USE OF PEPTIDE COMPOUNDS FOR PROMOTING SURVIVAL, GROWTH AND CELL DIFFERENTIATION

Abstract

The present invention relates to the use of peptide compounds for promoting survival and/or growth and/or differentiation of progenitor cells or stem cells.

Claims

1-17. (canceled)

18. A method for promoting survival and/or growth and/or differentiation of progenitor cells or stem cells comprising a step of culture of said progenitor cells or said stem cells in presence of at least one peptide compound comprising or consisting in: TABLE-US-00011 (SEQIDNO:1) X.sub.1-S-X.sub.2-W-S-X.sub.3-X.sub.4-S-X.sub.5, wherein: X.sub.1 is W or L-Nal2, X.sub.2 is G, V, A, S or Abu, X.sub.3 is S, D or A, X.sub.4 is C* or Abu, X.sub.5 is RS, RS-Nm, RSC*-G, R-Abu-Nm, R-Abu-C*-G, K-iPr-S-Nm or K-iPr-SC*-G, Abu corresponds to a 2-aminobutyric acid residue, iPr corresponds to an isopropyl-lysine residue, Nal2 corresponds to a 2-naphthylalanine, Nm indicates that the COOH end has been replaced by a NH.sub.2 end, * indicates that when both X.sub.4 and X.sub.5 represent or comprise a cysteine residue said cysteine residues can be linked by a disulfide bond.

19. The method according to claim 18, wherein said peptide compound comprises or consists in: TABLE-US-00012 (SEQIDNO:2) X.sub.1-S-X.sub.2-W-S-X.sub.3-X.sub.4-S-X.sub.5, wherein: X.sub.1 is W or L-Nal2, X.sub.2 is G, X.sub.3 is S, X.sub.4 is C* or Abu, X.sub.5 is RS, RS-Nm, RSC*-G, R-Abu-Nm, R-Abu-C*-G, K-iPr-S-Nm or K-iPr-SC*-G, Abu corresponds to a 2-aminobutyric acid residue, iPr corresponds to an isopropyl-lysine residue, Nal2 corresponds to a 2-naphthylalanine, Nm indicates that the COOH end has been replaced by a NH.sub.2 end, * indicates that when both X.sub.4 and X.sub.5 represent or comprise a cysteine residue said cysteine residues can be linked by a disulfide bond.

20. The method according to claim 18, wherein said peptide compound comprises or consists in: TABLE-US-00013 (SEQIDNO:3) W-S-G-W-S-S-[C/Abu]-S-R-S.

21. The method according to claim 18, wherein said peptide compound is selected from the group consisting of: TABLE-US-00014 (SEQIDNO:4) W-S-G-W-S-S-C-S-R-S, (SEQIDNO:5) W-S-G-W-S-S-C-S-R-S-Nm, (SEQIDNO:6) W-S-G-W-S-S-C-S-R-S-C-G, (SEQIDNO:7) W-S-G-W-S-S-C.sup.S-S-R-S-C.sup.S-G, (SEQIDNO:8) W-S-G-W-S-S-C-S-R-Abu-Nm, (SEQIDNO:9) W-S-G-W-S-S-C-S-R-Abu-C-G, (SEQIDNO:10) W-S-G-W-S-S-C.sup.S-S-R-Abu-C.sup.S-G, (SEQIDNO:11) W-S-G-W-S-S-C-S-K-iPr-S-Nm, (SEQIDNO:12) W-S-G-W-S-S-C-S-K-iPr-S-C-G, (SEQIDNO:13) W-S-G-W-S-S-C.sup.S-S-K-iPr-S-C.sup.S-G, (SEQIDNO:14) W-S-G-W-S-S-Abu-S-R-S, (SEQIDNO:15) W-S-G-W-S-S-Abu-S-R-S-Nm, (SEQIDNO:16) W-S-G-W-S-S-Abu-S-R-S-C-G, (SEQIDNO:17) W-S-G-W-S-S-Abu-S-R-Abu-Nm, (SEQIDNO:18) W-S-G-W-S-S-Abu-S-R-Abu-C-G, (SEQIDNO:19) W-S-G-W-S-S-Abu-S-K-iPr-S-Nm, (SEQIDNO:20) W-S-G-W-S-S-Abu-S-K-iPr-S-C-G, (SEQIDNO:21) L-Nal2-S-G-W-S-S-C-S-R-S, (SEQIDNO:22) L-Nal2-S-G-W-S-S-C-S-R-S-Nm, (SEQIDNO:23) L-Nal2-S-G-W-S-S-C-S-R-S-C-G, (SEQIDNO:24) L-Nal2-S-G-W-S-S-C.sup.S-S-R-S-C.sup.S-G, (SEQIDNO:25) L-Nal2-S-G-W-S-S-C-S-R-Abu-Nm, (SEQIDNO:26) L-Nal2-S-G-W-S-S-C-S-R-Abu-C-G, (SEQIDNO:27) L-Nal2-S-G-W-S-S-C.sup.S-S-R-Abu-C.sup.S-G, (SEQIDNO:28) L-Nal2-S-G-W-S-S-C-S-K-iPr-S-Nm, (SEQIDNO:29) L-Nal2-S-G-W-S-S-C-S-K-iPr-S-C-G, (SEQIDNO:30) L-Nal2-S-G-W-S-S-C.sup.S-S-K-iPr-S-C.sup.S-G, (SEQIDNO:31) L-Nal2-S-G-W-S-S-Abu-S-R-S, (SEQIDNO:32) L-Nal2-S-G-W-S-S-Abu-S-R-S-Nm, (SEQIDNO:33) L-Nal2-S-G-W-S-S-Abu-S-R-S-C-G, (SEQIDNO:34) L-Nal2-S-G-W-S-S-Abu-S-R-Abu-Nm, (SEQIDNO:35) L-Nal2-S-G-W-S-S-Abu-S-R-Abu-C-G, (SEQIDNO:36) L-Nal2-S-G-W-S-S-Abu-S-K-iPr-S-Nm, and (SEQIDNO:37) L-Nal2-S-G-W-S-S-Abu-S-K-iPr-S-C-G, wherein .sup.s indicates that cysteine residues are linked by a disulfide bond.

22. The method according to claim 18, wherein said peptide compound is: TABLE-US-00015 (SEQIDNO:6) W-S-G-W-S-S-C-S-R-S-C-G.

23. The method according to claim 18, wherein said peptide compound is: TABLE-US-00016 (SEQIDNO:7) W-S-G-W-S-S-C.sup.S-S-R-S-C.sup.S-G, wherein s indicates that cysteine residues are linked by a disulfide bond.

24. The method according to claim 18, wherein said peptide compound is: TABLE-US-00017 (SEQIDNO:15) W-S-G-W-S-S-Abu-S-R-S-Nm.

25. The method according to claim 18, wherein said progenitor cells and said stem cells are mammal cells.

26. The method according to claim 25, wherein said cells are human cells.

27. The method according to claim 18, wherein said stem cells are mesenchymal stem cells.

28. The method of claim 18, wherein said stem cells are embryonic stem cells.

29. The method according to claim 27, wherein said mesenchymal stem cells have been obtained from bone marrow, umbilical cord blood, fat tissue, dental pulp or muscle.

30. The method according to claim 18, wherein said peptide compound is used to promote differentiation of progenitor cells or stem cells into neural cells.

31. The method according to claim 18, wherein said peptide compound is selected from the group consisting of: TABLE-US-00018 (SEQIDNO:6) W-S-G-W-S-S-C-S-R-S-C-G, (SEQIDNO:7) W-S-G-W-S-S-C.sup.S-S-R-S-C.sup.S-G, and (SEQIDNO:15) W-S-G-W-S-S-Abu-S-R-S-Nm, wherein .sup.s indicates that cysteine residues are linked by a disulfide bond, for promoting differentiation of mesenchymal stem cells into neural cells.

32. The method according to claim 18, wherein said progenitor cells or said stem cells are grown in a culture medium containing the peptide compound, said culture medium further containing at least one growth factor.

33. The method of claim 32, wherein said growth factor is selected from the group consisting of bFGF, LIF, Activin-A, TGF, Wnt, Oct-4, Sox-2, BMP-4, Nanog, Shh, PDGF, retinoic acid, EGF, IGF1, IGF2, HGF, VEGF, insulin and transferrin.

34. The method according to claim 18, wherein said progenitor cells or said stem cells are grown in a culture medium containing the peptide compound, said culture medium further containing 0 to 20% of Fetal Bovine Serum (v/v).

35. The method according to claim 18, wherein mesenchymal stem cells are grown in a culture medium containing the peptide compound, said culture medium further containing bFGF and Fetal Bovine Serum.

36. The method according to claim 35, wherein said peptide compound is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 15.

37. The method according to claim 18, wherein said progenitor cells or said stem cells are grown on a support containing the peptide compound, said peptide compound being coated on the support.

Description

DESCRIPTION OF THE FIGURES

[0144] FIG. 1. Expression of the nestin protein in ESCs cultivated on gelatine during 7 days and treated with 0.5 mg/ml NX210 peptide. (A) Control untreated culture shows a greater number of Nestin+cells (brighest spots, white arrows) compared to the NX210-treated culture (B) with few Nestin+cells (white arrows).

[0145] FIG. 2. Expression of MAP2 in ESCs after 5 days in the presence of 0.5 mg/ml NX210 peptide. MAP2.sup.+ cells (white arrows) are only observed in presence of NX210 compared to control culture where no MAP2 expressing cells have been observed.

[0146] FIG. 3. Expression of the Nestin protein showing more neuronal precursors cells (white arrows) in treated-culture (B) compared to untreated conditions (A).

[0147] FIG. 4. Cell proliferation depending of the culture conditions.

[0148] FIG. 5. Gene expression analysis performed on hMSCs from patient n.sup.o 1 grown in presence of bFGF and ATRA.

[0149] FIG. 6. Gene expression analysis performed on hMSCs from patient n.sup.o 1 grown in presence of bFGF 10% without ATRA.

[0150] FIG. 7. Gene expression analysis performed on hMSCs from patient n.sup.o 2 grown in presence of bFGF 10% without ATRA.

[0151] FIG. 8. Immunocytochemical analysis of hMSCs after 12 days of culture in the different conditions.

[0152] FIG. 9. Fluorescence intensity of MAP-2 and Nestin immunolabelling.

EXAMPLES

Example 1: Early Differentiation

[0153] Materials and Methods

[0154] 129S v/J embryonic stem cells (ESCs) were seeded at a concentration of 0.710.sup.6 cells in 35 mm diameter Petri dishes covered with a gelatin substrate.

[0155] The culture medium used was the Glasgow's minimal essential medium (G-MEM) supplemented with N2 supplement.

[0156] To enable differentiation of ESCs into neuroepithelial progenitors, the culture medium was deprived of LIF (Leukemia Inhibitory factor) because LIF is a key factor of the self-renewal of ESCs.

[0157] The various tests were carried out in absence of serum because serum limits the possibility to specifically orientate the differentiation and it could hide, or at least reduce, the effects of the peptide compound of the invention.

[0158] ESCs were grown in the culture medium as defined above with addition of 0.5 mg/ml NX210 (SEQ ID NO: 6) at day 1, 3 and 5.

[0159] Then, cultures were fixed with 4% paraformaldehyde after 7 days of culture.

[0160] To analyze the differentiation of ESCs into neuroepithelial progenitors and into differentiated neurons, the various cell types were visualized by immunocytochemistry: [0161] using a primary anti-nestin antibody (nestin=intermediate filament protein), specific of neuroepithelial progenitors, and [0162] using a primary anti-MAP2 antibody (MAP2=microtubule associated protein 2), specific of dendrites, and thus specific of differentiated neurons.

[0163] The total number of cells was measured by labelling nuclei using bisbenzimide/Hoechst 33342, a fluorescent dye with an affinity for DNA that is commonly used to color nuclei in blue.

[0164] Results

[0165] After 7 days of culture, it has been observed that the population of ESCs expressing the nestin protein is reduced in the culture in the presence of NX210 (SEQ ID NO: 6) compared to the population of ESCs cells expressing the nestin protein in the culture in the absence of NX210 (SEQ ID NO: 6) (FIG. 1).

[0166] Indeed, in the absence of NX210 (SEQ ID NO: 6), it is observed some cells expressing the nestin protein (characteristic of neuroepithelial progenitors) but no cells expressing the MAP2 protein (that means no differentiated neurons), whereas in the presence of NX210, it is observed some cells expressing the MAP2 protein (FIG. 2).

[0167] Thus, the differentiation has reached rapidly a more advanced stage in the presence of NX210 (SEQ ID NO: 6).

[0168] These results demonstrate that NX210 (SEQ ID NO: 6) induces a preferential orientation of ESCs to the neuronal differentiation pathway in terms of kinetics.

Example 2: Selection of Neuroepithelial Progenitors

[0169] Materials and Methods

[0170] For these experiments, the differentiation of ESCs was preferably orientated to the neuronal differentiation pathway, according to the protocol adapted from Okabe et al., (Mechanisms of Development, 59:89-102, 1996).

[0171] In brief, to start the differentiation, the ESCs were grown on gelatin without LIF for two days. The cells were carefullly trypsinated to collect small ESC aggregates. Then, these aggregates were transferred on a Petri dish without any coating to produce non-adherent spherical cell bodies, namely the embryoid bodies (EB).

[0172] After 5 days of culture, the EB were transferred onto a new Petri dish covered with gelatin to enable cell adhesion.

[0173] After 24h of culture, the culture medium with fetal bovine serum 10% was replaced by a culture medium serum free to select neuroepithelial progenitors and the peptide NX210 (SEQ ID NO: 6) was added in the culture medium at a concentration of 0.5 mg/ml.

[0174] After 6 days of culture, the maximal content of cells expressing the nestin protein (a cytoskeletal protein specific of the neuroepithelial progenitors) was reached.

[0175] The cell population enriched for neuroepithelial progenitors was then trypsinated and seeded at a concentration of 110.sup.5 cells/cm.sup.2 on a substrate made up of a polyornithine and laminin mix.

[0176] After 2 days of culture in the presence of FGF2 (Fibroblast Growth Factor 2) to develop the pool of precursors, the cells were fixed and treated prior to Nestin immunostaining.

[0177] Results

[0178] More cells expressing the Nestin protein have been observed in the culture treated with NX210 (SEQ ID NO: 6) in comparison to the untreated culture (FIG. 3).

[0179] The production of a differentiation marker such as Nestin in the presence of NX210 (SEQ ID NO: 210) suggests an accelerated differentiation of ESCs into the neuronal pathway.

[0180] Thus, during the selection of the neuroepithelial progenitors, NX210 promotes a more efficient response to the specific stimulation by FGF2.

Example 3: Neural Differentiation of Human Mesenchymal Stem Cells

[0181] Materials and Methods

[0182] Adult human bone marrow mononuclear cells Ficoll-extracted from two donors were obtained from Lonza. Cells were passaged once a week. The human mesenchymal stem cells (hMSC) were selected by plastic adherence and expanded by plating 50 000 cells/cm.sup.2 in Modified Eagle Medium Alpha (MEMalpha, MacoPharma) supplemented with 10% Fetal Bovine Serum (FBS, PAA Laboratoires) and 0.1% Penicilline/Streptomycine (P/S, Lonza). The culture medium was changed after 3 days and then every day. After 2 to 5 weeks of culture at 37 C. in a humid environment and under 5% CO.sub.2, hMSC were harvested by trypsinization. The phenotype was confirmed by FACS analysis (BD Bioscience, BD LSR II) with CD73, CD90 and CD105 (positive), as well as CD34 and CD45 (negative) (Table 1).

TABLE-US-00008 TABLE 1 Immunophenotyping of hMSCs obtained from the two donors. Differences observed in the two patients are standard inter-individual variability. Cell surface marker Patient no1 Patient no2 CD105 42.5% 40.4% CD73 57.9% 86.8% CD90 58.3% 87.0% CD34 2.8% 11.5% CD45 12.8% 41.6%

[0183] Moreover, the self-renewal capacity of cells was confirmed by the colony forming unitsfibroblast assay (CFU-F). For this assay, hMSCs were seeded at two different densities: 20,000 cells/cm.sup.2 and 60,000 cells/cm.sup.2 in 25 cm.sup.2 flasks with the complete medium described above. Seven and 10 days later, the number of CFU-F colonies with more than 50 cells (large colonies), less than 25 cells (small colonies) and between 25 and 50 cells (medium size colonies) were counted.

TABLE-US-00009 TABLE 2 Results of the CFU-F assay. Average count of CFU-F at J7 Average count of CFU-F at J10 Number of <25 25-50 >50 <25 25-50 >50 Total at D 10 plated cells P1 P2 P1 P2 P1 P2 P1 P2 P1 P2 P1 P2 P1 P2 0.5 .Math. 10.sup.6 13 55 5.5 10.5 2.5 2 10 17.5 6.5 14.5 15 40.5 31.5 72.5 1.5 .Math. 10.sup.6 42.5 106.3 11 79 1 6 21.5 28 16.5 30 30 65 68 123 From D 7 to D 10, sample from patient no 2 forms a higher number of CFU-F than from patient 1 for each type of colony (small, medium, and large). At D 10, the total number of CFU-Fs is greater for patient no2 suggesting better proliferative capacities. Average count of CFU-F = measurements averaged over 2 to 3 counting. P1 = Patient no1. P2 = Patient no2.

[0184] To investigate the neural differentiation capacity of the peptides, 25 cm.sup.2 flasks and 12-wells culture plates were coated with sterile aqueous solutions of either NX210 or NS640 at a concentration of 80 g/cm.sup.2. Coatings were carried out at 37 C. and 5% CO.sub.2 over-night prior to cell plating.

[0185] On Day 0 (D0), hMSC were seeded at a concentration of 3,000 cells/cm.sup.2 in pre-coated 25 cm.sup.2 flasks or 12-well culture plates in complete medium and culture was placed at 37 C. in a humid environment and under 5% CO.sub.2. After 24h, medium was changed and replaced by different combinations of media containing either: MEMalphabFGF 12.5 ng/mLFBS 2%P/S 0.1%; MEMalphabFGF 12.5 ng/mLFBS 10%P/S 0.1%; MEMalphaFBS 2%P/S 0.1% (control condition); MEMalphaFBS 10%P/S 0.1% (control condition). The cells were placed at 37 C. in a humid environment and under 5% CO.sub.2, and medium was changed twice a week. On Day 8, medium was replaced by medium containing ATRA in the place of bFGF and thus was changed with: MEMalphaATRA 201 MFBS 2%P/S 0.1%; MEMalphaATRA 20 MFBS 10%P/S 0.1%; MEMalphaFBS 2%P/S 0.1% (control condition); MEMalphaFBS 10%P/S 0.1% (control condition). The cells were placed at 37 C. in a humid environment and under 5% CO.sub.2. Medium was changed twice a week. On Day 15 (D15), cells were harvested by trypsinization for mRNA extraction or fixed immunocytochemical analysis.

[0186] Assessment of differentiation was performed by gene expression after extraction of mRNA at various time point (DO, D1, D8 and D15), and immunocytochemical analysis performed at the end of the culture period (D15).

[0187] Immunocytochemical analyses were carried out on cells fixed in 4% paraformaldehyde and using standard protocols. After blocking with Triton 10.1%/BSA 5% during 30 min, cells were incubated with antibodies directed against Nestin (Millipore) or MAP-2 (Sigma) overnight at +4 C. After washing, appropriate FITC or Cyandidine-5 labelled secondary antibody are added. Nuclei were counterstained with Hoescht 33258 (Sigma). Obervations were performed using Leica TCS LSI system.

[0188] Total RNA was extracted using RNAeasy total purification kit following instructions of the manufacturer (Qiagen). cDNA were generated using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) as described by the manufacturer.

[0189] The expression levels of genes (GFAP, Musashi 1, Nestin, Beta3-Tubuline, MAP-2 and GAPDH) were analyzed by real-time PCR using TaqMan gene Expression Master Mix (Applied Biosystems) on the Applied Biosystems 7900 Real time PCR system (Life technologies). Samples were analyzed in triplicates. Data were normalized using the GAPDH gene as an internal control and relative quantification (RQ) of the genes in a sample was determined according to the equation 2.sup.ct.

[0190] Results

[0191] Despite inter-patient variations, number of MSCs is increased in presence of 10% FBS compared to 2% FBS. bFGF has a positive effect on cell proliferation whatever the FBS concentration. Moreover, add-on of ATRA (from J5) leads to small difference in the presence of 2% FBS and a decrease of the number of MSC in presence of 10% FBS (FIG. 4). NX210 or NS640 have no effect on proliferation whatever the conditions tested.

[0192] The protocols tested do not seem to favor a differentiation of MSCs in the glial pathway. The expression of GFAP was therefore not quantified subsequently.

[0193] Sequential differentiation by bFGF and then ATRA (from J5) in the presence of 2% FBS leads to an increase in the gene expression of Nestin (6.7 vs 1.8), III-Tubulin (22 vs 9.2) and MAP-2 (9.9 vs 3.5) compared to control conditions. NX210 or NS640 does not modify this increase. In the presence of 10% FBS, the increase in expression is less marked. NX210 and NS640 have a moderate effect that is observed by increasing MAP-2 and Nestin expression (1.21 and 1.46 vs 0.75, 2 and 1.5 vs 1.2 respectively) (FIG. 5).

[0194] For cells from patient n.sup.o 1, culture in presence of bFGF leads to a decrease in Musashi-1 and Nestin expression and to an increase in expression of III Tubulin and MAP-2 (respectively 9.3 and 2.3 with respect to the JO). At day 5, presence of NX210 and NX640 lead to an increase of Nestin expression compred to bFGF alone (2 vs 0.6 in the bFGF condition without NX210 or NX640 treatment) (FIG. 6).

[0195] Similarly to patient n.sup.o 1, culture of hMSCs from patient n.sup.o 2 in presence of bFGF leads to a decrease in Musashi-1 and Nestin expression and to an increase in expression of (3111 Tubulin and MAP-2 (respectively 9.3 and 2.3 with respect to the JO). Presence of NX210 leads to an increase of Nestin expression compred to bFGF alone (1.95 vs 0.6 in the bFGF condition without coating). Moreover, III-Tubuline is also maximized in presence of NX210 or NX640 after 5 days of culture. Whereas MAP-2 expression is decreased in with conditions bFGF alone, expression of this gene was increased at D5 in presence of NX210 (FIG. 7).

[0196] The results of the gene expression for Musashi-1, Nestin, III-Tubulin and MAP-2 are summarized in Table 3.

TABLE-US-00010 TABLE 3 Induction of gene expression. Musashi-1 Nestin III-Tubulin MAP-2 P1 P2 P1 P2 P1 P2 P1 P2 2% bFGF + ++ + + 3 FBS NX210 + bFGF ++ + ++ + + NS640 + bFGF ++ + + 10% bFGF + ++ + + + FBS NX210 + bFGF + + + ++ + + + NS640 + bFGF + + + + + P1 = Patient no1. P2 = Patient no2.

[0197] The addition of bFGF in the presence of 2% FBS increases the expression of Nestine and MAP-2 markers. In this condition, immunocytochemistry analysis showed a stronger staining when NX210 or NS640 are present. In 10% FBS condition, bFGF increases Nestine expression compared to the control condition, and this increase is maximized with NX210 or NX640 (increase of staining intensity). Only presence of NX210 and NX640 makes possible to observe MAP-2 protein presence (FIG. 8).

[0198] Gradations in the staining intensity corresponding to the expression of Nestin or MAP-2 were observed depending on the culture conditions (FIG. 9).