Appendix- I. Compositions of Buffers and Other Reagents:

Transcription

1 Appendix- I Compositions of Buffers and Other Reagents: Phosphate Buffered Saline (PBS) 10X, ph 7.4: NaCl 80.0g KCl 2.0g Na2HPO4 11.5g KH2PO4 2.0g The salts were dissolved in 800 ml of de-ionized water and the ph was adjusted to 7.4 with 1N HCl. The volume was made up to 1000 ml. A working solution was made by diluting the 10X stock (1:10) with de-ionized water. Sorenson s Buffer 10X, ph 6.8: A: Na2HPO 4.2H g B: KH2PO4 2.27g The salts were separately dissolved in 250ml of distilled water each. For 10X stock, 250ml of A and 190ml of B solutions were mixed. The ph was adjusted to 6.8. A working solution was made by diluting the 10X stock (1:10) with de-ionized water. Hank s Balanced Salt Solution (HBSS) 10X ph 7.4: NaCl 8.0g Glucose 1.0g NaHCO g Na 2 HPO g KH 2 PO g The salts were weighed separately and were dissolved in 50ml of distilled. The ph was adjusted to 7.4 and the volume was made up to 100ml with distilled water. A working solution was made by diluting the 10X stock (1:10) with distilled water. 179

2 CD34 Isolation Buffer: Na- Citrate 0.6g BSA 2g P & S 100µl The components were dissolved in sterile PBS (ph 7.4) and volume was made up to 100ml with sterile PBS. The buffer was filtered through 0.22µ filter. Tris Borate EDTA (TBE) 5X: Tris base 54.0g Boric acid 13.7g EDTA 4.69g The salts were dissolved in 800 ml of de-ionized water and the final volume was made up to 1000 ml with de-ionized water. The solution was autoclaved and was stored at room temperature. A working solution was made by diluting the 5 X stock (1:5) with deionized water. T 10 E 1 : Tris-HCl 1M (ph 8.0) EDTA 0.5M (ph 8.0) 1000μl of Tris-HCl + 200μl of EDTA were mixed and volume was made up to 100 ml with de-ionized water and the solution was autoclaved. T 10 E 0.1 : Tris-HCl 1M (ph 8.0) EDTA 0.5M (ph 8.0) 1000μl of Tris-HCl + 20μl of EDTA were mixed and volume was made up to 100 ml with de-ionized water and the solution was autoclaved. 180

3 RBC Lysis Buffer: Tris base 0.17M NH 4 Cl 0.16M 10ml of Tris + 90ml of NH 4 Cl were mixed and the ph was adjusted to % Buffered Paraformaldehyde: Paraformaldehyde powder was added to 80 ml 1X PBS (ph 7.4) and the solution was heated to ~ 80ºC for complete dissolution. The volume was made to 100 ml. This solution was always prepared fresh. 0.5% Triton X-100: 500μl of Triton X-100 was added to 100ml of 1X PBS and the solution was stored at room temperature. It was diluted, if required, with PBS before use. Lectins Stocks: The lectins stocks were diluted with PBS and stored at 4 0 C. Alpha Methyl Mannoside: 250mM stock was prepared by dissolving the sugar in distilled water (MW ). It was filtered through a 0.22µ filter and was stored at 4 0 C. The stock was diluted with culture medium to obtain the desired concentrations of the sugar. Heparin solution: Heparin sodium salt (20,000IU) was dissolved in 5ml of plain IMDM. It was filtered through a 0.22µ filter. The sterile solution was stored at 4 0 C. Tellesnicky s Fixative: 70% Ethanol 375ml Glacial Acetic Acid 18.75ml Formaldehyde 37ml The components were mixed and the volume was made up to 500ml with distilled water. The fixative was stored at room temperature. 181

4 Poly Ethylene Glycol (PEG) Stock for the preparation of GCT- CM: 0.1g of poly ethyl glycol was dissolved in 10ml of distilled water and was autoclaved. 1ml of PEG solution was added to 100ml of McCoy s medium and this medium was added to the confluent culture of GCT cells for the preparation of GCT-CM. MTT Extraction Fluid: HCl 0.04N in isopropanol The solution was prepared and was stored at room temperature. Hydrocortisone (10-6 M): 0.001g of hydrocortisone powder was dissolved in 5ml of plain IMDM and filtered through a 0.22µ filter. It was added to the medium just before use. Composition of Media: Thawing Medium for human cells: IMDM 80ml BSA 2.5g DNase (2000U/ml) 1ml The medium was pre-cooled before reviving the cells. Thawing Medium for murine cells: RPMI 90ml FBS 10ml DNase (2000U/ml) 1ml The medium was pre-cooled before reviving the cells. Methyl Cellulose (Stock): Methyl Cellulose Sterile D.W. IMDM (2X) 22gm 500ml 500ml 182

5 Methyl cellulose powder (4000cps) was dissolved in 500ml of boiling sterile distilled water. The mixture was stirred continuously on a heated magnetic stirrer for 30 minutes. After cooling, 500ml of pre-cooled sterile 2X IMDM was added to it and the solution was continuously stirred at 4 0 C for 4 hours. The stock was dispensed in sterile bottles and was stored at 4 0 C overnight. For longer storage, the bottles were stored at C. Stocks of the Additives for Methyl Cellulose: 1. Na-Pyruvate (10 µm): 0.011g of Na-pyruvate was dissolved in 10ml of plain IMDM, filtered through a 0.22µ filter and was stored at 4 0 C. 2. Holo-Transferrin: 0.36g of transferrin was dissolved in 10ml of plain IMDM, filtered through a 0.22µ filter and was stored at 4 0 C. 3. β-mercaptoethanol (5 x 10-5 M): 7µl of β-me was added in 20ml of plain IMDM, filtered through a 0.22µ filter and was stored at 4 0 C. Methyl Cellulose (Working Solution): Before use, the methyl cellulose stock bottle was thawed at 4ºC and working solution was prepared as follows: Following components were added to 100 ml of the methyl cellulose stock. Plain IMDM 46ml FCS 30ml Na Pyruvate 200 µl Transferrin 2ml β-mercaptoethanol 2ml BSA 2g The working solution was kept at 4 0 C. 183

6 Composition of Stains: Crystal Violet (Turk s Solution): Crystal violet stain 0.01g Glacial Acetic Acid 3ml Distilled water 100ml The components were mixed and the stain was stored at room temperature. Trypan Blue: Trypan Blue 0.4g The powder was dissolved in 100ml of HBSS and was stored at room temperature. Wright s Stain: Wright s powder 0.1g The powder was dissolved in absolute methanol by grinding with mortar & pestle. The mixture was allowed to stand at room temperature for 1-2 days, was filtered through Whatman filter paper and stored at room temperature. Giemsa Stain: Giemsa powder 1.0g Glycerol 66ml The powder was mixed with glycerol and the solution was heated at 56 0 C for 30 minutes. It was cooled and 66ml of methanol was added. The stock was diluted 1:10 with Sorenson s buffer (ph 6.8) before use. Oil Red O Stain: Stock Oil Red O 0.5% in isopropanol The stock stain was stored at room temperature for 2 days before use. A working solution was made by mixing 6ml of Oil Red O (Stock) + 4ml of distilled water. The stain was allowed to stand for 10 minutes at room temperature and was filtered through Whatman filter paper before use. 184

7 Calculation of Banana and Garlic lectins for Feeding of Mice: Banana lectin: Yield of BL was 10-15mg/ kg of banana. Taking the lower estimate, 10mg of BL/kg of banana Hence, 10ng ~ 1mg of banana/mouse/week Average weight of mouse ~ 20g For an human (average weight ~ 60kg), amount of BL ~ 3.0g Garlic lectin: Yield of GL was 30mg/ kg of garlic bulbs. Hence, 10ng ~ 0.34mg of garlic/mouse/week Average weight of mouse ~ 20g For an human (average weight ~ 60kg), amount of GL ~ 1.0g These concentrations of BL and GL are well within physiological limits. 185

8 Appendix II Reagents: Plant Lectins Used in the Study: Purification and characterization of the lectins under study was carried out at IISC, Bangalore by Prof. Surolia et al. The purified lectins were supplied to us as a part of the DBT-funded collaborative project between Dr. Mrs. V. P. Kale and Prof. Surolia. 1. Banana Lectin (BL): BL is isolated from banana fruit. It is a dimeric protein composed of two identical subunits of 15kDa. It readily agglutinates rabbit erythrocytes. 2. Garlic Lectin (GL): GL is isolated from the dried garlic bulbs. The affinity purified preparation revealed three peaks upon gel filtration with molecular masses of 136, 48 and 25 kda respectively. Peak 3 was used in the present study. The protein consists of 12.5 and 11.5 kda subunits. It strongly agglutinates rabbit erythrocytes. 3. Artocarpin Lectin (AL): AL is isolated from an extract of jackfruit seeds. The molecular weight of the lectin is a 14kDa. 4. Dolichos Lectin (DL): DL is isolated from the seeds of hyacinth beans. The lectin is of 67kDa molecular weight. Both BL and GL are proteins while AL and DL are glycoproteins. The lectins were dialyzed against phosphate-buffered saline (PBS, 10mM, ph 7.4) to remove the sugar used for the elution of lectins from the column and also the sodium azide used as a preservative by using Microcon centrifugal filters (cut off 3000 daltons Millipore) as per the manufacturer s protocol, followed by filtration through 0.2µ filters. The protein content of the lectin was estimated by using micro BCA kit (Pierce) and the aliquots were stored at 4 0 C. 186

9 Methods: 1. MTT (3, (4-5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) Assay: This is a quantitative colorimetric assay which measures viability and proliferation of cells. The principle of the assay is based on the capacity of mitochondrial succinate dehydrogenase enzyme in living cells to convert the colorless water insoluble substrate, MTT, into a violet coloured formazan salt and the amount formed is directly proportional to the cell number (471). This assay was carried out on TF-1 cell line to determine the concentration rage of the lectins that can be tolerated by the cells without obvious toxicity. The cells were washed twice with plain medium and were seeded in a 96 well plate at 2x10 4 cells per well. The cells were incubated with various concentrations of lectins ranging from 10ng/ml to 80ng/ml for 48 hours at 37 0 C in a 5% CO 2 atmosphere. 10µl of MTT solution (5mg/ml) was added to each well and the cells were incubated at 37 0 C for 4 hours. The reaction was terminated by 100 µl of MTT extraction fluid (0.04N HCL in iso-propanol). The stable colour complex was measured at 570nm-630nm by ELISA reader. 2. Coating of Culture Plate Surface with Lectins: To examine whether the lectins can exert their effect on the CD34 + cells when they were presented in matrix-bound state, we coated the surface of 24-well plates with the lectins (100pg, 200pg, 10ng and 20ng/ml). A solution of the lectins diluted in PBS (250µl) was added to the wells and the plates were incubated at 37 0 C for 2 hours. The lectin solution was removed and the wells were equilibrated with Stem- Pro medium. Then 2 x10 5 CD34 + cells suspended in the Stem Pro medium were seeded in each well and the plates were incubated for 10 days. After 10 days, the cells were harvested and were subjected to CFU assay. 3. Coupling of Tosyl-Activated Para-Magnetic Beads with Lectin: To examine whether lectins can be used as an HSPC-enrichment tool, tosyl-activated M- 450 para-magnetic beads were used for this purpose. These beads provide reactive sulphonyl esters that can react covalently with proteins or other ligands having primary 187

10 amino or sulphydryl groups. Once the beads are coupled with specific ligand, they can be mixed with a heterogenous cell population. the coated Dynabeads will bind to the target population during incubation and the cell-bound beads can be separated using magnet. Tosyl-activated beads were coupled with lectin (5µg lectin /10 7 beads) as per the manufacturer s protocol. The coupled beads were stored at 4ºC in buffer (PBS containing 0.1% BSA and 2mM EDTA, ph 7.4) until used. 4. Isolation of CD34 + Cells: CB-derived MNCs were suspended in 1ml of isolation buffer. Dynabeads M-450 coupled with anti CD34 antibody were washed and the beads were added to the cell suspension. The cell beads suspension was incubated at 4 0 C for 30 minutes with intermittent mixing. The beads with bound cells were isolated by keeping the tube on a magnetic separator and the beads were washed with the isolation buffer three times. 100 µl of DetachaBead solution was added to it and the tube was incubated at 37 0 C for 20 minutes with intermittent mixing. The cells were isolated by keeping the tube on the magnetic separator. The cells were washed and were used for the further assays. 5. Counting of Adipocytes Formed in CD34 + Cells: CD34 + cells were incubated either with BL or GL (100 and 200pg/ml) for 10, 20 and 30 days in Stem Pro medium. The cells were harvested after each time point and Turk s count was taken. Adipocytes were identified by their large size (as shown in results section). This phase contrast observation was confirmed by the Wright-Giemsa and Oil red O staining. 6. Wright - Giemsa Staining: CD34 + cells were stained after 10, 20 and 30 days of incubation with Wright and Giemsa stains to identify the type of the cells present at the end of the incubation period. Briefly, cytospin smears (Shandon Southern Products, Cheshire, UK) were prepared on a glass slide and were fixed in methanol for 10 minutes at room temperature. The smear was stained with Wright s stain by shaking gently for 4-5 minutes. The excess stain was removed by washing the slides with water and then they were stained with Wright s stain 188

11 (Wright s stain diluted 1:5 with Sorenson s buffer, ph 7.4) for 4-5 minutes. Again the slides were washed and were counterstained with Giemsa stain for 4-5 minutes. The slides were washed, dried and observed under an upright microscope under 100X oil immersion objective. 7. Oil Red O Staining: Oil Red O staining was carried out to confirm the identity of adipocytes formed in the culture. It was performed as per Lillie and Ashburin s method (472). Briefly, the cells were fixed in 10% buffered formalin for 10 minutes, washed with distilled water for 5-10 minutes and were stained with Oil Red O for 30 minutes, followed by 5-10 minutes of washing with distilled water. The plates were air dried and were observed for cells showing red colored intracellular oil droplets. 8. Cryopreservation and Revival of Cells: Freezing: Primary cells or the cell lines (harvested by trypsinization for the adherent cell lines) were suspended in the growth medium containing 20% FBS in a sterile conical flask and the flask was cooled. Equal volume of pre-cooled freezing mixture consisting of 20% DMSO, 20 % FBS and 60% plain medium was added drop wise to the cells, with constant mixing. Cells were dispensed in pre-cooled cryovials, and were frozen in a computer controlled portable programmable cryobath (Cryologic, Australia). The freezing program that was used is as follows: a cooling rate of 1ºC/minute down to - 40ºC followed by 10ºC/minute down to -90º C. The vials were then located in liquid Nitrogen (-196 ºC). Revival: Frozen cells were thawed rapidly by placing the vials in a water bath at 40 C and the contents of the vials were transferred to a sterile pre-cooled centrifuge tube as soon as the last ice crystal melted. Cold thawing medium (Appendix I) was added in a drop wise manner to dilute DMSO, and the tubes were centrifuged in a pre-cooled centrifuge to pellet the cells. The cells were washed once with growth medium and were seeded in culture vessels in their respective complete growth medium. 189

12 9. Preparation of Giant Cell Tumor (GCT) Conditioned Medium: TF1 cell line is a growth factor-dependent cell line and requires factors like GM-CSF or IL-3 for its continuous growth. GCT cell line is known to secret GM-CSF and, therefore, a conditioned medium of this cell line can be used for the maintenance of TF1 cells. GCT cell line was maintained using McCoy s medium containing 10% FBS. When the cells reached 70 80% confluence, 0.01% PEG in McCoy s medium was added. The cells were incubated for 5 days and the medium was collected. The conditional medium was centrifuged at 4 0 C at 2000 rpm for 10 minutes to remove the debris. The supernatant was collected, was filtered through 0.45µ filters and the aliquots were stored at C until used. 10. Nucleated and Viable Cell Counts: i) 90µl of cell suspension was mixed with 10µl of Turk s solution (Appendix I) and the cells were counted using Neubaure s counting chamber. ii) 45µl of cell suspension was mixed with 5µl of Trypan blue dye (Appendix I) and the cells were counted Neubaure s counting chamber. 190

13 Supplementary Results I] Determination of the Lectin Concentration to be used for Primary Cells: In the absence of any information about the biological activity of BL and GL in the literature, we screened these lectins for their toxicity using a CD34+ cell line, TF1, before their use on primary cells. The TF1 cells were incubated with various concentrations of the lectins (10 to 80 ng/ml) for 48 hours and the cells were subjected to MTT assay as described in appendix II. The stable color complex was measured at 570nm-630nm by ELISA reader. O.D. AT 570nm - 630nm CONTROL 10ng/ml 80ng/ml 0.0 BL GL AL DL Figure S1: TF1 cells were incubated with various concentrations of the lectins for 48 hours. In the figure data of only two concentrations have been depicted. 10ng/ml was the lowest concentration used and 80ng/ml was the highest. The cells were subjected to MTT assay to determine the proliferation of the cells in the presence as well as in the absence of the lectins. The cells incubated with the lectins proliferated to the same extent as controls indicating that the lectins did not have any toxic effect on TF1 cells when used up to 80 ng/ml concentration. As seen in the bar diagram, the growth of TF1 cells was not affected by the presence of the lectins, indicating that in the range used these lectins did not exert any toxic effects on the cells (Figure S1). II] Coating of a Plate Surface with Lectins: One of the concerns in using plant or animal products in the culture medium is their internalization and processing by the phagocytic cells, and the possibility of these processed antigens becoming immunogenic to the host. In the in vitro assays, BL and 191

14 GL showed a salutary effect on the HSPCs. It was, therefore, necessary to examine whether they exert similar effect when they are presented on a solid matrix. The surface of a 24 well plate was coated with the lectins as described and CD34 + cells were incubated in these coated wells for 10 days in a serum-free medium. After incubation the cells were subjected to CFU assay. A. B. CFU / 10 4 CELLS CONTROL BL 100pg BL 200pg BL 10ng BL 20ng CFU / 10 4 CELLS CONTROL GL 100pg GL 200pg GL 10ng GL 20ng 0 BFU(E) GM GEMM TOTAL 0 BFU(E) GM GEMM TOTAL Figure S2: CD34 + cells were incubated on the lectin-coated surface for 10 days. The cells were harvested and CFU assay was performed. More number of colonies formed from the cells incubated in the lectincoated well indicating both BL and GL exert their effect even in a matrix-bound form.. Cells incubated in the lectin-coated wells showed formation of more number of colonies as compared to the controls. No colony formation was observed in the controls. (Figure S2). All types of colonies were formed indicating that the effect of the lectins was comparable to that seen when the lectins were presented in a soluble form. These results indicate that these lectins can be used in the matrix-coated form as well. III] Determination of the Optimal Concentrations of the Lectins: As discussed in the results section, we observed that BL and GL showed better HSPC protection at lower concentrations used for the incubation. More numbers of GEMM colonies were formed in the cells treated with BL 100pg/ml concentration while GL 100pg and 200pg/ml concentrations indicating that lectin acted upon the multi-potent progenitors in vitro (Figure S3). 192

15 A. B. CFU / 10 4 CELLS Cells BL 100pg BL 200pg BL 400pg BL 10ng CFU / 10 4 cells Cells GL 100pg GL 200pg GL 400pg GL 10ng 50 0 BFU(E) GM GEMM TOTAL 0 BFU(E) GM GEMM TOTAL Figure S3: CD34+ cells were incubated with BL or GL for 10 days. The cells were harvested and were subjected to CFU assay. As seen in the bar diagram, significantly more numbers of colonies were formed in the cells incubated with the lower concentrations of BL (A) or GL (B). Presence of GEMM colonies in the lectin-treated cells indicated that the lectins protect multi-potent progenitors in vitro. In addition to BL and GL, we have used two other mannose-specific lectins, namely AL and DL in the present study. Though most of our work was concentrated on BL and GL, we also examined whether AL and DL also show HSPC protection activity. As was done with BL and GL, first we determined the the optimal concentration of AL and DL to be used as described earlier in the materials and methods section. The effect on the colony formation was found to be better with lower concentrations of the lectins (100pg/ml and 200pg/ml) than with the higher concentrations. At lower concentration both AL and DL exhibited HSPC protection/preservation activity (Fig S4). A. B. FOLD CHANGE AL100pg CONTROL 0.8 AL400pg AL200pg AL800pg FOLD CHANGE DL100pg DL200pg CONTROL 0.8 DL400pg DL10ng Figure S4: CD34 + cells were incubated with various concentrations of AL or DL for 10 days. The cells were harvested and CFU assay was carried out. More number of colonies were seen in the cells incubated with 100 pg/ml of both the lectins. 193

16 IV] AL and DL Protect Primitive Progenitors: CD34 + cells were incubated with AL or DL ( pg/ml) for 10, 20 and 30 days. After each time interval the cells were harvested and were subjected to LTC-IC assay. Significantly higher numbers of colonies were formed in AL-treated cells showing fold higher LTC-IC units than control (Figure S5 A). While DL treated cells showed 2-4 fold higher LTC-IC units as compared to control at all time intervals (Figure S5 B) A. B. CONTROL AL100pg/ml AL200pg/ml 10 8 CONTROL DL100pg/ml DL200pg/ml FOLD CHANGE 3 2 FOLD CHANGE DAYS 20 DAYS 30 DAYS (N=8) (N=5) (N=9) 0 10 DAYS 20 DAYS 30 DAYS (N=8) (N=4) (N=4) Figure S5: LTC-IC assay was carried out after 10, 20 and 30 days of incubation with DL. A: folds increase in LTC-IC units in AL treated cells and B: 2-4 fold increase in LTC-IC units was observed in DL treated cells than control ( p 0.05; p 0.01; p 0.001). CD34 + cells were incubated with AL or DL for 30 days and the cells were subjected to E- LTC-IC assay. A. B TOTAL ELT-IC UNITS / 10 4 CELLS TOTAL ELTC-IC / 10 4 CELLS CONTROL AL 100pg/ml AL 200pg/ml 0.0 CONTROL DL 100pg/ml DL 200pg/ml Figure S6: CD34+ cells were incubated either with AL or DL for 30 days and the cells were subjected to E-LTC-IC assay. E-LTC-IC units were seen only in the lectin sets.. In these samples no colonies formed in the control groups and thus statistical analyses were not possible. 194

17 As seen in the figure S6, both AL and DL showed more number of colonies as compared to control. A 1.5 and 2 fold increase in E-LTC-IC units was observed in CD34 + cells incubated with AL and DL respectively. V] Correlation of Lectin-Induced Adipogenesis with the Blood Group of the Cord Blood Sample used: We found very consistent observation of formation of adipocytes in the CD34 + cells treated with the lectins and this effect was over and above the basal level adipogenesis that was observed in the control cultures. As mentioned in the main text, we could not get the composition of StemPro medium, as it is the proprietary formulation of the company. Lectins have been known to interact with the carbohydrate moieties of blood group antigens and therefore, we analyzed our adipogenesis data with respect to the blood group of the sample used in the experiments. In this analysis, we examined the % of samples belonging to a particular blood group that showed significant level of adipocytes formation with respect to controls. % SAMPLES SHOWING ADIPOGENESIS O +VE A +VE B +VE AB +VE BL GL Figure: CD34 + cells were incubated with BL and GL for various time intervals. The bar diagram represents the % of samples having a particular blood group showing significantly elevated level of adipocytes formation with respect to controls. The samples having A+, B+ and AB+ blood groups showed a higher propensity of adipocytes formation as compared with the samples having O group. 195

18 As seen in the figure, the samples belonging to blood group A+, B+ and AB+ blood groups showed a higher propensity of BL-induced adipogenesis (more than 80% samples showed the formation of sufficient adipocytes), while the samples having A+ and AB+ blood groups showed GL-induced adipogenesis (70 to 75%). Only 57-58% samples belonging to O blood group showed significant adipogenesis. A clear cut correlation with a particular blood group was not observed indicating that the effect of the lectins was not mediated through these antigens. 196

19 Publication 1. Hinge A.S., Bajaj M.S., Limaye L.S., Surolia A. and Kale V.P. Oral administration of mannose-specific lectins leads to an enhancement in the hematopoietic stem and progenitor cell pool of mice. Manuscript under preparation Patents Filed 1. Kale V. P, Limaye L.S., Hinge A.S. and Surolia A. A method for preservation of Human Hematopoietic Stem or Progenitor Cells. International patent filed by Department of Biotechnology, New Delhi, India. 2. Kale V. P, Limaye L.S., Hinge A.S. and Surolia A. Adipogenic Differentiation of human hematopoietic cells Induced by Mannose Binding Dietary Lectins of plant origin. Indian patent filed by NCCS, Pune, India. 197