WO2012049369A1

WO2012049369A1 - Marker for cells

Info

Publication number: WO2012049369A1
Application number: PCT/FI2011/050888
Authority: WO
Inventors: Saara Laitinen; Eeva-Lotta Kilpinen; Reijo KÄKELÄ
Original assignee: Suomen Punainen Risti, Veripalvelu
Priority date: 2010-10-13
Filing date: 2011-10-13
Publication date: 2012-04-19

Abstract

The present invention is related to a lipid content of a cell and its use in determination of functional quality of the cell or a cell population thereof. In particular, the invention describes lipid contents in stem cells that can be used to evaluate the functional quality, such as age, immuno-modulatory and/or angiogenic and/or osteogenic potency, of the cell population in a therapeutic cell preparation. The invention further relates to a method of detecting relative amounts of lipids and/or lipid ratios for determination of functional quality of a cell or a population thereof.

Description

Marker for cells

FIELD OF THE INVENTION

The present invention is related to a lipid content of a cell and its use in determination of functional quality of the cell or a cell population thereof. In particular, the invention describes lipid content in stem cells that can be used to characterize the functional quality, such as age, immunomodulatory and/or differentiation potency, of the cell population in a therapeutic cell preparation. The invention further relates to a method of detecting lipids and/or lipid profiles for determination of functional quality of a cell or a population thereof. BACKGROUND OF THE INVENTION

Stem cells are characterized by their ability to renew themselves through mitotic cell division and to differentiate into a diverse range of cell types. The two main types of mammalian stem cells are embryonic stem cells and adult stem cells, such as hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells and tissue-specific stem cells. Induced pluripotent stem (iPS) cells are derived from adult tissues but converted to embryonic stem cell like cells.

Hematopoietic stem cells (HSC) are pluripotent (or multipotent) cells having ability to form all the blood cell types including myeloid and lymphoid lineages. HCSs are currently used for treating certain hematological and nonhematological diseases. HSCs can be derived for example from bone marrow and cord blood.

Mesenchymal stem cells, also called as mesenchymal stromal cells, (MSC) have the potential to differentiate into many cellular lineages and can be expanded in in vitro culture without losing their multipotency. Cell types into which MSCs have been shown to be able to differentiate in vitro and/or in vivo include osteoblasts, chondrocytes and adipocytes. Therefore, they present a valuable source for applications in cell therapy and tissue engineering. MSCs can be derived, for example, from bone marrow or cord blood. The exact definitions for MSC or cell lineages differentiated thereof are currently not finally established (Da Silva Meilleres et al., 2008 Stem Cells 26: 2287-99), but an example of a current set of criteria for undifferentiated MSC is described by Dominici et al., 2006, Cyto- therapy 8: 315-317. Transplantation of MSC offers a promising approach for treating certain nonhematological malignant and nonmalignant diseases and for stem cell-mediated tissue regeneration. In particular, they can be applied to in- duce immunosuppression (Nauta and Fibbe, 2007, Blood 1 10: 3499-3506). This can be done as supportive therapy in hematological stem cell transplantation in which immunologically-mediated graft-versus-host disease is a major complication. Imnnunonnodulation also has a great potential in autoimmune or immune-mediated diseases, such as multiple sclerosis, rheumatoid arthritis, or inflammatory bowel disease (Shi et al., 2010, Cell Research: 1-9). In addition to the immunomodulation, MSC can be therapeutically used, for example, to induce angiogenesis, a central feature for regenerative tissue repair, or to suppress Th-1 driven autoimmune or autoinflammation and inflammatory re- sponse, a therapeutic possibility for example in rheumatoid arthritis (Gonzalez et al., 2009, Arthritis Rheumatism 60 (4): 1006-1019).

Endothelial stem cells are multipotent stem cells and one of the three types of stem cells to be found in bone marrow.

Human embryonic stem cells (hESCs) are derived from the inner cell mass of 3-5 day-old blastocysts. hESCs are considered to be the building blocks for all types of cells in humans and thus have huge potential in applications of cell therapy and regenerative medicine. The early technologies for harvesting hESCs included destruction of the embryo, but there are now methods for harvesting hESCs which do not include the destruction of a human embryo (Klimankaya et al 2006; Nature 444: 481 -485).

Induced pluripotent stem (iPS) cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell by inducing a "forced" expression of certain genes. There are currently a number of ways to make iPS cells. Their therapeutic potential has been predicted to be enormous because patient's own cells can be induced and hence, ethical and histocompatibility problems can be avoided.

It is known that the growth and function of MSC, as well as their cell surface antigens depend to certain extend on the conditions of the in vitro cul- turing of MSC (Lee et al., 2009, Blood 1 13: 216-226). Also, the age of the do- nor of MSC, and/or culturing time or number of passages or divisions in vitro seem to have an effect on the functional properties of MSC. For example, Gnecchi and coworkers (Congress abstract presented in ISSCR 2010, San Francisco, CA, USA) have shown that the regeneration by MSC of heart muscle cells was not as efficient when MSC cells of older donors were applied as compared to those of young donors. Except for cumbersome functional testing or estimation of, for example, telomere length (Dominici et al., 2006 Cytothera- py 8: 315-317, Flores and Blasco, 2010, FEBS Letters 584(17): 3826-3830), not readily applicable or practical to routine clinical cell therapy, there is no useful marker or test system to estimate the age or functionality of MSC. As MSC have shown to be a promising option in cellular and regenerative therapy, there is need for practical biomarkers for their functional properties or potency.

Lipids are a molecular group with various functions related to energy storage, structural components and cell functional signalling. It is poorly known how molecular interactions of signalling proteins and lipids work on the functional control of cell.

A main risk in stem cell therapy is the ability of stem cells to maintain or re-adopt pluripotency allowing them to form stem cell-derived tumours, teratomas. Therefore, for the proper use and validation of stem cells obtained from various sources require a good understanding of the role of signalling in the differentiation process and during the in vitro culturing of the stem cells.

Thus, there is a need for new markers that individually or in combination with other markers or diagnostic means provide sensitive and specific information that can be used in evaluating the functional quality and/or assessing the status of the cell or a population thereof. It is advantageous to have a reliable method of determining the functional status of a stem cell or a population thereof, the results of which can then be applied in the in vitro culturing and storage of the cells and even in stem cell therapy, for example.

Stem cells, as well as their subcellular organelles and cellular fractions or microparticles (often called microvesicles; see Collino et al PLOS One 2010; 7: e1 1803) are surrounded by a semi-permeable membrane. These membranes are complex structures, consisting of proteins and in particular of a very high number of different lipid molecules and molecular classes (Vance, D. E., and Vance, J. E. (2002) Biochemistry of Lipids, Lipoproteins and Membranes. 4th ed., Elsevier), such as phospholipids, sphingolipids and sterols. The concentrations of the lipids of each cell type are maintained within certain limits, demonstrating how crucial lipid homeostasis can be for cell survival. Membrane lipids participate in cell recognition, signalling events, and protein traffic, domain assembly (rafts) and modulation of protein function. Recent developments in molecular analysis by mass spectrometry, especially elec- trospray-ionization technique, enable system scale analyses of detailed lipid profiles of cells (lipidomics) containing hundreds of individual lipid species. So far, no detailed analysis of lipids of MSC has been carried out or no specific li- pid markers for the functionality of MSC are available. Hence, lipids provide a novel family of marker molecules for functional properties of a cell.

BRIEF DESCRIPTION OF THE INVENTION

An object of the present invention is to provide a stem cell and/or a population thereof having specific lipid content and/or profile and a method for determining the lipid content and/or profile of the stem cell. Another object of the present invention is to provide a method to evaluate the functional quality of a stem cell or a population thereof by determining the lipid content or profile of a stem cell and/or a population thereof. Another object of the invention is a use of the lipid content or profile of a stem cell and/or a population thereof in evaluating the functional quality of the stem cell and/or the population thereof in a therapeutic cell preparation.

A further object of the invention is to provide a method of determining the functional quality of a stem cell or a population thereof in a subject comprising:

a) obtaining a sample from the subject

b) assaying the sample to determine the lipid content of the stem cell and/or the population thereof

c) optionally comparing the determined lipid content with a standard. A further object of the invention is to provide an in vitro method of determining the functional quality of a stem cell or a population thereof in a subject comprising:

a) determining the lipid content of the stem cell and/or the population thereof

b) optionally comparing the determined lipid content with a standard.

A further object of the invention is to provide a method of determining the functional quality of a stem cell or a population thereof in a sample comprising:

a) assaying the sample to determine the lipid content of the stem cell and/or the population thereof

b) optionally comparing the determined lipid content with a standard.

A further object of the invention is to provide a method of screening the functional quality of a stem cell or a population thereof in a subject com- prising:

a) obtaining a sample from the subject b) assaying the sample to determine the lipid content of the stem cell and/or the population thereof

c) optionally comparing the determined lipid content with a standard. A further object of the invention is to provide an in vitro method of screening the functional quality of a stem cell or a population thereof in a subject comprising:

a) determining the lipid content of the stem cell and/or the population thereof

b) optionally comparing the determined lipid content with a standard. An even further object of the invention is to provide a method of screening the functional quality of a stem cell or a population thereof in a sample comprising:

b) optionally comparing the determined lipid content with a standard.

The invention is based on the observation that quantitative and/or qualitative levels of certain lipid classes, species or molecular species or their ratio to other lipid classes, species or molecular species, in a stem cell or a population thereof are associated with the age of the donor of the cell and/or with the time period, or passage number for which, the cells have been cultured in vitro.

Accordingly, the present invention provides a novel and effective means for evaluating the functional quality of the stem cell and/or the population thereof in a therapeutic cell preparation. In other words, the present inven- tion provides a novel and effective means for assessing the quality of the stem cell and/or the population thereof in a therapeutic cell preparation.

The objects of the invention are achieved by the methods and uses set forth in the independent claims. Preferred embodiments of the invention are described in the dependent claims.

Other objects, details and advantages of the present invention will become apparent from the following drawings, detailed description and examples.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the profiles of lipid classes in MSC samples from young donors (A) and elderly donors (B). Figure 2 shows the principal component analysis (PCAbiplot) of lipid class compositions from all MSC lines (N= 10) and their passage numbers. PC 1 = principal component 1 , PC2 = principal component 2.

Figure 3 shows the molar percentages (of analyzed total lipids) of three simple sphingolipid classes of samples from young (panel A) and elderly (panel B) donors. The proportion of the sphingomyelin (SM) shown as an open bar decreased towards a higher cell passage.

Figure 4 A. Relative levels of the phosphatidylserine (PS) and phosphatidylinositol (PI) classes, shown as the mol% proportion changes in re- lation to the number of cell passage and to the age of the sample.

Figure 4 B illustrates the effect of the age of the sample on the ratio of phosphatidylinositol and phosphatidylserine (PI/PS).

Figure 5 illustrates the effect of the age of the sample on the proportion (mol %) of the phosphatidylcholine (PC) species. In panel A) cell sample 081 from a younger donor is shown and in the panel B) cell sample 271 from an elderly donor is shown.

Figure 6 shows the principal component analysis (PCA biplot) of all cell lines and passages combined with the phosphatidylcholine species. The result illustrates the associations (positive or negative) between lipid species PC38:04, PC36:04, PC36:02, PC36:01 , PC34:02, PC34:01 , and PC34:00 and the age of the sample. Especially the PC38:04 and 36:04 were enriched in the late passages. PC 1 = principal component 1 , PC2 = principal component 2.

Figure 7 illustrates the effect of the age of the sample on the proportion (mol %) of the phosphatidylethanolamine (PE) species. In panel A) cell sample 081 from a young donor is shown and in the panel B) cell sample 271 from an elderly donor is shown.

Figure 8 shows the principal component analysis (PCA biplot) of all cell lines and passages combined with the phosphatidylethanolamine species. The figure illustrates the associations (positive or negative) between lipid spe- cies PE40:06, PE38:04, PE36:04, PE36:02, PE36:01 , PE34:01 and some other minor species with the age of the sample. Especially the PE38:04 was enriched in the late passages. PC1 = principal component 1 , PC2 = principal component 2.

Figure 9 illustrates differences in phosphatidylcholine species when analyzed by electrospray mass spectrometry using positive ionization mode. Panels A and B (passages 4 and 14, respectively) represent specific tandem mass spectrometry scans (parents of m/z 184) detecting only those lipid species that release a choline fragment (polar head group of phosphatidylcholine). Panels C and D (passages 4 and 14, respectively) represent non-specific scans of lipids ionized positively, including phosphatidylcholines but also members from several other phospholipid classes. In both types of detection, the proportional (mol %) change between PC38:04 (m/z 810.8-81 1 .1 ) and PC34:01 (m/z 760.8-761 .0) was readily readable. A sample from elderly donor 271 is shown as an example.

Figure 10 illustrates differences in phosphatidylethanolamine species when analyzed by electrospray mass spectrometry. Panels A and B (passages 4 and 14, respectively) represent specific positive-ion-mode tandem mass spectrometry scans (neutral loss of m/z 141 ) detecting only those lipid species that release an ethanolamine group (polar head group of phosphatidylethanolamine) as a neutral fragment. Panels C and D (passages 4 and 14, respec- tively) represent non-specific scans of lipids ionized negatively, including phosphatidylethanolamines but also members from several other phospholipid classes. In both types of detection, the proportional (mol %) change between PE38:04 (m/z 766 or 768 in the negative and positive ion modes, respectively) and PE36:01 (m/z 744 or 746 in the negative and positive ion modes, respec- tively) was readily readable. A sample from elderly donors 271 is shown as an example.

Figure 1 1 shows the principal component analysis (PCA biplot) of all cell lines and passages combined with the phosphatidylcholine and phosphatidylethanolamine species. The figure highlights that the late passage stem cells were enriched with PC38:04 and PE38:04; and that the early passage stem cells instead were enriched with PC34:01 and PE36:01 . The positions of PC38:04 and PE38:04 species on this biplot, on the opposite side from the PC34:01 and PE36:01 (in relation to the origin in the middle of the plot; a crossing point of the guiding broken lines), shows that in the stem cells these polyunsaturated (:04) and monounsaturated (:01 ) lipid species had almost complete negative correlations (close to -1 ). PC 1 = principal component 1 , PC2 = principal component 2.

Figure 12 shows proliferation capacity of ten (10) cell lines during cell culture. Five young donor cell lines (A.) and five elderly donor cell lines (B.) were calculated in each passaging step (dot in the curve) starting at passage 4. The cumulative cell population doubling was counted (Y-axis) and compared to time (days at X-axis). This data show that elderly donor cell lines had more heterogeneity than young cell lines.

Figure 13 illustrates differences in the 20:4n-6 content of the BMSC of young donors when analyzed by gas chromatography. The average levels (± SD) of 20:4n-6 from passage 4 to passages 9-12 of the BMSC from five young donors (081 , 088, 089, 091 , 092) are shown. These data show that when cells are cultured several passages their 20:4n-6 content is increased.

Figure 14 illustrates how the total content of 20:4n-6 changes during the cell culture (from passage 4 to passage 12) of a one young donor. These data show how fatty acid precursor reservoirs in the BMSCs are developing to more pro-inflammatory direction when cells are cultured long time.

Figure 15 shows the principal component analysis (PCA biplot) of all cell lines and passages when fatty acids and lipid species were used as loadings together. PCA biplots demonstrate similarities and differences among BMSC samples in terms of the combined data of the individual fatty acids with the most prominent (A) phosphatidylcholine (PC) species and (B) phosphati- dylethanolamine (PE) species. Fatty acid abbreviations: [carbon number]:[double bond number]n-[position of the double bond calculated from the methyl end]. Phospholipid abbreviations: [CLASS code] [total acyl/alcenyl chain carbon number]:[total double bond number].

Figure 16 describes the immunosuppressive capacity of MSCs from two bone marrow donors using division index as indicator of the proliferation of stimulated T-cells. The division index is the average number of cell divisions that a cell in the original population has undergone. The lower the index, the higher is the immunosuppressive effect of MSC cells.

Figure 17 shows the ratio of PC38:04/34:01 lipid species (A.), PS38:04/36:01 lipid species (B.) and PI38:04/36:01 lipid species (C.) from the same samples studied in the immunosuppression assay (see Figure 16).

Figure 18 shows the molecular percentage of PE36:01 lipid species. Figure illustrates the differences between aged and young donor groups already in early passages.

DESCRIPTION OF THE INVENTION

The invention is based on the finding that quantitative and/or qualitative levels of certain lipid classes, species or molecular species or their ratio to other lipid classes, species or molecular species, in a sample of MSC are associated with the age of the donor of the MSC cell and/or with the time period, or passage number for which MSCs have been cultured in vitro. In particular, the levels of phosphatidylinositol (PI) species: PI38:04 and PI36:01 ; phosphatidyl- serine (PS) species PS38:04 and PS36:01 ; phosphatidylcholine (PC) species: PC38:04, PC36:04 and PC34:01 ; and/or phosphatidylethanolamine species (PE): PE38:04 and PE36:01 showed the association. Elevated levels of certain species in other lipid classes, in particular species 20:4n-6 and 22:n-3 may accompany the species changes found in the PI, PS, PC and/or PE classes, and can be used to complement the indicative marker combination. As aged or older stem cells are known to be functionally not as good as younger ones, the lipid profile based on the contents and/or levels of certain lipids according to the present invention, is a useful biomarker for the functional quality and/or clinical efficacy of a stem cell or a population thereof, especially in a therapeutic cell preparation. In a typical case, certain lipids or lipid classes are determined from a sample of stem cells, the quantitative levels of the lipids are measured and if the levels or ratios of particular lipid classes or species or molecular species are sufficiently low or high - i.e. outside the window of relevant controls obtained from samples with good functionality- the result indicates poor quality, functionality and usability in therapeutic treatments. Thus this approach offers new means for quality control. The cut-off value which would be indicative for a good functional quality may differ according to the exact conditions and cell type used and must be set beforehand by carrying out a relevant set of experiments.

The term "functional quality" refers to the functionality of a stem cell and/or a population thereof, including the age of the cell and/or a population thereof, and/or the immunomodulatory and/or angiogenic and/or osteogenic po- tency of a stem cell or a population thereof. In particular, the term refers to differentiation and proliferation capacity of a stem cell. Especially in a therapeutic cell preparation the functional quality of a stem cell or a population thereof is very important for the successful treatment result as well as minor ill or side effects.

The term "age of the cell and/or a population thereof " here refers to the age of the donor who donated the cell sample, and/or to the in vitro cultur- ing age, that is, the number of passages or cell divisions the sample has undergone in vitro - the latter regardless of the age of the donor.

Term "lipid class" here refers to classification based on the polar head group and backbone structures, i.e. glycerol or sphingosine of the lipid.

"Lipid species" or "lipid molecular species" refers to the fact that each lipid class consists of diverse molecular species where the number of carbon atoms and double bonds in the fatty acid moiety can vary. Lipid molecular species further refer to free fatty acids or liberated fatty acids in the sample. Lipid species further vary in they isobaric composition forming variation of lipid molecular species. (Shevchenko and Simons, 2010 Nature Re- views, Molecular Cell biology, 1 1 : 593-598).

The term "lipid profile" refers to a combination of contents or relative distribution of at least two of the lipid classes, species and/or molecular species in the sample.

Table 1 shows the abbreviations used for different lipids in the pre- sent invention.

Table 1

Cer = ceramide

DHA=docosahexanoic acid

EPA=eicosapentaenoid acid

GalCer = galactocylceramide

LysoPC = lysophosphatidylcholine

PC = phosphatidylcholine

PCe = phosphatidylcholine ether lipid (mainly alkyl-acyl species;

often marked just "alkyl")

PE = phosphatidylethanolamine

PEe = phosphatidylethanolamine ether lipid (mainly alkenyl-acyl

species; often marked just "alkenyl" or "plasmalogen")

PUFA = polyunsaturated fatty acid

PS = phosphatidylserine

SM = sphingomyelin

AA = arachidonic acid

The present invention relates to a method of determining a lipid con- tent or profile of a stem cell and its use in estimating the functional quality of a stem cell and/or a population thereof. The functional quality of a stem comprises age-related functional properties of the stem cell, such as the immunomodulatory potency and/or angiogenic potency and/or osteogenic potency, differentiation and/or proliferation capacity. The present invention relates also to a method to evaluate the functional quality of a stem cell and/or a population thereof by determining the lipid content or profile of the stem cell and/or the population thereof, and to a method of assessing the status of a stem cell and/or a population thereof by deter- mining the lipid content or profile of the stem cell and/or the population thereof.

The method of the invention involves determining the lipid content using methods described herein or otherwise known in the art and optionally comparing the result with a standard.

The methods of the invention may comprise steps, such as isolation of the cell population from various sources or taking an aliquot from a cell product earlier prepared; extraction of the lipid fraction; analysis of the lipid content of the lipid fraction by using mass spectrometry or chromatography; quantification of relevant lipids, lipid classes or lipid species; and comparison of the relative amount or specific ratio to reference values. The reference values will be determined from cell samples known to have sufficiently good quality, for example, to give required clinical response. In addition, the reference samples may be handled in similar ways or cultivated in sufficiently similar conditions. These steps are common and typical to various evaluation methods.

According to one embodiment, the invention relates to a method for evaluating the functional quality of stem cells of the possible donor candidate. Specifically, the invention relates to a method to evaluate functional quality of a stem cell and/or a population thereof by determining the lipid content of the stem cell and/or the population thereof. This data could be used to estimate the amount of cells needed for clinical stem cell transplantation if the said can- didate is used as the donor. The data also can be used as selection criteria for donation; individuals with too low a quality of stem cells may not be suitable for donation. In addition, the data can be applied to estimate the viability or quality of the cells during the storage, modification, cultivating and passaging, for example.

According to another embodiment, the present invention relates to a method of determining the functional quality of a stem cell or a population thereof in a subject comprising:

a) obtaining a sample from the subject

b) assaying the sample to determine the lipid content of the stem cell and/or the population thereof or determining the lipid content of the stem cell and/or the population thereof in a sample c) optionally comparing the determined lipid content with a standard. According to another embodiment, the present invention relates to an in vitro method of determining the functional quality of a stem cell or a population thereof in a subject comprising:

a) determining the lipid content of the stem cell and/or the population thereof

b) optionally comparing the determined lipid content with a standard.

According to another embodiment, the present invention relates to a method of determining the functional quality of a stem cell or a population thereof in a sample comprising:

a) assaying the sample to determine the lipid content of the stem cell and/or the population thereof or determining the lipid content of the stem cell and/or the population thereof in a sample

b) optionally comparing the determined lipid content with a standard.

The sample can be taken from blood, serum, plasma, bone marrow, cord blood, or any other appropriate tissue of the subject. In one embodiment of the invention, the sample is taken from blood, serum, plasma, bone marrow, cord blood, or any other appropriate tissue of the subject. In another embodi- ment, the sample is taken from blood, serum or plasma of the subject. In a further embodiment, the sample is taken from the subject without the assistance or exploitation of surgical procedures.

In addition, according to one embodiment, the invention relates to a method of screening the functional quality of a stem cell or a population thereof in a subject comprising:

a) obtaining a sample from the subject

b) assaying the sample to determine the lipid content of the stem cell and/or the population thereof or determining the lipid content of the stem cell and/or the population thereof in a sample

c) optionally comparing the determined lipid content with a standard.

According to another embodiment, the invention relates to an in vitro method of screening the functional quality of a stem cell or a population thereof in a subject comprising:

a) determining the lipid content of the stem cell and/or the popula- tion thereof

b) optionally comparing the determined lipid content with a standard. According to a further embodiment, the invention relates to a method of screening the functional quality of a stem cell or a population thereof in a sample comprising:

b) optionally comparing the determined lipid content with a standard. The sample can be taken from blood, serum, plasma, bone marrow, cord blood, or any other appropriate tissue of the subject. In one embodiment of the invention, the sample is taken from blood, serum, plasma, bone marrow, cord blood, or any other appropriate tissue of the subject. In another embodiment, the sample is taken from blood, serum or plasma of the subject. In a further embodiment, the sample is taken from the subject without the assistance or exploitation of surgical procedures.

Further, in the in vitro methods of the present invention any kind of a tissue sample suitable for such a method can be exploited.

The invention also relates to the use of lipid profile of a stem cell to estimate the functional quality such as, age-related functional properties of the stem cell.

The present invention relates also to a use of the lipid profile of a stem cell and/or a population thereof in evaluating the functional quality of the stem cell and/or the population thereof in a therapeutic cell preparation, and to a use of lipid profile of a stem cell and/or a population thereof in assessing the status of a stem cell and/or a population thereof in a therapeutic cell preparation.

In an embodiment of the present invention, the lipid profile of the stem cell and/or the population thereof comprises lipid classes PE and/or PC and/or PI and/or PS.

In one embodiment, the lipids measured are phosphatidylcholine species, PC38:04, PC36:04 and PC34:01 , and their levels or their relative ratio to each other or one or more other lipids can be used as a biomarker for functionality of a stem cell.

In another embodiment, phosphatidylethanolamine species, PE38:04 and PE36:01 , and/or their relative ratio to each other or to one or more other lipids are measured and used as a biomarker for functionality of stem cell.

In another embodiment, phosphatidylinositol (PI) species, PI38:04 and PI36:01 , and their levels and/or their relative ratio to each other or to one or more other lipids are measured and used as a biomarker for functionality of stem cell.

In another embodiment, phosphatidylserine (PS) species, PS38:04 and PS36:01 , and their levels and/or their relative ratio to each other or to one or more other lipids are measured and used as a biomarker for functionality of stem cell.

In a further embodiment, phosphatidylinositol (PI) class as a whole and/or phosphatidylserine (PS) class as a whole and/or their relative ratio to each other or one or more other lipids are measured and used as a biomarker for functionality of stem cell.

In another embodiment, arachidonic acid (AA) class as a whole or its relative ratio to one or more other lipids are measured and used as a biomarker for functionality of stem cell.

In a further embodiment, arachidonic acid (AA) species 20:4n-6, and its level and/or relative ratio to one or more other lipids are measured and used as a biomarker for functionality of stem cell.

In a further embodiment, one or more of the lipids in the lipid content and/or profile are selected from the list:

a. PE38:04

b. PE36:01

c. PC34:01

d. PC38:04

e. PC36:04

f. PS38:04

g. PS36:01

h. PI38:04

i. PI36:01

j. 20:4n-6 (= AA)

In one embodiment, the ratio of contents of at least two lipids is measured.

In another embodiment, the ratios measured are PI/PS, and/or PS38:04/PI38:04, PS38:04/PI36:01 and/or PS36:01/PI36:1 , and/or PC38:04/PC34:01 and/or PC36:04/PC34:01 and/or PC38:04/PC36:04 and/or (PC38:04+PC36:04)/PC34:01 and/or (PC38:04+PC34:01 )/PC36:04 and/or (PC36:04+PC34:01 )/PC38:04 and/or PE38:04/PE36:01 and/or PS38:04/PS36:01 and/or PI38:04/PI36:01 and/or other combinations thereof. In a further embodiment, the ratios measured are PI/PS, specifically PS38:04/PI38:04, PS38:04/PI36:01 and/or PS36:01/PI36:1 , and/or PC38:04/PC34:01 and/or PC36:04/PC34:01 and/or (PC38:04+PC36:04)/PC34:01 and/or PE38:04/PE36:01 and/or PS38:04/PS36:01 and/or PI38:04/PI36:01 .

In one embodiment of the invention, the determination step or stage of the method comprises detecting the presence or absence of one or more of the lipid classes, lipid species and/or lipid molecular species. In another embodiment of the invention, the determination step or stage of the method comprises quantifying the amount of one or more of the lipid classes, lipid species and/or lipid molecular species.

In one embodiment of the present invention, the stem cell is any type of stem cell selected from mesenchymal stem cell, hematopoietic stem, endothelial stem cell, embryonic stem cell or induced pluripotent stem cell. In another embodiment of the invention, the stem cell is selected from mesen- chymal stem cell, hematopoietic stem, endothelial stem cell or induced pluripotent stem cell. In a further embodiment of the invention, the stem cell is a hESCs harvested solely by a method that does not include the destruction of a human embryo. In an even further embodiment, the stem cell is a mesenchymal stem cell.

In one embodiment of the present invention, the stem cell or population thereof is obtained from a cell culture.

The invention can be used, for example, to estimate the clinical quality, usefulness or efficacy of a stem cell or a population thereof in a preparate or composition. Immunomodulation and angiogenic ability are the major features assumed for therapeutic stem cells and the present invention provides a novel means to estimate these functional properties. Determination of a lipid biomarker, readily detectable using relevant analytical tools well known in the art, can be much more reliable than cumbersome functional assays. It should also be noted that the indicative ratios of phosphatidylcholine and/or phosphatidylethanolamine species (e.g. ratios of above mentioned species a.-j.) can be analyzed quickly and cost-efficiently by using any elec- trospray-ionization mass spectrometer, including the cheapest single- quadrupole and ion trap equipments. In addition, preseparation of the extracted lipid mixture by means of liquid chromatography is not necessary. Direct in- fusion of the sample into the device and collecting the data for one minute will already produce useable results. Thus using these biomarker tools does not require any substantial equipment investments. If the phospholipid classes PS and PI are used as additional criteria, they can alternatively be analyzed by using conventional thin-layer chromatography or liquid chromatography. Analysis methods for PUFAs so far have based mainly on gas chromatography, but it is possible to develop antibodies against these lipids and/or enzymatic assays to determine content of these lipids in a sample.

The invention can be applied to estimate the quality of a cell preparation after manipulations, or during a manipulation process, or after storage of the preparation. The invention can also be used to evaluate the quality of stem cells of a subject, a donor and/or a patient.

The invention furthermore can be used as a test measurement when screening optimal in vitro culturing conditions for stem cells; lipid profile provides a practical endpoint parameter for screening assays. The conditions resulting in sufficiently good or better lipid profiles, as compared to the relevant standard are favoured ones for further development, whereas those leading to worse lipids profiles are not worth further development.

It is clear that methods based on the present invention can be developed and used for other cell preparations and/or other cell types as well. Examples of these cells can be, but is not limited to, hematopoietic stem cells, therapeutic dendritic cells, natural killer type of cells, and/or T lymphocytes, such as regulatory T cells or cytotoxic T cells. In particular, the lipid content or profile can be measured from suborganelles of the above mentions cells or cellular fractions or microparticles (microvesicles) derived from the cells. In this invention microvesicles refer to exosomes and/or microparticles or similar structures derived from the plasma membrane and/or endocytotic pathway of the cells.

The invention will be described in more detail by means of the following examples. The examples are not to be construed to limit the claims in any manner whatsoever. EXAMPLES

Materials and Methods

The materials and methods described herein are common to examples 1-7.

Characterized (according to minimal criteria defined by ISCT, Domi- nici et al., 2006, Cytotherapy 8: 315-317) human bone marrow derived MSCs were isolated as previously described (Leskela et al., 2003, Biochemical and Biophysical Research Communications 31 1 : 1008-1013). MSCs were harvested after informed consent from five older donors (samples 164, 172, 194, 268, 271 ) (from 62 to 82 years of age, mean 74.6 years) and five young adult donors all under 25 years (samples 81 , 88, 89, 91 , 92).

MSCs were cultured in minimum essential alpha-medium (aMEM) supplemented with 20mM HEPES, 10% FCS, 2mM L-glutamine and 1 x penicillin-streptomycin (all from Gibco). The medium was renewed twice a week until 70-80% confluent and the cells were subcultured further at density of 1000 cells/cm². Cells were cultured until senescence was reached. For lipid analysis cells were plated on 75 cm² cell culture flasks and cultured until near confluen- cy or up to 3 weeks (late passages) changing media twice a week.

For lipid analysis adherent cells were washed with ice-cold phosphate-buffered saline (PBS), scraped on ice and transferred to silylated tubes. Cells were pelleted in silylated vials and stored at -70°C for later use.

The lipids of the bone marrow derived MSC samples were extracted according to Folch et al., 1957, J Biol. Chem, 226: 497-509, spiked with internal standards, evaporated to dryness under nitrogen, and dissolved in 1 :2 chloroform/methanol. Several internal standards were used to correct for the effects of polar head group and acyl chain length on the instrument response according to previously reported procedures (Koivusalo et al., 2001 , J Lipid Res 42: 663-72., Kakela et al., 2003, J Neurochem 84: 1051-65, Hermansson et al., 2005, Anal Chem 77: 2166-2175). The PC, PE and PS were standardized by using 2-3 different diunsaturated lipid species having 28-44 carbons in the acyl chains, PI by a 37:4 species, lysolipids by 14:1 and 22:1 lysoPC species, and the sphingolipids by SM 25:0 and Cer 17:0. The concentrations of PL standards were determined by phosphate analysis (Bartlett and Lewis, 1970, Anal Chem 77: 2166-2175), and those of SL species according to Naoi et al.,1974, Anal Biochem 58: 571-577. Just prior to the mass spectrometry 1 % NH₄OH was added and the lipid extracts with the internal standards were infused to the elecrospray source of a Quattro Micro triple quadrupole mass spectrometer (Micromass, Manchester, UK) at the flow rate of 8 μΙ/min. The collision energy of the instrument was set to 25-65 eV, and negative and positive ion modes were used. Argon was used as the collision gas. The PC, lysoPC and SM (precursor of 184), PE (neutral loss of 141 ), PS (neutral loss of 87), PI (precursor of 241 ) and Cer (precursor of 264) species were selectively detected using head-group specific MS/MS scanning modes (Brugger et al., 1997, Proc Natl Acad Sci 94:2339-44, Sullards and Merrill, 2001 , Sci STKE 67:PL1 .). After the identification steps, quantification of the molecular species found in the mass spectra extracted by MassLynx software (Micromass, Man- Chester, UK) was carried out based on the internal standards using the LIMSA software (Haimi et al., 2006, Anal Chem 78: 8324-8331 ). The relative concentrations of the lipid classes were obtained by summing of the concentrations of the individual molecular species in a class.

Multivariate principal component (PC) analysis was applied to com- pare lipid classes or species composition of the MSC samples from different cell lineages and passages (Kvalheim and Karstang, 1987, J Clin Invest 1 10: 3-8). Prior to the analysis, the molar percentages of the lipid classes or species were logarithmically transformed to prevent the abundant components with large variance from dominating the analysis. The samples positioned in multi- dimensional space were plotted in two new coordinates (PC1 and PC2) calculated to describe the largest and second largest variance of the data among the samples. The computations were performed by using SIRIUS program package (Pattern Recognition Systems, Bergen, Norway).

The changes in each lipid percentage were analyzed with linear mixed effects models, fitting a model with fixed terms for age and passage and a random effect for cell line (repeated measures type analysis). The effect of each term for every lipid was estimated via normal F-test p-values and visualized with interaction plots depicting the mean values in each passage and group. Software for statistical analysis was computing language R, version 2.12, using package nlme for mixed effects analysis.

The materials and methods described below refer to example 8. Fatty acid derivatization was performed as follows. Aliquots (150 μΙ) of the lipid extracts were transferred into inert glass vials and the solvent evaporated in room temperature with nitrogen, and the methylation reagent, 1 % H2SO4 in methanol (containing hexane as cosolvent) was added immediately. Then the solutions were heated for 120 min at 95°C under nitrogen atmosphere and the formed fatty acid methyl esters (FAME) extracted with hexane in two steps. The dried and concentrated FAME solutions (40 μΙ in insert vial) were analysed by a gas-liquid chromatograph (GC) using flame ionization detection (FID) (6890N network GC, Agilent, USA) and DB-wax capillary columns (30 m, ID 0.25 mm, film 0.25 μηη, J&W Scientific, USA). A volume of 5 μΙ was injected and the split ratio was 1 :20. The injector and detector were set at 250°C. Helium was used as a carrier gas (1 .8 ml/min). The initial oven temperature of 180°C was held for 8 min, programmed to rise 3°C /min to final temperature of 210°C, which was kept for 25 min. Agilent Chemstation software was used for peak area integration. The identification of the FAME was based on retention time, mass spectra acquired earlier for similar samples, and comparisons with authentic (Sigma, St. Louis, MO) and natural standards of known composition. Quantifications were based on FID responses corrected according to the theoretical response factors (Ackman 1992) and calibrations with quantitative authentic standards. The fatty acid components present in the samples with more than 1 mol% were included in the calculations. The trace components were found to introduce more noise than real information into the data (especially in the case of the diluted late passage samples) and thus were removed. The fatty acid proportions were calculated as molar %, and the fatty acids were marked by using the abbreviations: [carbon number]:[number of double bonds] n-[position of the first double bond calculated from the methyl end] (e.g. 22:6n-3).

Statistical Analysis was performed as follows: The fatty acid profiles were studied at first using 1 ) conventional mol% bar profiles. The fatty acids were studied individually or grouped according to their common structural features e.g. double bond content and position. Next, to analyze relationships between different BMSC samples and the fatty acids therein, the data were subjected to multivariate principal component analysis (PCA) using SIRIUS 6.0 and 7.1 software packages (Pattern Recognition Systems, Bergen, Norway) (Kvalheim & Karstang 1987). This unsupervised method of mathematical modelling for exploratory analysis is useful to identify patterns or capture trends in high dimension data when there are correlated variables. PCA also helps in expressing the data in such a way that it highlights 7 compositional similarities and differences by reducing the number of dimensions of the data without much loss of information. For PCA, the percentage data were log-transformed for sufficient normality. The samples positioned in multidimensional space were plotted in two new coordinates (principal components, PC1 and PC2) calculated to describe the largest and second largest variance of the fatty acid or phospholipid data among the samples. Subsequently the fatty acid and the previously determined phospholipid data were studied in combination by PCA. When fusing the fatty acid and phospholipid data for PCA, the values for fatty acids and phospholipid classes were expressed as mol% per the total of all detected lipids, and the values for each phospholipid species as mol% in its own class. The abbreviation for individual phospholipid species were: [CLASS code] [total acyl chain carbon number]:[total double bond number].

Materials and methods described below refer to example 9.

MSCs were co-cultured with mononuclear cells (MNC) in order to measure their capability to inhibit T-cell proliferation. Mesenchymal stem cells were grown until 80% confluent, trypzinized and resuspended in RPMI medium supplemented with 5 % fetal bovine serum and 1x penicillin-streptomycin. 3x10⁴ MSCs were plated in triplicates on cell culture treated 48 multidish plates (Nunc, Thermo Fisher). The MSCs were allowed to attach for two hours before adding CFSE labeled mononuclear cells.

MNCs were isolated from buffy coats from healthy anonymous blood donors (Finnish Red Cross Blood Service) and cryo-preserved in liquid nitrogen for later use. Prior to use MNCs were thawed gently and labelled with CFSE (5(6)-Carboxyfluorescein diacetate /V-succinimidyl ester). Cells were filtered with 30 μιτι sterile syringe filter (Becton Dickinson, Franklin lakes, NJ, USA). The filtered cells were resuspended in 0.1 % Human serum albumin (HSA, Sanquin)-PBS at the concentration of 20x10⁶ cells/ml. Same volume of freshly diluted 5 μΜ CFSE-solution (Molecular probes) in 0.1 % HSA-PBS was added. Cells were vortexed immediately and incubated 5 minutes at room temperature. Labelled cells were washed three times with at least 10x volume of 0.1 % HSA-PBS and resuspended in RPMI growth medium at the concentration of 5x10⁶. 1 .5x10⁶ CFSE-labelled cells were added on top of the attached MSCs in 300 μΙ. To activate T-cell proliferation 100ng/ml CD3 antibody clone Hit3a (BioLegend, San Diego, CA, USA) diluted in RPMI growth medium was added. Non-stimulated CFSE- labelled cells as well as stimulated and non- stimulated non-labelled MNCs were used as controls. RPMI growth medium was added to wells to achieve final volume of 650μΙΛ/νβΙΙ. Plates were placed in humidified incubator (+37 °C 5% CO₂) for four days after which the cell proliferation was analyzed by using flow cytometry (FACSAria, Becton Dickinson) and FlowJo software (version 7.6.1 ). EXAMPLE 1

Profiles of phospholipid and sphingolipid classes

The lipid profiles of both phospholipid and sphingolipid classes showed variations dependent on the source (elderly versus young) and be- tween early and late passages. The lipid class profiles of bone marrow MSCs were studied using conventional mol% bar profiles (Figure 1A Lipid profiles of young donors and Figure 1 B Lipid profiles of elderly donors). To analyze relationships between the different cell samples and the lipid classes, the data were subjected to multivariate principal component analysis (PCA) (Figure 2). PCA indicated that the levels of several lipid classes were associated with the number of passages or to the age of the donor (elderly versus young) (Figure 1 and 2).

EXAMPLE 2

The profile of simple sphingolipid classes

The differences in the profiles of three simple sphingolipid classes

(SM = sphingomyelin; Cer = ceramide; GalCer = galactocylceramide) were studied in relation to the age of the samples (elderly vs. young donor; early vs. late passages). Especially, the proportion (mol%) of sphingomyelin decreased towards late passages (Figure 3). EXAMPLE 3

The differences in the relative amounts of the phospholipid classes, phosphatidylserine (PS) and phosphatidylinositol (PI) were studied in relation to the age of the samples (elderly vs. young; early vs. late passages) (Figure 4A). A significant difference was seen in the ratio of PI/PS proportion (mol %) (Figure 4B). The ratio increased towards the later passages, for example, in sample 89, the PI/PS -ratio at P4 was 0.5 and increased up to 1 .5 at P9. The ratio also was higher in elderly patients already in early passages, typically around 1 whereas in the young samples it was around 0.5 (Figure 4B).

EXAMPLE 4

Changes in molar percentage of PC species

The alterations in profiles of individual molecular species in each class were studied. The profile of phosphatidylcholine (PC) species showed several changes in a number of species. The most significant alterations were found in the mol% of PC38:4, PC36:4 and PC34:1 . The proportion (mol %) of PC38:4 and PC36:4 increased towards the late passages whereas the proportion (mol %) of PC34:1 decreased in both elderly (Figure 5A, Donor 081 ) and young (Figure 5B, Donor 271 ). To analyze relationships between different BMMSC samples and the PC species the data were subjected to multivariate principal component analysis (PCA). The results demonstrated a tight association (either a negative one: 34:01 vs. 38:04 or 34:01 vs. 36:04 or a positive association, 38:04 vs. 36:04) between the PC species. The 38:04 and 36:04 had high values in the late passages, and the 34:01 was prominent in early pas- sages (Figure 6). In addition figure 18A shows significant difference (p-value 0.0487) in PC34:01 between young and aged groups.

EXAMPLE 5

Changes in molar percentage of PE species

Further the profile of phosphatidylethanolamine (PE) species showed significant alterations in the PE38:04 and PE36:01 species. The relative amount (mol %) of PE38:04 increased towards the late passages whereas the proportion (mol %) of PE36:01 decreased in both elderly (Figure 7A, Patient 081 ) and young (Figure 7B, Patient 271 ). To analyze relationships between different BMMSC samples and the PE diacyl species the data were sub- jected to multivariate principal component analysis (PCA). The results demonstrated a negative association between PE36:01 and PE38:04, the former being high in the early passages and the latter in the late passages (Figure 8). The relative amount (mol%) of PE36:01 was increased in young donors in both early and late passages compared to aged donors (Figure 18B). EXAMPLE 6

The use of the ratio of mass spectrometric peaks

This approach showed that, for example, the ratio between PC38:04 and PC34:01 as detected by mass spectrometric peaks was many fold in samples from the late passages as compared to earlier passage (Figure 9). In ad- dition, a similar result could be achieved also for the PE (normal diacyl species) 38:04 and 36:01 (Figure 10). The association between the age of samples and phospholipid species profile was confirmed by PCA analysis (Figure 1 1 ). Table 2 shows the ratios of lipid species in all ten samples (Table 2). _dlon e_ryi Table 2 shows the ratios of mol% values between PC38:04/PC34:01 and/or PC36:04/PC34:01 and/or (PC38:04+PC36:04)/PC34:01 and/or PE38:04/PE36:01 .

Table 2

PC38:04/ PC36:04/ (PC38:04+PC36:04) PE38:04/

PC34:01 PC34:01 /PC34:01 PE36:01

Donor start end start end start End start end

081 0.25 0.63 0.12 0.29 0.37 0.92 1.64 3.54

_Q 088 0.24 0.77 0.12 0.38 0.36 1.15 1.62 3.27 c

o 089 0.34 0.58 0.16 0.28 0.50 0.85 1.71 2.80 3

CO

C 091 0.21 0.57 0.21 0.29 0.53 0.90 2.17 2.74 o

>- 092 0.34 0.64 0.17 0.31 0.50 0.95 2.1 1 3.08

164 0.42 0.64 0.18 0.32 0.60 0.96 2.08 3.40

172 0.45 0.60 0.20 0.29 0.65 0.89 2.56 3.33 o

194 0.55 0.84 0.26 0.44 0.81 1.28 2.54 5.85

268 0.31 0.50 0.17 0.26 0.48 0.74 2.02 3.36

LU 271 0.30 0.67 0.17 0.67 0.46 1.00 2.33 4.79

EXAMPLE 7

The growth curves of the cell lines studied during the experimentation

Stem cell growth rate (cumulative cell doublings, Y-axis vs. days in culture, X-axis) was studied throughout the culture from each passage step (Figure 12). These results show that the cell doubling capacity degreases during cell culture. It also shows that the cell lines from elderly donors are more heterogeneous compared to young donors.

EXAMPLE 8

The fatty acid composition of the cell samples

The BMSCs were further studied for fatty acid profiles of total lipids by using gas-liquid chromatography (GC). Consecutive cell passages (from p4 even up to p14) from 10 donors (5 young and 5 aged individuals) were analyzed and the compositional differences among them studied by multivariate statistics (Figure 15). These results combined with the results from phospholip- id profile confirm the result from the membrane lipid profile. The suggested lipid markers for cell functionality do correlate with the precursor fatty acids of the active signaling lipids known to be important in immune modulation, cell differentiation and many other cellular functions (Figure 13-14).

EXAMPLE 9

The immunosuppression efficacy of the cells

The immunosuppressive efficacy of stem cells from early (passage 4 and 5) and late passages (passage 1 1 ) was measured in co-culture with mononuclear cells. Cells from early passages were more immunosuppressive compared to cells from late passages (figure 16).

EXAMPLE 10

The lipid marker of the cells

The lipid species content and ratios were examined form the same stem cells used in example 9. Ratios of PC38:04/PC34:01 , PI38:04/PI36:01 and PS38:04/PS were changed (Figure 17) as predicted in relation with the cell functionality (Figure 16). This confers the link between the marker and functionality.

Claims

1 . A method to evaluate functional quality of a stem cell and/or a population thereof by determining the lipid content of the stem cell and/or the population thereof.

2. A method of determining the functional quality of a stem cell or a population thereof in a subject comprising:

a) obtaining a sample from the subject

c) optionally comparing the determined lipid content with a standard.

3. An in vitro method of determining the functional quality of a stem cell or a population thereof in a subject comprising:

a) determining the lipid content of the stem cell and/or the population thereof

b) optionally comparing the determined lipid content with a standard.

4. A method of determining the functional quality of a stem cell or a population thereof in a sample comprising:

b) optionally comparing the determined lipid content with a standard.

5. A method of screening the functional quality of a stem cell or a population thereof in a subject comprising:

a) obtaining a sample from the subject

c) optionally comparing the determined lipid content with a standard.

6. An in vitro method of screening the functional quality of a stem cell or a population thereof in a subject comprising:

a) determining the lipid content of the stem cell and/or the population thereof

b) optionally comparing the determined lipid content with a standard.

7. A method of screening the functional quality of a stem cell or a population thereof in a sample comprising:

b) optionally comparing the determined lipid content with a standard.

8. The method according to any one of the preceding claims in which the stem cell or population thereof is obtained from a cell culture.

9. The method according to any one of claims 1 to 8, wherein the lipids determined comprises lipid classes and species: PE and/or PC and/or PI and/or PS.

10. The method according to any one of claims 1 to 8 wherein one or more of the lipids determined are selected from the list:

a. PE38:04

b. PE36:01

c. PC34:01

d. PC38:04

e. PC36:04

f. PI38:04

g. PI36:01

h. PS38:04

i. PS36:01

j. 20:4n-6

1 1 . The method according any one of claims 1 to 8 wherein the ratio of contents of at least two lipids is measured.

12. The method according to claim 1 1 wherein the ratios measured are PI/PS and/or PC38:04/PC34:01 and/or PC36:04/PC34:01 and/or (PC38:04+PC36:04)/PC34:01 and/or PE38:04/PE36:01 and/or PI38:04/PI36:01 and/or PS38:04/PS36:01 .

13. The method according to claim any one of claims 1 to 8 wherein the method comprises a step for comparing the lipid content with a standard.

14. Use of lipid content of a stem cell and/or a population thereof in evaluating the functional quality of the stem cell and/or the population thereof in a therapeutic cell preparation.

15. The use according to claim 14 wherein the lipids determined comprise lipid classes PE and/or PC and/or PI and/or PS.

16. The use according to claim 14 wherein one or more of the lipids determined are selected from the list:

a. PE38:04

b. PE36:01

c. PC34:01

d. PC38:04

e. PC36:04

f. PI38:04

g. PI36:01

h. PS38:04

i. PS36:01

j. 20:4n-6.

17. The use according to claim 14 wherein the ratio of contents of at least two lipids is measured.

18. The use according to claim 17 wherein the ratios measured are PI/PS and/or PC38:04/PC34:01 and/or PC36:04/PC34:01 and/or (PC38:04+PC36:04)/PC34:01 and/or PE38:04/PE36:01 and/or PI38:04/PI36:01 and/or PS38:04/PS36:01 .