BACKGROUND OF THE INVENTION
Stem cells show differential characteristics as they are able to sustain themselves and differentiate into one or more cell type. Although research into stem cells and their applications is still in its early stages, adult stem cells in bone marrow have been used in transplants for more than 30 years. Nevertheless, in recent years, stem cell technology has made large advances such that stem cells are currently considered as a promising source of tissue and organs, with an important therapeutic potential for repair and regeneration of tissues.
The use of stem cells is an alternative therapy for several human diseases, particularly those in which there is a loss of functional cells, including chondral, bone and muscular lesions, neurodegenerative diseases, immunologic rejection, heart disease and skin disorders (see U.S. Pat. Nos. 5,811,094, 5,958,767, 6,328,960, 6,379,953, 6,497,875).
In addition to cell therapy applications, stem cells have potential applications in the research and development of new drugs. On the one hand, the study of mechanisms implicated in the proliferation and differentiation of stem cells is of great value in the process of searching for and characterizing new genes involved in a wide range of biological processes, including cell development and differentiation and neoplastic processes (Phillips et al., 2000; Ramalho-Santos et al., 2002; Ivanova et al., 2002). On the other hand, stem cell technology allows specialized cells to be generated and the development of cell models for human and animal diseases, in which the efficacy and toxicity of new active ingredients can be determined in the preclinical phase (see U.S. Pat. No. 6,294,346).
An adult somatic stem cell is an undifferentiated cell which is found in a differentiated tissue and which has the capacity to proliferate and differentiate into one or more cell types. Adult stem cells are present in different adult tissues, their presence being extensively reported in bone marrow, blood, cornea, retina, brain, muscle, skeleton, dental pulp, gastrointestinal epithelium, liver and skin (Stem Cell Book, 2001). By their nature, autologous adult stem cells are incompatible and their use does not raise any ethical concerns.
An adult stem cell should be able to give rise to fully differentiated cells with mature phenotypes which are integrated into the tissue where they are found and which are able to carry out the specialized functions of the given tissue. The term phenotype refers to observable characteristics of the cell; such as characteristic morphology, interactions with other cells and with the extracellular matrix, cell surface proteins (surface markers) and characteristic functions (Stem Cell Book, 2001).
Different populations have been described of adult stem cells able to contribute to the repair of different tissues. Among these populations, those of mesodermic origin are of particular interest because they offer the theoretical possibility of regenerating a large number of clinically very relevant connective tissues such as bone, cartilage, tendons, skeletal muscle, heart muscle, vascular endothelium, subdermal fat and bone marrow stroma. The first cell population of this type isolated was the so-called mesenchymal stem cells (MSC), which are found in bone marrow stroma (Friedenstein et al., 1976; Caplan et al., 1991; Pittenger et al., 1999). These cells have been extensively characterized and studies performed with these cells have shown that they can differentiate into different mesenchymal cell lines such as adipocytes (Beresford et al., 1992), chondrocytes (Johnstone et al., 1998), myoblasts (Wakitani et al., 1995) and osteoblasts (Haynesworth et al., 1992). Likewise, they also have the capacity to differentiate into neurons (Sanchez-Ramos et al., 2000).
The ideal source of adult stem cells is one in which they can be obtained by an easy, non-invasive process and one that allows a sufficient number of cells to be isolated. In particular, a source should provide stem cells that can be easily isolated from a living subject without significant risks and discomfort and the source should allow a high yield to be obtained with minimal contamination from other cell types, without excessive cost of isolation and culture.
The process of obtaining bone marrow is painful and the yield is very low, a substantial increase in the number of cells being necessary by ex vivo expansion, to obtain a clinically relevant amount. This step increases cost and makes the procedure time consuming, as well as increasing the risk of contamination and loss of material. For these reasons, it would be very desirable to be able to isolate multipotent cells from mesenchymal tissues other than bone marrow. In particular, given their surgical accessibility, it would be convenient to be able to isolate cells from non-osteochondral mesodermal tissues such as, but not limited to, skin, fat and muscle tissue. In the present invention, we refer to these non-osteochondral mesodermal tissues as “soft tissues”.
The presence of different populations of multipotent stem cells in soft tissues derived from the embryonic mesoderm has been reported by several authors. For example, it has been reported that multipotent cells can be obtained from skeletal muscle and other connective tissue of mammals (Young et al. 1993, Rogers et al. 1995). Multipotent cells have also been obtained from human lipoaspirated tissue (Zuk et al., 2001). Another example of multipotent cells isolated from adult connective tissue is the so-called Multipotent Adult Progenitor Cells (MAPC) obtained from bone marrow (Verfaillie et al., 2002). In principle, all these isolated cell populations could be used in the repair and regeneration of connective tissue in a similar fashion to the MSC of bone marrow (Caplan et al., 2001). However, except for MAPC, none of these populations has been, until present, sufficiently characterized at the phenotype level. Therefore, although the presence of multipotent stem cells has been described in different connective tissues, in the current state of the art, it is not possible to identify and unequivocally distinguish between different multipotent cell types obtained from soft tissue, or to obtain a substantially pure population.
Currently, phenotype characterization of stem cells comprises determination of markers such as cell surface receptors, among others; and the determination of their capacity for differentiation in in vitro cultures. Each cell type has a certain combination of surface markers, that is, it has a certain profile of expression that characterizes that particular cell type, distinguishing it from others.
Different combinations of surface markers have been used for identifying and isolating substantially pure populations of hematopoietic stem cells from the bone marrow of mice, such as: [Linneg/low, Thy1.1 low, c-Kithigh, Sca-1+], [Lin−, Thy1.1 low, Sca-1+, rhodamine 123low] (Morrison, S. J. et al., 1995) or [Lin−, CD34−/int, c-Kit+, Sca-1+] (Osawa, M. et al., 1996). Likewise, similar combinations of markers have been used for enriching populations of human hematopoietic stem cells [Lin−, Thy1+, CD34+, CD38neg/low] (Morrison, S. J. et al., 1995).
Currently, it is not known how many markers associated with compromised and differentiated cells are also present in the different mesenchymal stem cell populations. For example, a commonly used marker for enriching mesenchymal stem cells is CD44 (hyaluronic acid receptor). Nevertheless, CD44 is also present in different types of compromised and differentiated cell types. The uncertainty about which markers are associated with the stem cells to allow them to be distinguished from those cells that show a greater degree of differentiation, along with the low percentage of stem cells present in adult cells, has made it difficult to identify and purify populations of adult mesenchymal stem cells.
A significant disadvantage in using adult stem cells resides in the fact that most of the current sources for obtaining stem cells are contaminated with other cell types, complicating the process of identification, isolation and characterization of the populations of stem cell with the objective of using them for therapeutic or other ends. Thus, there is an interest in obtaining a population of multipotent stem cells isolated in a substantially pure form.
The characterization of a multipotent adult stem cell population from non-osteochondral mesenchymal tissue will allow a method for identification and isolation to be designed, as well as the identification of growth factors associated with self-regeneration. Moreover, there may be growth factors associated with the initial phases of differentiation, knowledge of which would allow more efficient in vivo and ex vivo differentiation, as well as for exercising control over the proliferation of stem cells.
The present invention provides a multipotent adult stem cell population from non-osteochondral mesenchymal tissue, preferably from adipose tissue, isolated and characterized by means of immunophenotype markers present on the cell surface, showing their multipotent nature.
- BRIEF DESCRIPTION OF THE INVENTION
Similarly, the present invention provides a method for the identification and isolation of a population of multipotent stem cells from non-osteochondral mesenchymal tissue, dependent on a pattern of characteristic immunophenotype markers, allowing a composition of substantially homogeneous multipotent stem cell markers to be obtained.
A first aspect of the invention relates to an isolated multipotent adult cell from non-osteochondral mesenchymal tissue, characterized in that it is positive for the following markers: CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 and in that it lacks expression of the following markers: CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and CD133.
A preferred aspect of the invention relates to an isolated multipotent adult cell from non-osteochondral mesenchymal tissue obtained by a method that comprises:
- a. Collecting a non-osteochondral mesenchymal tissue;
- b. Obtaining a cell suspension by enzymatic digestion;
- c. Sedimentating the cells and resuspending the cells in an appropriate culture medium; and
- d. Culturing of the cells and elimination of those that show no adhesion.
In which said cell is characterized by being positive to the following markers: CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105, and lacking expression of the following markers: CD11 b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and CD133.
In a most preferred aspect of the invention, the non-osteochondral mesenchymal tissue is a connective tissue, preferably the adipose tissue.
In a still more preferred aspect of the invention, the cells of the present invention can be genetically modified.
A second aspect of the invention relates to cell(s) that express(es) at least one characteristic of a specialized cell, derived from an isolated multipotent adult stem cell of the present invention in which said cell preferably expresses at least one characteristic of an epithelial or endothelial cell or of an adipocyte or a myocyte or a chondrocyte or an osteocyte or a neuron or an astrocyte or an oligodendrocyte or a hepatocyte or a cardiomyocyte or a pancreatic cell.
A third aspect of the invention relates to an isolated population that comprises cells from non-osteochondral mesenchymal tissue characterized in that they are positive for the following markers: CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105, and lack expression of the following markers: CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and CD133.
A fourth aspect of the present invention relates to a method for identifying a population of multipotent adult cells, in which said population consists of isolated cells of the present invention, which method comprises:
- 1) Incubating the cells with labelled specific binding compounds for one or more of the markers that characterize said population; preferably said specific binding compounds are antibodies; and
- 2) Detecting the presence or absence of binding to these specific binding compounds.
A fifth aspect of the present invention relates to a method for isolating a population of multipotent adult cells of the present invention or of a cell object of the present invention, which method comprises:
- 1) Collecting non-osteochondral mesenchymal tissue;
- 2) Obtaining a cell suspension by enzymatic digestion;
- 3) Incubating said cell suspension with labelled compounds that bind specifically to one or more of the surface makers whose presence or absence characterizes said cells; and
- 4) Selecting those cells that have a profile of expression of markers characteristic of that of the cells object of the present invention.
In a preferred aspect of the invention, said method of isolation consists of making a negative selection, excluding those cells that bind to labelled compounds or which bind specifically to CD11b or CD14 or CD15 or CD16 or CD31 or CD34 or CD45 or CD49f or CD102 or CD104 or CD106 or CD133 and a subsequent positive selection of those cells that present binding to labelled compounds that bind specifically to CD9 or CD10 or CD13 or CD29 or CD44 or CD49a or CD51 or CD54 or CD55 or CD58 or CD59 or CD90 or CD105.
A preferred aspect of the invention relates to a population of multipotent adult stem cells from non-osteochondral mesenchymal tissue obtained according to a method of isolation of the invention.
A sixth aspect of the invention relates to a cell composition substantially homogeneous that comprises a cell or cell population object of the present invention. Additionally, said pharmaceutical composition may comprise a pharmaceutically acceptable vehicle or carrier or excipient.
A preferred aspect of the present invention comprises a pharmaceutical composition object of the invention for therapeutic use. In an additional aspect, said pharmaceutical composition is used for the repair and regeneration of tissues.
BRIEF DESCRIPTION OF THE FIGURES
A seventh aspect of the invention relates to a method for evaluating in vitro or in vivo the cell response to biological or pharmacological agents, or to combinatorial libraries of said agents, which comprises:
- 1) Isolating said cell population according to a method of the present invention from an individual or a statistically significant population thereof;
- 2) Optionally differentiating the cells isolated to a specific type of cell;
- 3) Expanding the cells being cultured;
- 4) Optionally differentiating the expanded cells to a specific type of cell;
- 5) Bringing the culture into contact with one or more biological agents or pharmacological agents or with a combinatorial library of said agents; and
- 6) Assessing the possible biological effects of said agents on the cultured cells.
FIGS. 1 a-1 d show histograms of fluorescence immunocytometry corresponding to the profile of surface markers obtained from cells isolated from liposuction samples of a healthy donor. The results show the evolution of the markers studied over time in the culture and in each case it is indicated to what time during the culture period the cells analyzed belong. FIG. 1 a shows the expression of markers on Day 0. FIG. 1 b shows the expression of markers on Day 7 of culturing. FIG. 1 c shows the expression of markers after 4 weeks of culturing and FIG. 1 d shows the expression of markers after 3 months of culturing.
FIGS. 2 a-2 d show the histograms of fluorescence immunocytometry corresponding to the profile of surface markers obtained from cells isolated from liposuction samples from a second healthy donor. The results show the evolution of the markers studied over time in the culture and in each case it is indicated to what time during the culture period the cells analyzed belong. FIG. 2 a shows the expression of markers on Day 0. FIG. 2 b shows the expression of markers on Day 7 of culturing. FIG. 2 c shows the expression of markers after 4 weeks of culturing and FIG. 2 d shows the expression of markers after 3 months of culturing.
FIGS. 3 a-3 d show the histograms of fluorescence immunocytometry corresponding to the profile of surface markers obtained from cells isolated from liposuction samples from a third healthy donor. The results show the evolution of the markers studied over time in the culture and in each case it is indicated at what time in the culture period the cells analyzed belong. FIG. 3 a shows the expression of markers on Day 0. FIG. 3 b shows the expression of markers on Day 8 of culturing. FIG. 3 c shows the expression of markers after 4 weeks of culturing and FIG. 3 d shows the expression of markers after 3 months of culturing.
DESCRIPTION OF THE INVENTION
FIGS. 4 a-4 d show microphotographs of cells incubated in osteogenic medium for 3 weeks. FIG. 4 a shows mesenchymal stem cells from human bone marrow (positive control). FIG. 4 b shows the cells incubated in osteogenic medium for the first week. FIG. 4 c shows the cells incubated in osteogenic medium for the second week. FIG. 4 d shows the cells incubated in osteogenic medium during the third week.
With the objective of designing a method for identification and isolation that allows a defined population of multipotent stem cells to be obtained from a soft tissue, the phenotyping of the human mesenchymal cells obtained from subdermal adipose tissue was done and their evolution was studied during the expansion of the cells in vitro, as well as their capacity for differentiating into different cell lines.
Firstly, expression of a series of surface markers on the adult stem cells from subdermal adipose tissue was monitored by flow cytometry when newly isolated and during the development of the culture in vitro. To do this, a series of commonly used markers were used to identify stem cells, as well as to characterize differentiated cells, including but not limited to: integrins, hematopoietic markers, growth factor receptors and extracellular matrix receptors (see Example 1).
The characterization of multipotent adult stem cells from non-osteochondral mesenchymal tissue by means of determining their immunophenotype profile allows us to define said population in terms of the presence or absence of a certain set of surface markers. These markers are epitopes that can be identified with specific antibodies, constituting a valuable tool that allows us to identify the population, as well as design a strategy for isolation or purification thereof.
The cells of the present invention are characterized in that they are positive for the following markers: CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 and in that they lack expression of the following markers: CD11 b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and CD133.
Once characterized, the cells of the invention were induced to differentiate in vitro into cells that express at least one characteristic of a specialized cell, with the objective of showing their multipotent nature. The methods that can be used to induce differentiation of stem cells of the present invention into different specific cell types are known by those skilled in the art and some of them are explained in detail in Examples 2, 3 and 4 below.
The phenotype characterization of a cell population by surface markers can be performed either by individual staining of the cells (flow cytometry) or by making histological cuts of the population in situ, done in accordance with normal methods.
The determination of the expression profile of surface markers by antibodies (immunophenotype characterization), may be direct, using a labelled antibody or indirect, using a second labelled antibody against the primary specific antibody of the cell marker, thus achieving signal amplification.
On the other hand, the presence or absence of binding to the antibody may be determined by different methods that include but are not limited to immunofluorescence microscopy and radiography. Similarly, it is possible to carry out the monitoring of the levels of binding of the antibody by flow cytometry, a technique that allows the levels of fluorochrome to be correlated with the quantity of antigens present on the cell surface bound specifically to the labelled antibodies.
The differential expression of a series of surface markers on a cell population provides a method for identification and isolation of said population.
In the assay of identification and isolation, the cell population comes into contact with a specific reagent, whether labelled or not, depending on whether the assay is performed by a direct or indirect detection method, respectively. The term “specific reagent” refers to a member of a specific binding pair. As members of a specific binding pair, binding pairs of antigens and antibodies, pairs comprising MHC antigens and T-cell receptors, complementary nucleotide sequences, as well as pairs of peptide ligands and their receptor are included. The specific binding pairs include analogues, fragments and derivatives of the specific member of the binding pair.
The use of antibodies as reagents with affinity is of particular interest. The production of specific monoclonal antibodies will be evident to any ordinarily skilled person in the art. In experiments of identification or separation of cell populations, the antibodies are labelled. To do this, markers are used that include but are not limited to: magnetic particles, biotin and fluorochromes that will allow identification or separation of that cell type to which the antibody has bound. Thus, for example, the analysis of the cell population by flow cytometry allows different antibodies labelled with fluorochromes that emit at different wavelengths to be used in the same sample. Thus, we can know the specific profile of the population for these surface markers, as well as carry out a separation for the set of markers used.
The separation of the populations that present the phenotype of interest can be carried out with affinity separation techniques, which include magnetic separation (using magnetic particles coated with specific antibodies), affinity chromatography, cytotoxic agents bound to monoclonal antibodies or used along with monoclonal antibodies and panning with the antibody attached to a solid support, as well as by other techniques that are appropriate. A more precise separation would be obtained by flow cytometry, a technique that allows the separation of cell populations according to the intensity of staining, along with other parameters such as cell size and cell complexity.
Isolation of Stem Cells from Soft Tissue and Characterization of Surface Markers
The following examples are presented to illustrate the invention, but they in no way limit it.
The isolation of multipotent stem cells from soft tissue was performed by selecting those cells with a capacity for proliferation and differentiation, characterized in that they show adhesion to the plastic container of the cell culture. Then, the cells were characterized by monitoring by flow cytometry of the expression of a series of surface markers on the recently isolated cells and during the course of the culture development in vitro.
The isolation of the multipotent stem cells was carried out from subdermal adipose tissue, obtained by liposuction from three health donors (donors 1, 2 and 3).
First, the sample from the subdermal adipose tissue was washed with phosphate buffered saline solution (PBS). To achieve destruction of the extracellular matrix and the isolation of the cells, an enzymatic digestion was performed with type II collagenase in saline solution (5 mg/ml) at 37° for 30 minutes. The collagenase was deactivated by adding an equivalent volume of DMEM medium, with 10% fetal bovine serum. This cell suspension was centrifuged at 250 g for 10 minutes to obtain a cell deposit. NH4Cl was added at an end concentration of 0.16 M and the mixture was incubated for 10 minutes at room temperature to induce the lysis of the erythrocytes present. The suspension was centrifuged at 250-400 g and resuspended in DMEM-10% FBS with 1% ampicillin-streptomycin. Finally, the cells were plated, inoculating 20-30,000 cells per cm2.
The cells were cultured for 20-24 hours at 37° C., under an atmosphere with 5% CO2. After 24 hours, the culture was washed with PBS to remove the cells and the remains of the tissue in suspension. The cells selected by adherence were cultured in DMEM+10% fetal bovine serum (FBS).
After isolation, the stem cells isolated were characterized from one of the donors, in function of the presence/absence of a series of surface markers. To do this, the expression of the following surface markers was monitored by flow cytometry:
Integrin: CD11b, CD18, CD29, CD49a, CD49b, CD49d, CD49e, CD49f, CD51, CD61, CD104.
Hematopoietic markers: CD3, CD9, CD10, CD13, CD14, CD19, CD34, CD38, CD45, CD90, CD133.
Growth factor receptors: CD105, NGFR
Extracellular matrix receptors: CD15, CD31, CD44, CD50, CD54, CD62E, CD62L, CD62P, CD102, CD106, CD146, CD166
Others: CD10, CD13, CD36, CD55, CD56, CD58, CD59, CD95, HLA-I, HLA-II, β2-microglobuline.
The immunophenotype characterization of the cells were performed on recently isolated cells and also on day 7, after 4 weeks and after 3 months of culture, of the samples from the three healthy donors. Taking into account that the selection is performed by adherence to the plastic of the culture, cells from the cell fraction adhered after less than 24 hours in the culture since isolation are considered as recently isolated cells.
The cells to be characterized were collected by means of gentle digestion with trypsin, washed with PBS and incubated for 30 minutes at 4° C. with fluorescein (FITC) or phycoerythrin (PE) labelled antibody markers against each one of the surface markers to be analyzed. The cell markers were washed and immediately analyzed using the Epics-XL cytometer (Coulter). As controls, cells stained with unspecific antibodies of the corresponding isotopes labelled with FITC or PE were used.
FIGS. 1 a-1 d, 2 a-2 d and 3 a-3 d show the histograms grouped by donor for a better visualization of the evolution of the markers studied during the culturing, indicating in each case to what time in the culture period the analyzed cells belong.
The analysis of surface markers at different times allowed their presence or absence to be determined, as well as their behavior during the culture process. The results obtained show that the cell populations isolated from the different healthy donors show a homogeneous behavior in their phenotype characterization.
From the analysis of the profile of expression of surface markers (FIGS. 1 a
, 2 a
and 3 a
), 3 criteria were used to determine which markers define the cell population and allow it to be identified and differentiated with respect to other types of cell. These criteria are:
- 1. Discard those markers that vary from one sample to the other or over time during culturing.
- 2. Verify that those that are positive are also positive at time zero (recently isolated cells).
- 3. Select them as a function of their biological relevance, discarding markers characteristic of specific cell types (for example, CD3 is a marker exclusive to lymphocytes).
- Example 2
In Vitro Differentiation of Multipotent Stem Cells from Human Non-Osteochondral Mesenchymal Tissue into Bone Phenotype Cells
Applying these criteria, the multipotent stem cell isolated from non-osteochondral mesenchymal tissue, of the present invention, is characterized in being positive for CD9<+>, CD10<+>, CD13<+>, CD29<+>, CD44<+>, CD49A<+>, CD51<+>, CD54<+>, CD55<+>, CD58<+>, CD59<+>, CD90<+> and CD105<+>; and for lacking expression of CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and CD133.
In the differentiation assay, characterized human cells were used. The cells were isolated from the three samples of lipoaspirate analyzed, each corresponding to a healthy donor. A sample of Mesenchymal Stem Cells (MSC) of human bone marrow was used as a the positive control.
The cells isolated were seeded at a density of 10,000 cells/cm2
onto 6-well plates (one plate per sample), and were incubated in standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic). After two days of culturing, the culture medium of one of the wells (control) is replaced with fresh medium, and the remaining wells by osteogenesis inducing medium, which contains the standard culture medium with the following added:
- Dexamethasone 100 nM,
- Ascorbic acid 50 μM; and
- β-Glycerophosphate 10 mM.
The cells are cultured for 3 weeks under normal conditions, changing the medium every 2-3 days. After three weeks, the presence of mineralized deposits of calcium phosphate can be seen, which indicates the presence of osseus nodules. These nodules are detected by staining with Alizarin red (Standford et al., 1995). Specifically, the medium is eliminated, the cells are washed twice with PBS and fixed with 70% cold ethanol for 30 minutes at room temperature. The fixed wells are then washed with PBS and stained with Alizarin red (40 mM, pH 4.1) for 10 minutes at room temperature. The stained cells are washed with abundant water, and the precipitates of calcium phosphate, which appear strongly stained red, are examined under the microscope.
- Example 3
In Vitro Differentiation of Multipotent Stem Cells from Human Non-Osteochondral Mesenchymal Tissue into Muscle Phenotype Cells
FIGS. 4 a-4 d show microphotographs of the osteoinduced cells stained with Alizarin red. Although the formation of calcium phosphate is quicker in the sample corresponding to MSC from bone marrow which acts as a positive control (FIG. 4 a), in the three samples from the adipose tissue, the formation of large quantities of bone matrix can be discerned, although with differing intensity in each of the samples. All wells in which osteogenesis was induced showed the same behavior and in the control wells (not submitted to osteogenic stimuli) the formation of bone matrix was not detected. No relationship was seen between the amount of bone matrix formed and the time that each sample was being cultured after isolation from the tissue (between 3 and 9 weeks).
In the differentiation assay, characterized human cells were isolated from the three liposuction samples each corresponding to a healthy donor, as well as a sample of Mesenchymal Stem Cells (MSC) of human bone marrow, which was used as the positive control.
The cells isolated were seeded at a density of 10,000 cells/cm2
into standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic). After two days of culturing, the culture medium of one of the wells (control) is replaced with fresh medium, and the remaining wells by myogenesis inducing medium (Wakitani et al., 1995), which contains the standard culture medium with the following added:
- Ascorbate-2-phosphate 0.1 mM,
- Dexamethasone 0.01 μM,
- ITS+1 (Sigma-Aldrich), and
- 5-Azacytidine 3 μM.
- Example 4
In Vitro Differentiation of Multipotent Stem Cells from Human Non-Osteochondral Mesenchymal Tissue into Neuronal Phenotype Cells
After 24 hours, the medium is replaced by standard culture medium, and the cells are cultured for 2-3 weeks, changing the medium every 2-3 days. After this time, the cells acquire an elongated phenotype, form fibrillar structures and some cell fusions can be seen. To detect the myoblast phenotype, the cells obtained are fixed with paraformaldehyde (PFA) at 4% and incubated with an antibody against the heavy chain of myosin, which is the specific antigen for muscle.
In the differentiation assay, characterized human cells were used. These cells were isolated from the three samples of lipoaspirate analyzed, each corresponding to a healthy donor, as well as a sample of Mesenchymal Stem Cells (MSC) from human bone marrow, which was used as the positive control.
The cells isolated were seeded at low density into standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic), supplemented with 10 ng/ml bFGF and incubated for 24-36 hours to yield a large number of cells. The wells are then washed and neuron-inducing medium is added (Black and Woodbury, 2001), the medium comprising:
- α MEM,
- BHA 200 μM,
- L-Glutamine 2 mM,
- Forskolin 10 μM,
- 2% DMSO,
- Hydrocortisone 1 μM,
- Insulin 5 μg/ml,
- ClK 25 mM, and
- Valproic acid 2 mM.
A few hours after induction, a morphological change can be observed; the cells acquire a rounded shape and very refringent, with prolongations with a similar appearance to axons and dendrites of nerve cells. After 3 days, the cells obtained are fixed with PFA at 4% and incubated with antibodies against neuron specific antigens NF-200 and TuJ1.
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