until the blue line runs out of the gel 14 Blotting  Use methanol resistant cloves for all following steps!  Prepare the transfer buffer and store in freezer (100 mL 10 x transfer buffer + 200 mL Methanol + 700 mL dH2O)  equilibrate nitrocellulose membrane, sponge (black ones) and filter paper in 1 x transfer buffer (for each gel, you need 2 sponges and 4 filter papers)  Carefully open gel chamber and equilibrate gel in 1x transfer buffer  Create a transfer sandwich as follows:  White side of retainer  Black Sponge  2 Filter papers  Nitrocellulose membrane  SDS Gel  2 Filter papers  Black Sponge  Black side of retainer  Check out that there are no air bubbles between the gel and the membrane  Relocate the sandwich to the transfer apparatus (arrange black side of the retainer to black side of the transfer apparatus  Add cooling pack and fill with transfer buffer  Transfer for 1h at 100V Blocking and antibody incubation  Stain the membrane with Ponceau-S solution. The dye is used to check whether protein has been transferred to the membrane.  Wash membrane with water and detect with BioRad Imager (14C.424, will be done by us)  Cut membrane at 70kDa  Wash in 1x TBS-T until red staining is gone  Block membrane in 5ml TBS-T with 5 % milk for 1h  Seal membrane with primary antibody (in blocking solution), 1ml per membrane  Pank4 1:1000 rabbit 82 kDa  GAPDH 1:500 mouse 36 kDa  incubate membrane on a shaker overnight at 4°C  Wash membrane 3x for 10min with TBS-T  Incubate membrane for 1h at room temperature with secondary antibody diluted in blocking solution, 5ml per membrane o Goat anti rabbit 1:5000 o Goat anti mouse 1:5000  Wash membrane 3x for 10min with 1x TBS-T  Prepare ECL solution, mix both substrates 1:1 (2ml per membrane) and incubate membrane for 1min  Detect at BioRad Imager (14C.424, will be done by us)  Strip upper membrane part with 5ml stripping buffer 10min at room temperature on shaker  Wash with TBS-T  Block membrane in 5ml TBS-T with 5 % milk for 1h  Seal membrane with primary antibody (in blocking solution), 1ml per membrane  MF20 1:1 mouse 223 kDa  incubate membrane on a shaker overnight at 4°C 15   Wash membrane 3x for 10min with TBS-T  Incubate membrane for 1h at room temperature with secondary antibody diluted in blocking solution, 5ml per membrane o Goat anti mouse 1:5000  Wash membrane 3x for 10min with 1x TBS-T  Prepare ECL solution, mix both substrates 1:1 (2ml per membrane ) and incubate membrane for 1min  Detect with BioRad ChemiDoc Imager (14C.424, will be done by us) Buffers (will be provided until otherwise stated): Buffer Components [g/mol] concentration comments Bis-Tris buffer Bis-Tris 209,24 1,25 mol/l In aqua dest 3,5x, pH 6,5 (for resolving and stacking gel) 20x TBS buffer pH 7,6 Tris base 121,14 0,5 mol/l In aqua dest NaCl 58,44 1,5 mol/l 1x TBS-T buffer TBS buffer 20x Dilute with aqua dest 0.1% Tween-20 1x PBS buffer, pH 7,4 NaCl 58,44 0,14 mol/l In aqua dest KCl 74,55 0,0027 mol/l Na2HPO4*2H2O 141,96 0,01 mol/l KH2PO4 136,09 0,0018 mol/l 20x MOPS running buffer MOPS 209,27 1 mol/l In aqua dest (for SDS-Page) Tris base 121,14 1 mol/l EDTA 292,24 0,02 mol/l SDS 288,38 2% 1x MOPS running buffer MOPS running buffer 20x 0,05 mol/l Dilute with aqua dest (for SDS-Page) Sodium bisulfite 104,061 0,005 mol/l Sodium bisulfite Sodium bisulfite 104,06 1 mol/l In aqua dest 10 x Blotting-Buffer Tris base 121,14 0,25 mol/l In aqua dest Glycine 75,07 1,92 mol/l 16 1 x Blotting-Buffer 10 x Blotting-Buffer 100 mL 10 x Buffer Methanol 20% + 200 mL Methanol + 700 mL dH2O WB blocking solution (Milk) Milkpowder 5% In TBS-T buffer TBS-T buffer 4x SDS loading buffer Bis-Tris 209,24 0,5 mol/l In aqua dest (Laemmli Puffer) HCl 36,46 0,3 mol/l Glycerol 92,09 2% SDS 288,38 0,14 mol/l EDTA 292,24 0,002 mol/l DTT 154,2 0,4 mol/l Coomassie Blue G 250 854.025 0,03% ß- Mercaptoethanol 78,13 25% RIPA buffer pH 8,0 Sodium chloride 58,44 0,15 mol/l In aqua dest Triton X-100 625 1% Sodium deoxycholate 414,55 0,5% SDS 288,38 0,1% Tris base 121,14 0,05 mol/l References Barr, F.G., Galili, N., Holick, J., Biegel, J.A., Rovera, G., and Emanuel, B.S. 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Select “Analyse” > “Tool” > “Grid” and select points and “ok” 19 6. Select “Plugins” > “Analyze” > “Cell Counter” 7. Press “Initialize”, select “Type 1” and mark all green nuclei, use grid for orientation 8. To count all nuclei within MF20 positive cells select the overlay picture. Select “Analyse” > “Tool” > “Grid” and select points and “ok” 9. Select “Plugins” > “Analyze” > “Cell Counter” 10. Press “Initialize”, select “Type 1” and mark all nuclei within red myotubes, use grid for orientation 20 Protocol: Myotube measurement with ImageJ/Fiji 1. Open all overlay images in ImageJ/Fiji 2. Select “Analyze” > “Set Scale” and make following setting: 264 px, 100, unit: µl, global press OK 3. Select the “straight line tool”, draw a line at the thickest part of a myotube and press “M” on the keyboard. Repeat for all myotubes in one picture. 4. Save messurements via “File” > “Save As…” in the same folder as the image. Name the new file as in this example : Result_Foldername.txt 5. Select the image again and burn in the messurements via “Image” > “Overlay” > “To ROI Manager”. Select all points in the list via Strg/Ctrl+A and press “Flatten. A new picture will be crated. Save this new picture via “File” > “Save As…” > “.tif” and save as “Foldername_ measurement.tif” in the same folder as the image. 6. Close the image and clear the results (“Analyze” > “Clear Results”. Start with the next image 21