Use of annelid haemoglobin for maintaining stem cells in the undifferentiated state

Abstract

A method for maintaining stem cells in the undifferentiated state by mixing the stem cells with at least one extracellular hemoglobin, globin or globin protomer from annelids.

Claims

1. A method for maintaining stem cells in an undifferentiated state, comprising a step of mixing said stem cells with at least one extracellular hemoglobin, globin or globin protomer from annelids in amount sufficient to maintain the stem cells in an undifferentiated state, wherein the stem cells are animal progenitor cells or animal mesenchymal stem cells, and said annelids are from the family Nereididae.

2. The method of claim 1, wherein the extracellular hemoglobin, globin or globin protomer is present in a composition comprising a buffer solution.

3. The method of claim 1, wherein the annelid is Nereis sp.

4. The method of claim 1, wherein the annelid is Nereis virens.

5. The method of claim 2, wherein the extracellular hemoglobin, globin or globin protomer is present in the composition at a concentration of between 0.001 and 10 mg/ml.

6. The method of claim 1, wherein the stem cells are human stem cells.

7. A method for preserving stem cells in the undifferentiated state, comprising a step of mixing said stem cells with a buffer solution comprising at least one extracellular hemoglobin, globin or globin protomer from annelids in amount sufficient to preserve the stem cells in an undifferentiated state, wherein the stem cells are animal progenitor cells or animal mesenchymal stem cells, and said annelids are from the family Nereididae.

8. The method of claim 7, wherein the buffer solution comprises sodium chloride, calcium chloride, magnesium chloride, potassium chloride, sodium gluconate and sodium acetate, and has a pH of between 6.5 and 7.6.

9. The method of claim 2, wherein the buffer solution comprises sodium chloride, calcium chloride, magnesium chloride, potassium chloride, sodium gluconate and sodium acetate, and has a pH of between 6.5 and 7.6.

10. The method of claim 2, wherein the extracellular hemoglobin, globin or globin protomer is present in the composition at a concentration of between 0.20 and 1.50 mg/ml.

11. The method of claim 8, wherein the buffer solution has a pH of 7.10.5.

12. The method of claim 1, wherein the stem cells are epidermal progenitor cells.

13. The method of claim 1, wherein the stem cells are maintained in an undifferentiated state for at least 13 days.

14. The method of claim 1, comprising mixing the stem cells with the at least one extracellular hemoglobin, globin or globin protomer from annelids, and maintaining the stem cells in an undifferentiated state while the stem cells are cultured in vitro.

15. A method for maintaining stem cells in an undifferentiated state, comprising a step of mixing said stem cells with at least one extracellular hemoglobin, globin or globin protomer from annelids in amount sufficient to maintain the stem cells in an undifferentiated state, wherein the stem cells are epidermal progenitor cells, and said annelids are from the family Nereididae.

16. The method of claim 1, wherein the stem cells are epidermal progenitor cells or mesenchymal stem cells.

17. The method of claim 1, wherein the stem cells are mesenchymal stem cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Determination of the percentage occupation of the CFE dishes by the clones after fixing and staining of the clones obtained after 13 days of culture, using the Image J software, for the population enriched in stem cells. The results are the means of the triplicates per condition.

(2) FIG. 2: Results of counting the number of clones present in the CFE dishes after fixing and staining of the clones obtained after 13 days of culture for the population enriched in stem cells. The results are the means of the triplicates per condition.

EXAMPLES

Materials and Methods

(3) Active Agent

(4) The extracellular hemoglobin from annelids, in this case from Nereis virens, in solution in a buffer solution comprising 90 mM of NaCl, 23 mM of Na-gluconate, 2.5 mM of CaCl.sub.2, 27 mM of Na-acetate, 1.5 mM of MgCl.sub.2, 5 mM of KCl, and at a pH of 7.10.5, which can contain between 0 and 100 mM of antioxidant of ascorbic acid and/or reduced glutathione type, is used at the tested concentrations of 0.25, 0.75 and 1.25 mg/ml. This hemoglobin is available under the reference Hemarina-M201 from the company Hemarina.

(5) CFE Clonogenicity Test (2D)

(6) Principle: The CFE clonogenic efficiency test makes it possible to evaluate the frequency of keratinocytes capable of generating a cell clone within a given population. It is considered that a strong clonogenic potential is associated with stem/progenitor cells, whereas differentiated keratinocytes lose this potentiality. This culture test consists in seeding keratinocytes at low density (from 100 to 1000 cells per dish), so as to obtain well-individualized clones, which can be easily counted and are easy to distinguish from a qualitative point of view. The culturing is carried out in medium containing serum on a feeder layer of irradiated fibroblasts.

(7) Protocol: The extracellular hemoglobin from Nereis virens (Hemarina-M201) was tested independently on various cell populations: Test on a bank of preamplified run-of-the-mill keratinocytes: immediately after thawing, the cells were seeded in a proportion of 100 cells per Petri dish under CFE conditions in the presence of Hemarina-M201 or the absence of the latter (control) in the culture medium. Test on cell populations freshly extracted from a breast skin plasty and subsequently enriched in or depleted of epidermal stem and progenitor cells: On D0, the cells were seeded in a proportion of 1000 cells per Petri dish under CFE conditions in the presence or absence (control) of Hemarina-M201 in the culture medium.

(8) On D3 and D8 of culture, the media were changed in the presence or absence (control) of Hemarina-M201 in the culture medium. The culture was continued up to D13 and then stopped and fixed and the cell clones were stained and analyzed. Each condition was tested in triplicate.

(9) Results

(10) Evaluation of Hemarina-M201 on a Population of Run-of-the-Mill Keratinocytes from Two Banks:

(11) For the first bank, the number of clones present in the CFE dishes after 13 days of culture is not significantly different between the control, Hemarina-M201 at 0.25 mg/ml, Hemarina-M201 at 0.75 mg/ml and Hemarina-M201 at 1.25 mg/ml conditions (FIG. 2). Hemarina-M201 does not therefore influence the viability of said clones.

(12) On the other hand, the percentage occupation of the CFE dishes by the clones after 13 days of culture is significantly different between the control condition and the Hemarina-M201 at 0.25 mg/ml, Hemarina-M201 at 0.75 mg/ml and Hemarina-M201 at 1.25 mg/ml conditions: it is lower for the conditions with Hemarina-M201, in a dose-dependent manner (FIG. 1). The higher the concentration of Hemarina-M201, the lower this percentage occupation of the CFE dishes.

(13) Hemarina-M201 therefore influences cell differentiation. The number of viable clones in the presence of Hemarina-M201 is the same at the various times as at time T0, but, in the absence of Hemarina-M201, the cells have differentiated, as shown by the area of occupation of the dishes, which increases.

(14) The results obtained with the other bank are similar to those obtained with the first bank.

(15) In conclusion, whatever the cell bank studied, the number of cell clones obtained does not appear to drastically change given the standard deviations.

(16) These results indicate that the Hemarina-M201 hemoglobin prevents cell differentiation of the stem cells of run-of-the-mill keratinocytes derived from the banks, while at the same time preserving their viability.

(17) Evaluation of Hemarina-M201 on Populations of Freshly Extracted Keratinocytes Enriched in or Depleted of Stem Cells and Progenitor Cells:

(18) Population Enriched in Stem Cells and Progenitor Cells:

(19) The percentage occupation of the CFE dishes by the clones after 13 days of culture is significantly lower for the Hemarina-M201 at 0.75 mg/ml and Hemarina-M201 at 1.25 mg/ml conditions, compared with the control and with Hemarina-M201 at 0.25 mg/ml.

(20) Run-of-the-Mill Population of Freshly Isolated Keratinocytes:

(21) The number of clones present in the CFE dishes after 13 days of culture is not significantly different between the control, Hemarina-M201 at 0.25 mg/ml and Hemarina-M201 at 0.75 mg/ml conditions, but is significantly higher than the Hemarina-M201 at 1.25 mg/ml condition.

(22) The percentage occupation of the CFE dishes by the clones after 13 days of culture is significantly lower for the Hemarina-M201 at 0.25, 0.75 and 1.25 mg/ml conditions, in a dose-dependent manner, compared with the control.

(23) Population Depleted of Stem Cells and Progenitor Cells:

(24) The number of clones present in the CFE dishes after 13 days of culture is not significantly different between the control and Hemarina-M201 at 0.25 mg/ml conditions. On the other hand, the Hemarina-M201 at 0.75 and 1.25 mg/ml conditions have significantly lower numbers of clones, in a dose-dependent manner.

(25) The percentage occupation of the CFE dishes by the clones after 13 days of culture is significantly lower for the Hemarina-M201 at 0.25, 0.75 and 1.25 mg/ml conditions, in a dose-dependent manner, compared with the control.

(26) In conclusion, whatever the cell population studied (run-of-the-mill population, or population enriched in or depleted of stem cells and progenitor cells), the Hemarina-M201 hemoglobin blocks the differentiation potential of the stem cells giving keratinocytes. The number of clones does not change in the presence of Hemarina-M201 and their viability remains intact.

CONCLUSION

(27) These studies show that the Hemarina-M201 hemoglobin has an action on the clonogenic potential of the cell populations tested. Hemarina-M201 prevents the differentiation of the stem cells, while at the same time preserving their viability.