WO2011134707A1

WO2011134707A1 - Method for modifying the proliferative activity and differentiation capacity of multipotent mesenchymal stromal cells

Info

Publication number: WO2011134707A1
Application number: PCT/EP2011/053368
Authority: WO
Inventors: Elena Romualdovna Andreeva; Liudmila Borisovna Buravkova; Anatoly Ivanovich Grigoriev; Umberto Orazio Giuseppe Maugeri; Yulia Vladimirovna Rylova
Original assignee: State Scientific Center Of The Russian Federation Institute Of Biomedical Problems Of The Russian Academy Of Sciences; Fondazione Salvatore Maugeri Clinica Del Lavoro E Della Riabilitazione; Sib Laboratories Limited
Priority date: 2010-04-26
Filing date: 2011-03-07
Publication date: 2011-11-03
Also published as: RU2418066C1

Abstract

The present invention concerns the field of stem cells and in particular regards an in vitro method for expanding a population of stem cells, while at the same time inhibiting their differentiation.

Description

METHOD FOR MODIFYING THE PROLIFERATIVE ACTIVITY AND DIFFERENTIATION CAPACITY OF MULTIPOTENT MESENCHYMAL STROMAL CELLS

Field of the invention

The present invention concerns the field of stem cells and in particular regards an in vitro method for expanding a population of stem cells, while at the same time inhibiting their differentiation.

State of the art

Invention belongs to the biotechnology field and relates to progenitor cell cultures.

In order to obtain the expansion of progenitor cells, cytologists have been faced with the problem of obtaining the required amount of multipotent mesenchymal stromal cells (MMSCs) within a short time period with low heterogeneity and high viability. These MMSC cells are required for applications related to regenerative medicine.

Mesenchymal stromal precursor cells may be isolated from different tissues. They present a very small cell population characterized by a large proliferative potential, the ability of self-renewal by maintaining the undifferentiating state, and also the possibility of differentiating into several cell types under the influence of specific stimuli. The basis of their reparative potential lies in the ability of MMSCs to differentiate at least into the tissues of mesenchymal origin.

Up to now, bone marrow has been considered as the main source of stem cells of the adult organism. Bone marrow contains hemopoietic stem cells and their more committed progeny as well as stroma with the so called MMSCs or precursor cells of adult organism (Caplan Al. Mesenchymal stem cells. J Orthop Res. 1991 #9 Vol.5, p.641 -650; Friedenstein AJ. Precursor cells of mechanocytes. Int. Rev. Cytol. 1976 #47, p.327-359).

Presently it has been proved that MMSCs exist not only in the bone marrow, but practically in all of the organism's tissues, for example, in skin, fat tissue, in the epithelium of the small intestine, and other tissues (Zuk, P.A., Zhu, M., Mizuno H., et al., Multiliniage cells from human adipose tissue: implications for cell-based therapies. // Tissue Eng. - 2001 -Vol.7 - P. 21 1 -226; Zuk PA, Zhu, M., Mizuno H. et al Human adipose tissue is a source of multipotent stem cells. // Molecular biology of the cell - 2002 - Vol.13 - P.4279-4295).

Adipose tissue is considered as one of the alternatives to bone marrow for gathering MMSCs for their further use in therapeutic purposes. Subcutaneous fat, like bone marrow, is a derivative of mesenchyma and contains stroma which could be easily isolated. Besides, the procedure of sampling of fat tissue is less traumatic and more easily accepted by patients than the removal of a sample of bone marrow. Many researchers have shown that cells isolated during enzyme treatment of fat tissue and further expanded in vitro are able to differentiate into different cell types under the influence of corresponding stimuli (Zuk PA, Zhu, M., Mizuno H. et al Human adipose tissue is a source of multipotent stem cells. // Molecular biology of the cell - 2002 - Vol.13 - P. 4279- 4295; Katz AJ, Tholpady A, Tholpady SS, Shang H, Ogle RC. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells. Stem Cells. 2005 # 23 Vol.3, p.412-423; Ryden M, Dicker A, Gotherstrom C, Astrom G, Tammik C, Amer P, Le Blanc K. Functional characterization of human mesenchymal stem cell-derived adipocytes. Biochem Biophys Res Commun. 2003 #31 1 Vol.2. p391 -397). The obtained data prove that MMSCs isolated from adipose tissue and cultured in vitro could be used in regenerative medicine. For this purpose, the methods of MMSCs isolation differ for different laboratories. The method proposed in the publication by Zuk is the most popular (Zuk PA, Zhu, M., Mizuno H. et al Human adipose tissue is a source of multipotent stem cells. // Molecular biology of the cell - 2002 - Vol.13 - P. 4279-4295) for MMSCs isolation from fat tissue. This method includes the lipoaspiration of the tissue, its processing by collagenase, to obtain a cellular pellet which is washed, centrifugated and plated onto conventional tissue culture plates in control medium. The cells obtained during extraction from fat are called multipotent mesenchymal stromal cells isolated from lipoaspirate or from adipose tissue (hereinafter - AT- MMSCs).

AT-MMSCs have a very high potential in regenerative medicine but the problem to be overcome is that of accumulating a sufficiently large cell population with a high level of progenitor characteristics in a short period of time..

It is therefore object of the present invention the development of a method for the modification of the proliferative activity of non-differentiated AT-MMSC cells which results in the accumulation of these cells.

Summary of the invention

The present invention concerns an in vitro method for expanding a population of stem cells, while at the same time inhibiting the differentiation of the stem cells, comprising the steps of:

a) pre-cultivation of the stem cells under hypoxia conditions and, b) cultivation of the stem cells under more stringent hypoxia conditions.

As will be further described in the detailed description of the invention, the method of the present invention has the advantages of obtaining a large number of non-differentiated stem cells in a short period of time.

Brief description of the drawings

The characteristics and advantages of the present invention will be apparent from the detailed description reported below, from the Examples given for illustrative and non-limiting purposes, and from the annexed Figures 1 -3, wherein:

Figure 1 : shows the growth of N-AT-MMSC and Hyp-AT-MMSC incubated in the medium with decreased 0₂ as described in Example 2, where:

A. Number of population doublings within 4 passages in N-AT-MMSCs, 20% and N-AT-MMSCs cultures under decreased 0₂ content.

B. Number of population doublings within 4 passages in Hyp-AT-MMSC, 5% and Hyp-AT-MMSC cultures under decreased 0₂ content. The number of population doublings was determined at the end of each passage.

Figure 2: shows the growth of AT-MMSCs cultured in a medium with different 0₂ content, at 1 -4 passages as described in Example 3.

Figure 3: shows the immunophenotype of AT-MMSCs cultured under decreased oxygen content in the medium as described in Example 3.

Detailed description of the invention

In the present invention, "expanding a population of cells" is intended in terms of cell growth and is used in the contexts of cell development and cell division (reproduction). When used in the context of cell division, it refers to growth of cell populations, where one cell (the "mother cell") grows and divides to produce two "daughter cells".

The purpose of invention is to speed-up the getting of low-differentiated AT- MMSCs by the modification of proliferative activity and differentiation capacity of these cells by means of pre-incubation of AT-MMSC in hypoxic gaseous phase.

Composition of gaseous phase is one of the most important factors determining the viability of cells. In modern research practice incubation of cells is usually performed with use of medium in which content of oxygen is equal to content of oxygen in atmosphere. Decrease of oxygen level in the medium allows making the incubation conditions close to physiological ones. The technical result is the speeding-up of the AT-MMSC mass accumulation by means of increased proliferative activity, without loss of cells viability.

The stem cells according to the present invention can be selected from the group consisting in hematopoietic cells, neural cells and oligodendrocyte cells, skin cells, hepatic cells, muscle cells, bone cells, pancreatic cells, chondrocytes, stroma cells, mesenchymal cells, and multipotent mesenchymal stromal stem cells (MMSCs).

According to a preferred embodiment the in vitro method according to the present invention is a method for expanding while at the same time inhibiting the differentiation of a population of multipotent stem cells.

According to a more preferred embodiment the in vitro method according to the present invention is a method for expanding while at the same time inhibiting the differentiation of a population of multipotent mesenchymal stromal stem cells (MMSCs).

A further advantage of the in vitro method according to the present invention is a method for expanding while at the same time inhibiting the differentiation of a population of stem cells, wherein said stem cells derive from bone marrow. A still further advantage of the in vitro method according to the present invention is a method for expanding while at the same time inhibiting the differentiation of a population of stem cells, wherein said stem cells derive from adipose tissue.

A still further advantage of the in vitro method according to the present invention is a method for expanding while at the same time inhibiting the differentiation of a population of stem cells which derive from adipose tissue, wherein said adipose tissue is subcutaneous fat.

In a further aspect, the invention provides an in vitro method which is a method for expanding while at the same time inhibiting the differentiation of a population of stem cells comprising the steps of:

a) pre-cultivation of the stem cells under hypoxia conditions and, b) cultivation of the stem cells under more stringent hypoxia conditions, wherein said pre-cultivation of the stem cells of step a) is under hypoxia conditions with 5% 0₂.

a) pre-cultivation of the stem cells under hypoxia conditions and, b) cultivation of the stem cells under more stringent hypoxia conditions, wherein said cultivation of the stem cells of step b) is under hypoxia conditions with 1 -3% 0₂.

a) pre-cultivation of the stem cells under hypoxia conditions and, b) cultivation of the stem cells under more stringent hypoxia conditions, wherein said cultivation of the stem cells of step b) is under hypoxia conditions with 1 % 0₂.

a) pre-cultivation of the stem cells under hypoxia conditions and, b) cultivation of the stem cells under more stringent hypoxia conditions, wherein said cultivation of the stem cells of step b) is within 1 -2 passages. EXAMPLES

Example 1.

AT-MMSCs extraction from lipoaspirate

MMSCs were isolated from human lipoaspirate, obtained after lipoaspiration in patients under clinical supervision. After extraction the material was stored in a refrigerator at 4°C.

Materials:

Sterile test-tubes, 50 ml (Nunc, Denmark)

Sterile pipettes, 10 and 25 ml (Nunc, Denmark)

Cell filters, sterile, 100 nm (Nunc, Denmark)

Petri dishes, 60 and 90 mm, sterile, (Nunc, Denmark) Culture flasks, 25 cm² and 75 cm², (Nunc, Denmark)

Non-sterile tubes for flow cytometer

Sterile tips for 200-1000 μΙ (Eppendorf, Germany)

Growth culture medium: DMEM with low content of glucose (1 mg/ml), penicillin

100 u/ml, streptomycin 100 mg/l, amphotericin B 50 mg/ml, L-glutamine 2 mM, sodium bicarbonate 1 g/l

Fetal bovine serum (FBS) (Hyclone, USA)

Trypsin- EDTA 0,25-0,04% (Gibco, UK)

D-PBS (Gibco, UK)

Collagenase IA (Sigma-Aldrich, USA)

Complete culture medium: growth medium +10% FBS

Equipment:

Eppendorf 5804R centrifuge

Safety cabinet (Sampo, Russia)

Waterbath (Elmi, Latvia)

Weigh-scales (Ohaus, Germany)

Pippete-aid

Automatic pipettes, set (Eppendorf, Germany)

Inverted microscope, phase-contrast (Leica, Germany)

Flow cytometer BeckmanCoulter Epix XL (BeckmanCoulter, USA)

C0₂-incubator (Sanyo, Japan)

Standard conditions of incubation: 5% C0₂ + 20% 0₂ + 75% N₂, 37°C, 100% humidity.

Multigas incubator (Sanyo, Japan)

Hermetic camera for different gaseous mixtures (5% C0₂ + 1 -3-5% 0₂ + 90-

94% N₂) (StemmCell Technologies, USA)

Procedure:

Lipoaspirate was put into the 50-ml tube (about 1/3 of tube volume) and, after adding of 50 ml D-PBS, gently shaken.

It was centrifuged for 5 minutes at 1500 rpm and 18°C. The erythrocytes pellet can be identified at the bottom of the tube, above this pellet the debris - buffer layer and then lipoaspirate. Lipoaspirate is covered by fat layer from destroyed adipocytes.

Lipoaspirate was carefully transferred to the clean sterile tube (50 ml), and then tissue was repeatedly washed with centrifugation under the following conditions: 10 minutes, 1000 rpm, 18°C.

Lipoaspirate was carefully transferred to the preliminary weighed 50 ml sterile tube, weighed and then the collagenase IA solution was added.

Enzyme solution was added to the preliminary weighed lipoaspirate up to the final concentration 0,075%.

It was incubated at waterbath for 30 minutes at 37 ^<€, with periodical shaking - once every 5 minutes.

Collagenase IA was inactivated by the addition of complete culture medium up to 50 ml.

Centrifugation for 5 minutes, 1500 rpm, at 18°C.

Supernatant was poured out and the cell pellet was re-suspended in 10 ml of complete medium bringing it to a total volume of 50 ml.

Centrifugation for 5 minutes, 1500 rpm, at 18°C.

Supernatant was poured out and the cell pellet was re-suspended in 10 ml of complete culture medium.

Cell filter was placed in the 50 ml tube, and the cell and tissue debris suspension was passed through it. The growth medium was added to the tube, up to a total volume of 50 ml.

Centrifugation for 5 minutes, 1000 rpm, at 18°C.

Supernatant was poured out and the cell pellet was re-suspended in 10 ml of growth medium.

An aliquot of medium with cells was taken, and the number of nucleated cells was calculated in hemacytometer.

Cell suspension was diluted in order to obtain a seeding density of 2χ10^5"3χ10⁵ cm². The cells were plated in the culture flasks and left in the C0₂-incubator for 24 hours.

Supernatant with unattached cells was taken, and D-PBS was washed out 2 times.

Required amount of complete medium was added.

Medium was replaced every 3 days.

Growth of cells was controlled with use of a microscope.

Cells were subcultured after 70-80% confluence was reached.

Example 2.

Cell culture at different O? conditions

Incubation conditions with lower content of oxygen in the medium were performed as following. Part of the cells just after isolation from tissue was placed into multigas incubator Sanyo (Japan) where the corresponding oxygen concentration (5%) was maintained. Cells were constantly cultured at the lower oxygen content conditions, and the kept at normal oxygen conditions not more than 30 minutes during medium replacement.

Immediately after isolation all cells were separated into 2 populations and placed in normal oxygen conditions (95% atmosphere, 5% C0₂, N-cells or N- AT-MMSCs) or hypoxia (5% 0₂, 5%C0₂, 90% N₂, Hyp-cells, Hyp-AT-MMSCs). Cell culture and subculture after isolation were constantly performed at the higher atmosphere conditions.

Comparative study of proliferative activity, viability and immune phenotvpe

AT-MMSCs were harvested under oxygen stress 5%, 3% and 1 % after preliminary expansion of cells under 20% and 5% O₂.conditions. The comparative assessment of proliferative activity was performed within 4 passages (from 1 st to 4th).

Pre-cultivation in 5% 0₂ is performed for enrichment of cell population with poorly differentiated precursor cells just after isolation of MMSCs, before reaching the monolayer by cells.

Table 1 . shows the cell culturing conditions of the experiments performed with different 0₂ concentrations in the gaseous phase of culture medium on the morpho-functional characteristics of AT-MMSCs.

AT-MMSCs were isolated according to the method described above. The isolated cells were pre-cultivated in the corresponding conditions (20% or 5% 0₂) and N-AT-MMSCs or Hyp-AT-MMSCs were obtained accordingly.

Cells were then divided into two batches: cells of the first batch were subcultured at initial conditions (20% or 5% 0₂), and cells of the second batch were placed at lower 0₂ content conditions according to the Table 1 .

The number of cells was estimated during 4 passages at all different AT- MMSCs cultivation conditions by calculating the cell population doublings within each passage, and then this data was summed up to calculate the cell population doublings for all period of culturing.

The results show that the number of N-AT-MMSCs at 20% and the number of the same cells placed into the hypoxic conditions (Fig. 1 a) reveal that proliferative activity of AT-MMSCs at lower 0₂ was increased 1 ,5-2 times. The cell number of Hyp-AT-MMSCs 5%, Hyp-AT-MMSCs 3% and Hyp-AT-MMSCs 1 % was approximately the same, but was more than twice increased in compare with N-AT-MMSCs 20% (Fig. 1 b). On the whole, the number of Hyp- AT-MMSCs was higher than in N-AT-MMSCs transferred to the hypoxic conditions. Example 3

The number of population doublings was determined at the end of each passage and then this data was summed up. The results of the comparison population doublings of N-AT-MMSCs 20%, Hyp-AT-MMSCs 5% and the same cells under lower 0₂ conditions within each of the studied passages are shown on Fig. 2.

As it can be seen from the diagrams shown on fig. 2, the increase in cell number of N-AT-MMSCs 20% was not high and did not differ in 1 -3 passages. In the 2-3 passages considerable increase in cell number was observed in Hyp-AT-MMSCs 5% cultures, and also in N-AT-MMSCs 20% and Hyp-AT- MMSCs 5% cultures placed into lower 0₂ conditions. The population doublings under 3% and 1 % 0₂ in 1 -3 passages exceeded this value in AT- MMSC cultures under 5% 0₂, and in 4th passage was 1 ,5 - 2 times lower than under 5% 0₂.

The results of assessment of viability of AT-MMSCs cultured at different 0₂ concentrations are shown on Tables 2 and 3.

Table 2. Viability of AT-MMSCs cultured at different 0₂ concentrations after the first harvesting passage.

Table 3. Viability of AT-MMSCs cultured at different 0₂ concentrations after the second harvesting passage.

N-AT- N-AT- N- AT- N- AT- Hyp- Hyp- AT- Hyp- AT- MSCs, 20% MSCs, MMSCs, MSCs, 1% AT-MSCs, MMSCs, MMSCs,l%

5% 3% 5% 3%

apoptosis 0,79 7,3 6,02 10,5 1,82 0,47 0,72 necrosis 2,39 10,2 34,08 14,39 3,54 2,56 3,36 living 96,82 82,5 59,9 75,11 94,64 96,8 95,92

The considerable decrease of the living cells were revealed after the first harvesting passage in N-AT-MMSC 1 % and after the second harvesting passage in N-AT-MMSC 3% and N-AT-MMSC 1 % populations.

Immunophenotyping of AT-MMSCs cultivated at different 0₂ concentrations in the medium did not reveal considerable differences in CD73 antigen expression (Fig. 3), in all cases from 90 to 100% of cells were CD73-positive. Expression of CD54-antigen was varying greatly on AT-MIMSCs, and no any dependence on 0₂ concentration or passage number was revealed. The portion of CD90-positive cells among AT-MMSCs cultured at 5% 0₂ within 2-4 passages was higher for sure than among Hyp-AT-MMSCs 3% and Hyp-AT- MMSCs 1 %.

Comparison of morpho-functional characteristics of AT-MMSCs cultured at 3% and 1 % 0₂ after pre-cultivation at 20% and 5% 0₂ shows the following:

- Proliferative activity of AT-MMSCs under 5, 3, 1 % 0₂ is higher than at 20% of 0₂. The most considerable increase in growth is observed in 2-3 passages. Proliferative activity of AT-MMSCs pre-cultured in 20% oxygen conditions after transfer to hypoxia (1 -3-5% 0₂) is lower than proliferative activity of AT- MMSCs pre-cultured in 5% 0₂.

- Comparison of AT-MMSCs growth during their cultivation at different lower 0₂ concentrations showed that the number of population doublings within 4 passages at 1 -3-5% 0₂ is practically equal, but at 1 % and 3% 0₂ after splash of proliferative activity in 1 -3 passages the cells growth in 4th passage abruptly decreased whereas at 5% 0₂ AT-MMSCs maintained sufficiently high proliferative activity also in further passages. - AT-MMSCs placed into the lower 0₂ conditions had high viability (90% and higher). Considerable decrease of the portion of alive cells was marked at N- AT-MMSC, 3% and N-AT-MMSC, 1 % population. It could be supposed that AT-MMSCs pre-cultivated at normal oxygen conditions were less stable to the pronounced decrease of 0₂ concentration in the medium than AT-MMSCs pre- cultivated in hypoxia.

- The most N-AT-MMSCs and Hyp-AT-MMSCs expressed mesechymal stromal cells markers: CD73 and CD90. The share of CD90-positive cells was highest among Hyp-AT-MMSCs, 5%.

After induction of adipogenic differentiation in N-AT-MMSCs and Hyp-AT- MMSCs it was revealed that decrease in 0₂ tension had slowed down the formation of lipid droplets in cultured cells and this effect had been more pronounced in 1 % and 3% 0₂.

Thus, decrease of oxygen concentration in the culture medium lower than 5% does not cause damage of AT-MMSCs functions. But obtained data allow to suppose that effects of 0₂ decreasing up to 5% and 1 -3% rather differ. AT- MMSCs reacted on the decreasing of 0₂ in cultivation medium up to 5% by way of smooth increase of cell number within 1 -3 passages with maintenance of sufficiently high proliferative activity in the further. At 5% 0₂ most of AT- MMSCs showed mesenchymal marker CD90. Besides, at common high viability of AT-MMSCs, while transferring N-AT-MMSCs into 3% and 1 % 0₂ the portion of dead cells in the population was increased greatly.

Based on the obtained results, two possible protocols of cultivation could be proposed:

To create cultivation conditions close to physiological ones which would provide stable growth of AT-MMSCs with expressed mesenchymal phenotype and high viability, it is necessary to use smooth hypoxic conditions by decreasing of 0₂ concentration up to 5%.

In those cases when it is necessary to accumulate cell mass quickly, more strict hypoxic conditions can be used within 1 -2 passages - decreasing of 0₂ up to 3% and 1 %. At this, it is necessary to take into account that AT-MMSCs pre-cultured at normoxic oxygen conditions before placing into hypoxia, have lower viability, so it is more preferable to use AT-MMSCs constantly cultivated at 5% 0₂.

The investigations showed that AT-MMSCs cultured at standard (20% 0₂) and decreased up to 5% oxygen differ in their morpho-functional characteristics. For example, AT-MMSCs constantly cultivated in the medium with lower 0₂ content have higher proliferative activity that makes the method of cells growth under decreased 0₂ very attractive from the point of view of preparation of considerable cells amount for the requirements of cells therapy and regenerative medicine.

From the above description and the above-noted examples, the advantage attained by the product described and obtained according to the present invention are apparent.

Claims

1 . An in vitro method for expanding a population of stem cells, while at the same time inhibiting the differentiation of the stem cells, comprising the steps of:

c) pre-cultivation of the stem cells under hypoxia conditions and, d) cultivation of the stem cells under more stringent hypoxia conditions.

2. The method according to claim 1 , wherein said stem cells are multipotent stem cells.

3. The method according to claim 3, wherein said stem cells are multipotent mesenchymal stromal stem cells (MMSCs).

4. The method according to claims 1 or 2, wherein said stem cells derive from bone marrow.

5. The method according to claims 1 to 3, wherein said stem cells derive from adipose tissue.

6. The method according to claim 5, wherein said adipose tissue is subcutaneous fat.

7. The method according to claims 1 to 6, wherein said pre-cultivation of the stem cells of step a) is under hypoxia conditions with 5% 0₂.

8. The method according to claims 1 to 6, wherein said cultivation of the stem cells of step b) is under hypoxia conditions with 1 -3% 0₂.

9. The method according to claims 1 to 6, wherein said cultivation of the stem cells of step b) is under hypoxia conditions with 1 % 0₂.

10. The method according to claims 8 or 9, wherein said cultivation of the stem cells of step b) is within 1 -2 passages.