WO2009092789A1 - Medium for propagating and expanding stem cells - Google Patents

Abstract

The present invention relates to a growth medium for in vitro stem cell expansion comprising: Selenium 5ng/ml to 0.1mg/mL, Transferrin 5mg/ml to 100mg/mL, Insulin 2.5μg/ml to 1mg/mL, Pyruvate, preferably sodium pyruvate, 0.05 to 1 mM, L-glutamine 0.5 to 10 mM, Nucleosides 1 to 100 μg/ml at least one amino acid, preferably non-essential amino acid 5 to 1000 μg/ml, Iscove's modified Dulbecco ' s medium (IMDM) up to 1L.

Description

Medium for propagating and expanding stem cells

The present invention relates to a plain medium for cultivating stem cells, which in combination with cytokines allow the propagation and the expansion of different types of stem cells derived from alternative sources that retain the capacity to transdifferentiate into different cell lineages.

Stem cells are commonly defined as cells, which exist for the lifetime of an organism and are able to undergo symmetric and/or asymmetric divisions, to give rise to further stem cells (for preservation of the stem cell pool) and to more differentiated cells with defined lifetime (for organ-specific functions) . Due to this unique propertystem cells are ideal vehicles for somatic gene therapy. Indeed, once stably transfected, they would maintain the transgene for the lifetime of the tissue and the organism, and would carry the transgene expression into the differentiated cells. Stem cells may be totipotent (e.g. embryonic stem cells), pluripotent (e.g. hematopoietic stem cells, neural glial stem cells, hepatocyte stem cells, chondrocytic stem cells) or unipotent (e.g. keratinocytic stem cells, muscle precursor cells, tracheal epithelial precursor cells) .

It is an object of the present invention to provide a growth medium for the in vitro expansion of stem cells.

The present invention relates to a (plain) growth medium for in vitro (adult) stem cell expansion comprising:

Selenium 5ng/ml to O.lmg/mL,

Transferrin 5mg/ml to lOOmg/mL,

Insulin 2.5μg/ml to lmg/mL,

Pyruvate, preferably sodium pyruvate, 0.05 to 1 mM, L-glutamine 0.5 to 10 mM,

Nucleosides 1 to 100 μg/ml at least one amino acid, preferably non-essential amino acid

5 to 1000 μg/ml,

Iscove's modified Dulbecco ' s medium (IMDM) up to IL. The medium according to the present invention can be used in combination with a specific cocktail of cytokines to expand and/ or to cultivate different types of stem cells while maintaining intact their differentiation potentiality towards specific cell types. The transferrin present in the plain medium according to the present invention is of human origin, whereby the insulin is synthetically produced, preferably with recombinant methods.

Stem cells and in particular adult stem cells can usually not be cultured with (basic) IMDM. However, it could be shown that the addition of further components results in a medium, which can be used to expand and propagate adult stem cells.

The adult stem cells, which can be propagated with the medium according to the present invention, may be obtained preferably from cord-blood, placenta or amniotic fluid. Thereby the inventive medium ("HMF-SCm") shows several advantages in comparison to similar products known in the art:

1. High rate of pure stem cells/erythroid cells harvesting

2. No addition of sera required

3. Suitable for different type of SCs

4. Suitable for different cell types

5. No need of enrichment steps

6. No need of cell-derived feeding layers

7. No need to feed the cells every day

8. High and stable cell viability

9. High rate of cryo-preservation with different freezing media: cell phenotype and functionality mantained after repeated (up to three times) cycles of freezing/ thawing

10. High transdifferentiation potentialities

11. Stable undifferentiate state of SCs and erythroblasts with high cell proliferation

12. Absence of phenol red and therefore suitable for studies with minimal quantity of steroid hormones

13. Medium stability at -20⁰C and -180⁰C: 2 years

14. Medium stability at 4°C: 1 week

HMF-SCm fulfils quality criteria, which result in:

1. A strong improvement of stem cells expansion to quantities suitable for large transdifferentiation studies (50-100*10⁶ SCs) involving human stem cells derived from minimal quantities of source tissues such as cord-blood/amniotic fluid (50-100 mL) or from a placenta

2. A high-rate stem cells expansion in a very defined and restricted time (e.g. 10 days) 3. Possibility to obtain at the same time significant amounts of HSC and MSCs from the same source (e.g. 10 days) according to the use of different cocktails as described in this patent

4. Re-vivo use according to the GMP ("Good Manufacturing Practice") standard

Such characteristics make HMF-SCm particularly indicated either for research studies or for clinical trials based on the ex-vivo expansion of SCs potentially followed by re-vivo reinjection .

As mentioned above the medium of the present invention can be used for the specific expansion and the subsequent transdifferentiation of different sub-types of stem cells such as hematopoietic, mesenchymal or amniotic-derived stem cells.

1. Hematopoietic stem cells (HSCs) : these cells can be isolated from bone marrow, peripheral blood, placenta and cord-blood to be subsequently expanded and trans-differentiated in vitro into either white or red blood cells. The in vitro expansion of specific blood cell types may play a major role in the cure of several cancers, such as acute and chronic leukaemia, myelodys- plastic syndromes, aplastic anaemia, myelo- and lymphoprolifer- ative disorders, phagocyte disorders and other genetic disorders, such as congenital thrombocytopenia. In particular, it can help to optimize immunotherapies based on the generation of high amounts of autologous dendritic cells (DCs) . Indeed, such cells are present in minimal amount (less than 0.01-0.1% of the total number of the blood cells) in human blood and their in vitro generation requires a high amount of precursors (monocytes) that presently can be obtained exclusively through patient's leukapheresis, a very discomfortable procedure that may hinder the eligibility of the patients for such treatment. A major issue to be faced in order to design an optimal protocol for the expansion of the HSCs is to set the parameters describing their phenotype. Because HSCs look and behave in culture like ordinary white blood cells, this has been a difficult challenge and this makes them difficult to identify by morphology (size and shape) . Even today, scientists must rely on cell surface proteins, which serve, only roughly, as markers of white blood cells. The challenge is formidable as about 1 in every 10,000 to 15,000 bone marrow cells is thought to be a stem cell. - A -

In the blood stream the proportion falls to 1 in 100,000 blood cells. HCSs are ultimately responsible for the constant renewal of blood. The transplantation of bone marrow to restore a healthy blood system in leukaemia patients is limited by the unavailability of HSCs in quantity and purity that are crucial for successful transplantation. Because of their relative rarity

(one in every 10,000 bone marrow cells) and the difficulty of separating them from other components of the blood, bone marrow transplants are generally impure. Obtaining purified hematopoietic stem cells is a major challenge, and purification in a clinical setting is expensive and difficult.

HMF-SCm, the medium of the present invention, has been designed to selectively support the expansion of the HSCs and to allow the recovery of about 5-10*10⁷ pure HSCs within 10 days of cell culture. The harvested cells can be frozen and cryostored for further use or immediately transdifferentiated into a specific cell type.

2. Mesenchymal stem cells (MSCs). Several studies have demonstrated that multipotent MSCs, under specific permissive conditions, can be induced to differentiate into bone, adipose, cartilage, muscle, and endothelium. In particular, MSCs, because of their unique immunologic characteristics that suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo, may survive longer in a xenogeneic environment. Data from preclinical transplantation studies suggested that MSCs infusions not only prevent the occurrence of graft failure but also have immunomodulatory effects. Nevertheless, MSCs are a rare population (approximately 0.001-0.01%) of adult human bone marrow and of the adult peripheral blood and their number significantly decreases with age. Moreover, MSCs are also relatively few in term cord blood. In this context, a recent study showed that a population of MSC-like cells exists within the umbilical vein endothelial/sub endothelial layer. However, term CBs compared with preterm ones are a poor source of MSCs. As shown by Miao et al . , (2006), it is possible to isolate a population of pluripo- tent cells from the human term placenta, a temporary organ with fetal contributions that is discarded postpartum and that exhibits many markers common to bone marrow mesenchymal stem cells

(BMSCs) . Placental MSCs (pMSCs) may therefore provide an ethically uncontroversial and easily accessible source of multipotent cells for future experimental and clinical applications.

Using routine cell culture techniques, pMSCs can be successfully isolated and expanded in vitro. Although the initial cell culture consists of both fibroblastoid and non-fibroblastoid cell types, only the fibroblastoid population remain after enzymatic digestion and passaging. In their undifferentiated state MSCs are spindle-shaped and resemble fibroblasts. There are no markers, which specifically and uniquely identify MSCs and therefore they are defined by their immunophenotypic profile as well as by their characteristic morphology; indeed, while MSCs express neither haemopoietic markers (e.g. CD45, CD34, CD14) nor endothelial markers (e.g. CD34, CD31, vWF) , they do express a large number of adhesion molecules (e.g. CD44 and integrins) , some stromal cell markers (e.g. SH-2, SH-3 and SH-4) and some cytokine receptors (e.g. IL-IR, TNF-R) .

Currently, the presence of MSCs in umbilical CB is still contested, and in reports that document isolation of such cells, low yield and inter-individual variation are typical limitations. On the contrary, pMSCs can be easily isolated and expanded without morphological and characteristic changes in medium supplemented only with FBS. Therefore, the placenta may prove to be an attractive and rich source of MSCs. The presence of stem cells in the placenta could have tremendous implications. The initial data on the differentiation capabilities of pMSCs are promising, and there may be important therapeutic uses for these cells. Along with the ease of accessibility, lack of ethical concerns, and abundant cell number, pMSCs may be an attractive, alternative source of progenitor or stem cells for basic research.

HMF-SCm, the medium of the present invention, has been designed to selectively support the expansion of the pMSCs and to allow the recovery of about 4-8*10⁷ pure pMSCs within 10 days of cell culture. The harvested cells can be frozen and cryostored for further use or immediately transdifferentiated into a specific cell type.

3. Amniotic stem cells. Amniotic epithelial cells develop from the epiblast by eight days after fertilization and prior to gastrulation, opening the possibility that they might maintain the plasticity of pre-gastrulation embryo cells. Amniotic epi- thelial cells isolated from human term placenta express surface makers normally present on embryonic stem and germ cells. In addition, amniotic epithelial cells express the pluripotent stem cell specific transcription factors octamer-binding protein 4 (Oct-4), and Nanog. Under certain culture conditions, amniotic epithelial cells form spheroid structures, which retained stem cell characteristics. Amniotic epithelial cells do not require other cell-derived feeder layers to maintain Oct-4 expression, do not express telomerase and are non-tumorigenic upon transplantation. Based on immunohistochemical and genetic analysis, amniotic epithelial cells have the potential to differentiate to all three-germ layers-endoderm (liver, pancreas) , mesoderm (cardi- omyocytes) , and ectoderm (neural cells) in vitro. Amnion is derived from term placenta, following live birth, thus it may be a useful and non-controversial source of stem cells for cell transplantation and regenerative medicine.

The medium (HMF-SCm) of the present invention is particularly suited to culture human hematopoietic, mesenchimal and fibroid-like stem cells from cord-blood, placenta and amniotic fluid as well as to culture human erythroid cells from cord-blood and placenta without the need of intermediate enrichment steps, such as the use of magnetic beads. Hematopoietic cells are cultured in absence of feeding layers and/or a coating mix. The mesenchimal/fibroid-like stem cells are cultured in absence of feeding layers but on a coating mix, prepared according to the protocol described herein. The different types of SCs are separately cultured in presence of different growing factors (GFs) mixtures, developed in order to guarantee the maximum harvesting rate of the selected cells. All cell types cultured in HMF-SCm are stable upon cryopreservation in glycerol or DMSO between -80 und -180⁰C. These cells can be kept in culture for a maximum period of 20 days as pluripotent stem cells or expanded for a maximum of two months when cultured in presence of a cocktail that cause their differentiation towards erythroid cells under the shape of immature erythroblasts . In this case, the addition of the appropriate maturation cocktail causes the terminal differentiation of the said erythroblasts into fully enucleated erythrocytes. According to the protocol designed for the expansion of pluripotent SCs and/or of the immature precursors of a specific cell lineage, the cells must be fed every second day with the cocktail required for their optimal proliferation/expansion. Such procedure guarantees both cell expansion and cell viability, which tends to remain over 85% of the total amount of cells present in the culture.

The amount of a mixture of selenium, transferrin and insulin in the medium of the present invention is preferably 1 to 20% (V/V) .

According to a preferred embodiment of the present invention, the expansion of the pMSCs can be further increased by the addition of LDL, preferably human LDL, to a final concentration comprised between 1 and 100 μg/ml . Moreover LDL addition helps to increase the cell viability while decreasing the rate of the spontaneous differentiation.

According to another preferred embodiment of the present invention the medium comprises further an iron salt, preferably ferrous sulphate, in an amount of 1 to 100 nM when used for the expansion of the HSCs.

According to the present invention a medium comprising the following components is particularly preferred:

Selenium 70μg/mL,

Transferrin 50mg/mL,

Insulin 0.5mg/mL,

Pyruvate, preferably sodium pyruvate, 0. ImM, L-glutamine 2mM,

Nucleosides lOμg/ml

Non-essential amino acid lOOμg/ml,

Iscove's modified Dulbecco ' s medium (IMDM) up to IL.

According to the most preferred embodiment of the present invention the medium comprises:

Selenium 70μg/mL,

Transferrin 50mg/mL,

Insulin 0.5mg/mL,

Pyruvate, preferably sodium pyruvate, 0. ImM,

L-glutamine 2mM,

Nucleosides lOμg/ml, Non-essential amino acid lOOμg/ml,

Iron Sulphate 40 nM, human LDL 40μg/mL Iscove's modified Dulbecco ' s medium (IMDM) up to IL.

In order to allow the expansion of different types of stem cells, the plain medium described above may be added up with at least one cytokine selected from the group of SCF, GM-SCF, TPO and Fit-3 in a final concentration comprised between 1 and 200μg/mL. It is particularly preferred to add to the medium:

SCF lOOμg/mL

GM-CSF 3.5μg/mL

TPO 7μg/mL

Flt-3 20μg/mL

Another aspect of the present invention relates to the use of a medium according to the present invention for cultivating/expanding hematopoietic, amniotic and/or mesemchymal stem cells.

Yet another aspect of the present invention relates to a method for culturing hematopoietic, amniotic and/or mesenchymal stem cells comprising the steps of:

- Providing a stem cell source, and

- incubating said source with medium according to the present invention.

The present invention is further illustrated in the following figures and example, however, without being restricted thereto .

Fig. 1 shows cell phenotypes of HSC, derived from CB, and cultured in different media and GF mixtures.

Fig. 2 shows hemoglobin accumulation by HSCs differentiated into erythrocyte in different media and with different cytokines (GFl vs GF2) .

EXAMPLE :

Materials and Methods

SC expansion

The Sc source for the experiments described below were:

1. Term (38-40 weeks gestation) placentas

2. 5OmL of term CB

3. 5OmL of amniotic fluid obtained from healthy donor mothers. CB-derived cells were ficolled and put in culture. The placenta tissue was washed in PBS, mechanically minced, enzymatically digested with 0.25% trypsin-EDTA, filtered and centrifuged. The cells were finally collected and the erythrocytes lysed. The homogenate was subsequently pelleted, resuspended in HMF-SCm medium added up with different SC cocktails and cultured at 37°C with a water-saturated atmosphere and 6.5% CO₂. Medium was replaced every second day.

The growth kinetics of SCs is assessed every day for two weeks .

1. HMF-SCm:

SCs transdifferentiation

The differentiation potential of HSCs towards erythroid cells has been investigated as follows.

After two days expansions, the HSCs were differentiated into erythroid precursors upon incubation with 1 U/mL human EPO, 100 ng/mL SCF, 10^"6M Dex and 40 ng/mL IGF-I and after six days were further differentiated into fully mature erythrocyte upon incubation with 10 U/mL Epo, 10 ng/mL insulin, 3xlO^"6 M ZK112.993, and 1 mg/mL iron-saturated human transferrin. Alternatively, cells were cultured four days in HMF-SCm + HMF-GFs and terminally differentiated into erythrocytes by incubation with 10 U/mL Epo, 15 ng/mL insulin, 100 ng/mL IL-3, and 1.5 mg/mL iron-saturated human transferrin.

Other differentiation potentials of SCs were investigated in cell type-specific media.

Differentiation into endothelial-like cells was induced by culturing semi-confluent cells in presence of 2% fetal calf serum and 50 ng/mL vascular endothelial growth factor (VEGF) , while the endothelial phenotype is detected by staining with antibody against Von Willebrand Factor (vWF) .

For neuron-like or astrocyte-like cells differentiation, cells were cultured in the presence of 2% dimethyl sulfoxide (DMSO) and lOOxlO^"6 M butylated hydroxyanisole (BHA) . The differentiated cells were identified by staining with antibodies against neuron specific enolase (NSE) and glial fibrillary acid protein (GFAP) .

The stem cells differentiation into smooth muscle cells was induced by culturing them in the Quantum 212 (PAA) plus Supple- mentalPack (PromoCell, C-39262), whereas their differentiation into skeletal muscle cells is induced by culturing them in the Quantum 212 (PAA) plus SupplementMix (PromoCell, C-39360) .

SmMC were detected by ASMA, smoothelin, calponin, caldesmon and SM22. SkMC were detected by anti-skeletal myosin.

Stem cells were cultured in the Quantum 212 plus Supplement- Mix (PromoCell, C-39262) or in the Quantum 212 plus Supplement- Mix (PromoCell, C-39360) for about two weeks, washed with PBS, and incubated with a bath solution consisting of 155 inM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, and 10 mM Hepes. Contraction was elicited by incubation with 10^"5M carbachol, and relaxation was subsequently elicited by addition of 10^"4M atropine. The ability of differentiated muscle cells to contract in response to 10^"11M, 10^"8M, and 10^"5M carbachol was evaluated to- gether with their ability to respond to atropine, investigated by incubation in 10^"4M atropine for 45 min followed by addition of carbachol through the use of the 3D collagen culture kit (Chemi- con, ECM675) .

The stem cells were induced to differentiate into myocytes by culturing them in D-MEM: M199 (4:1), supplemented with 15% horse serum, 5% FBS, 5mM of sodium butyrate (or 2% DMSO) and 10 mM of BDM (2 , 3-butanedione monoxime; Sigma B0753-100G) , in order to impair myofibrillogenesis . The myocytes were detected by desmin (Chemicon, MAB3430) and connexin 43 (Chemicon, AB1728) .

FACS

Stem cells were detected by the evaluation of the following markers expression:

ABC-G2, SSEA-4, CD34, CDl 17, CD133, CD45, CD9, CD166, CDlO, CD3, CD19, CD3, CD44, CD105, CD73, CD13, CD90, HLA-DR, HLA-ABC, CD29 and CD14.

Erythroid cells were detected by the evaluation of the following markers expression:

CD71, GPA, CD34, CD36, CD117, CD14, CD3 and CD45,

Hemoglobin accumulation

To quantify the hemoglobin accumulation, every day 50 μL of cell suspension were applied in triplicate to a 96 well plate and centrifuged for 5 min at 1200 rpm, washed once with PBS, eventually resuspended in 30 μL of milliQ water and stored at - 80⁰C.

At the end of each set of experiments, the plates were stained as described in the following protocol: the plates were thawed and 125 μL of citrate phosphate buffer (IV of 0.1 M citrate mixed with 2V of Na₂HPO₄) mixed with 0.5 mg/ mL O-phenylenediamide and H₂O₂ (lμL/mL) were added to each well.

As soon as the reaction mix in the wells became yellow, the reaction was stopped by adding 25 μL H₂SO₄ (8 N) to each well. The extinction was measured by the ELISA photometer at 492 nm and counter-measured at 620 nm, a mean to correct artifacts, due to the presence of particles and/or bubbles. Values were obtained from triplicate determinations, averaged and normalized to cell number and cell volume.

Proliferation assay

Cells were thawed and co-cultured at a final concentration of l*10⁴ cells/well in a 96-well U-bottom plate in HMF-SC + HMF- GFs, supplemented with 10% AlamarBlue at a final volume of 150 μL . Proliferation caused reduction of the dye changing from oxidized (non-fluorescent, blue) form to reduced (fluorescent, red) form. For 2-7 days, every 4-12h, the absorbance was measured in a plate-reader at 595 nm with reference wavelength at 620 nm.

Western blot

Cytoplasmatic fraction

The cells were lysed in Frackelton buffer for 20 min in ice and centrifuged at lOOOg 10 min at 4°C.

After collecting the supernatant, the protein concentration was measured with the Bradford assay and blue sample buffer was added to each sample in a ratio of 1:4.

Nuclear fraction

The cells were lysed in NP40 buffer for 5 min in ice and centrifuged at 80Og 3 min at 4°C.

After collecting and resuspending the pellet, the protein concentration was measured with the Bradford assay and blue sample buffer was added to each sample in a ratio of 1:4.

After preparing a polyacrylamide gel, the proteins were run and blotted on a nitrocellulose membrane.

Thereafter, the membrane was washed in TBS-T and incubated at first Ih in the blocking solution, made up of TBS-T IX, added up with 0.05% Tween-20 and milk 5% and then in the primary antibody, diluted in TBS-T w/o milk, added up of 3% BSA and 0.02% NaN₃, O/N.

Afterwards the membrane was washed 3X in TBS-T and incubated with the secondary antibody for 40 min at RT.

Finally, the protein expression was detected with the ECL kit.

Immunofluorescence (IF)

The cells were washed with cold PBS twice for 5 min; fixed with MetOH + 5 mM EDTA (-20⁰C), for 7 min; washed with cold PBS pH=8 twice for 5 min and with Q/W IX 5 min, before permeabiliz- ing them with Triton 0.05% 10 min at RT and washing them with Q/ W 3X 5 min.

Thereafter the cells were incubated with a blocking solution made of 0.2% gelatin, 0.3% Triton, 50 mM NH₄Cl and 10% horse serum in IX CB for 30 min at 37⁰C and with the primary antibody, diluted in 0.2% gelatin, 0.3% Triton, 50 mM NH₄Cl and 10% horse serum in IX CB for Ih at 37°C, before washing with Q/W 3X 5 min and incubating with the secondary antibody, diluted in 0.2% gelatin, 0.3% Triton, 50 mM NH₄Cl and 10% horse serum in IX CB for Ih at RT. Thereafter the cells were washed with Q/W 3X 5 min; incubated with a blocking solution made of 10% horse serum, 1% BSA, 0.02% NaN₃ in PBS pH=8 for 30 min at RT and washed with cold TBS pH=8 3X 5 min. Finally, the cells were incubated with the primary antibody diluted in 1% BSA in TBS pH=8 (40 μL) O/N, washed with cold TBS pH=8 3X 5 min, incubated with the secondary antibody (1:1500) diluted in 1% BSA in TBS pH=8 for Ih at RT and washed with cold TBS pH=8 3X 5 min.

At the end, the coverslip was mounted with ProLong Antifade kit.

Model of heart failure

Myocardial neovascularization was assessed by capillary density; LV remodelling was evaluated by the percent of fibrosis area. To evaluate LV function, transthoracic echocardiography was performed immediately before as well as 5 and 28 days after MI. Under general anesthesia with ketamine and xylazine, LV end- diastolic and end-systolic dimensions (LVEDD and LVESD, respectively) and fractional shortening (FS) were measured at the mid- papillary muscle level. Regional wall motion score (RWMS) was evaluated.

Immediately after the final echocardiography on day 28, the rats underwent cardiac catheterization for more invasive and precise assessment of global LV function. A 2.0F micromanometer- tipped conductance catheter was inserted via the right carotid artery into the LV cavity. LV pressure and its derivative (LV dP/dt) were continuously monitored with a multiple recording system.

Heart rate, LV end-diastolic pressure (LVEDP), LV ejection fraction (EF) , and the maximum and minimum LV dP/dt (+dP/dt and - dP/dt, respectively) were recorded continuously for 20 minutes. All data were acquired under stable hemodynamic conditions.

Upon killing, only those rats with infarct size >25% of the left ventricle area, were included in the study.

Tissue Harvesting

All rats were killed 28 days after transplantation with potassium chloride. At necropsy, hearts were sliced into 4 transverse sections from apex to base, embedded in OCT compound, snap- frozen in liquid nitrogen, and stored at -80⁰C.

Rat hearts in OCT blocks were sectioned, and 5 μm serial sections were collected on slides, followed by fixation with 4% paraformaldehyde at 4°C for 5 min and stained immediately.

Morphometric Evaluation of Capillary Density and Infarct Size

Histochemical staining with isolectin B4 was performed, and capillary density was morphometrically evaluated by histological examination of 5 randomly selected fields of tissue sections, recovered from segments of LV myocardium, subserved by the occluded left anterior descending coronary artery. Capillaries were recognized as tubular structures positive for isolectin B4. To elucidate the severity of myocardial fibrosis, Masson trichrome staining was performed on frozen sections from each tissue block, and the stained sections were used to measure the average ratio of fibrosis area to entire LV area (percent fibrosis area) . Masson trichrome staining was performed with a kit (Dako) .

Morphological and Histological Studies

After completion of all measurements, the heart was arrested with potassium chloride and rapidly excised. The coronary arteries were perfused with 100 mL 10% formaldehyde, and the heart was fixed in diastole position with an intraventricular pressure of 30 mmHg in formaldehyde solution. The fixed heart was sliced into 5 mm thick slices and photographed. Cross-sectional areas of the ventricular muscle, scar area, and scar thickness were measured. A cube of tissue of approximately 5 mm from the center of the infarct zone was embedded in paraffin and cut into 10 μm sections. Serial cutting sections were used for immunohistochemical studies to localize the transplanted cells, infiltrated cells, and contractile protein positive cells.

For angiogenesis analysis, the myocardial biopsies were sectioned and immunohistochemically stained for factor VIII and α- smooth muscle actin. The number of large blood vessels (both factor VIII and smooth muscle actin positive) and capillaries (factor VIII positive only) were counted in a blind trial and compared between the groups . Results

SCs expansion after 10 days culture from fresh isolated cells from placenta and cord-blood

Cells isolated from cord blood (CB) or placenta, were cultured in presence of HMF-GF mix, on MASC coated dishes for 1 day. After 24h, the non-adherent cells (HSCs) were transferred into non-coated dishes and cultured for additional nine days. The adherent cells (MSCs/Fibroid-like SCs) were further cultured on coated dishes. The medium + GFs was replaced every second day.

After ten days, about 8*10⁷ HCSs and about 5*10⁷ MSCs/Fibroid-like SCs were harvested and their phenotype was analysed by FACS.

Non-adherent cells derived from placenta and cultured in uncoated dishes were positive for both HSCs markers and MSCs markers. Adherent cells derived from placenta and cultured in coated dishes were mildly positive for HSCs markers and high positive for MSCs markers. All these data together argued that placenta was a better source for MSCs than for HSCs and that non adherent cells, derived from placenta still expressed many MSCs markers, along with HSCs ones.

Table 1. Analysis of the phenotype of cells derived from placenta and cultured with HMF-SC, added up with HMF-GFs mix, in presence (MSC/fibroid-like SCs) and in absence (HSCs) of the coating solution according to the present invention.

Non adh. Adh HSC ABC-G2 21.7 12.2

ABC-G2+CD34 20.9 11.5

ABC-G2+CD133 21.4 12.2

CD34 45.9 18.9

CD133 24.4 16.4

CD34+CD133 22.7 13.8

CD117 43 33

CD34+CD29 20.5 18.8

CD34+CD105 22.7 14.8

CD34+ HLA-DR 9.5 5.5

CD34+CD44 23.6 9.8

CD34 + HLA-ABC 23.8 16.8 MSC CD105 30 48.6

HLA-ABC 60.5 73.6

CD105 + HLA-

20.6 45.9 ABC

HLA-DR + HLA-

4.7 4.5 ABC

CD105 + HLA-DR 7.4 4.8

CD44 94.7 98.4

CD29 41.3 55.2

CD44+CD29 40.5 41

HLA-DR+CD29 6.1 4.4

HLA-DR+CD44 5.4 5.1

HLA-DR 10.1 9.6

CD3 7 2.8

CD14 15 16.7

CD19 5.5 5.5

T-cell contamination (CD3⁺ cells) constituted less than the 7%, B-cell contamination (CD19⁺ cells) less than the 6% and monocyte contamination (CD14⁺ cells) less than the 17% of the final cell number upon completion of the culture. Less than the 11% of the cells present upon completion of the experiment bore HLA-DR on their surface.

Non-adherent cells derived from CB and cultured on uncoated dishes were strongly positive for the HSC markers but almost completely negative for the MSCs ones. On the opposite, adherent cells derived from CB and cultured on coated dishes while being almost completely negative for HSCs markers became strongly positive for the MSCs ones. All these data together argue that CB may represent an excellent source for both MSCs and for HSCs. Moreover, CB-derived HSCs accumulate in the non-adherent portion and the MSC/fibroid-like SCs accumulate in the adherent portion. Therefore, CB is a suitable source for both kinds of SCs, which may become easily separated upon completion of the culture according to their different adherence properties.

T-cell contamination (CD3⁺ cells) constituted less than the 7.5%, B-cell contamination (CD19⁺ cells) less than the 6% and monocyte contamination (CD14⁺ cells) less than the 17% of the fi- nal cell number upon completion of the culture. Less than the 11% of the cells present upon completion of the experiment bore HLA-DR on their surface (Table 2) .

Table 2. Analysis of the phenotype of cells derived from CB and cultured with HMF-SC, added up with HMF-GF mix, in presence (MSC/fibroid-like SCs) and in absence (HSCs) of the coating solution according to the present invention. non adh adh

HSC ABC-G2 31.7 12.2

ABC-G2+CD34 30.9 11.5

ABC-G2+CD133 31.4 12.2

CD34 45.9 18.9

CD133 44.4 16.4

CD34+CD133 42.7 13.8

CD117 63 33

CD34+CD29 30.5 18.8

CD34+CD105 28.7 14.8

CD34+ HLA-DR 9.5 5.5

CD34+CD44 33.6 9.8

CD34 + HLA-ABC 43.8 16.8

MSC CD105 9.4 45.6

HLA-ABC 10.5 73.6

CDl 05 + HLA-ABC 0.6 35.9

HLA-DR + HLA-ABC 4.7 14.5

CD105 + HLA-DR 7.4 4.8

CD44 3.7 98.4

CD29 1.3 55.2

CD44+CD29 0.5 41

HLA-DR+CD29 0.1 4.4

HLA-DR+CD44 2.4 5.1

HLA-DR 10.1 9.6

CD3 7 2.8

CD14 15 16.7

CD19 5.5 5.5 SCs expansion after 10 days culture from frozen cells from placenta and cord-blood

In order to determine whether cells derived from CB or placenta, cultured in HMF-SC for ten days and stored deep-frozen (-180⁰C) in DMSO 10% or Glycerol 10% freezing solution, were able to maintain their original phenotype upon thawing, such cells were deep-frozen, thawed and put in culture in presence of the right medium for 24h before being analysed by FACS.

The following six batches were analysed:

1. CB-derived HSCs, frozen in DMSO

2. CB-derived HSCs, frozen in DMSO

3. Placenta-derived HSCs, frozen in DMSO

4. Placenta-derived HSCs, frozen in Glycerol

5. CB-derived HSCs, frozen in DMSO (2^nd freezing/thawing cycle)

6. Placenta-derived HSCs, frozen in DMSO (3^rd freezing/thawing cycle)

After thawing, all the cells maintained either their HSC or MSC phenotype as well as a high proliferation level (CD117⁺) . In comparison with the contamination levels by T- and B- cells as well as by monocytes observed upon harvesting of the primary culture before the freezing step, T-cell contamination (CD3⁺ cells) decreased below 5% of the total amount of cells; B- cell contamination (CD19⁺ cells) below the 15% and monocyte contamination (CD14⁺ cells) showed the strongest decrease (below the 7.5% of the total amount of cells) .

Table 3A. Analysis of the phenotype of HSCs derived from CB or placenta, frozen in DMSO or in Glycerol, thawed and re-cultured with HMF-SC, added up with HMF-GF mix.

CB CB Placenta

(DMSO) (DMSO) (DMSO)

ABC-G2 20 23 22.8

ABC-G2+CD34 19.4 1,7 19.3

ABC-G2+CD133 19.8 20.3 22.1

CD34 23.5 3.6 44.2

CD133 33.3 29.4 33.8

CD34+CD133 21.8 2.7 32.1 CD117 65.1 55.9 58.8

CD36 17.5 4.9 6.7

GPA 3.9 1.8 2.9

CD3 0.1 0.5 4.6

CD14 0 6.5 7

CDl9 0 11.4 14.3

HLA-DR 0 11.1 13.4

Placenta CB CB

(Glycerol) (Glycerol) (DMSO)

ABC-G2 16.5 33.6 37.6

ABC-G2+CD34 12.7 30.5 31.8

ABC-G2+CD133 11.5 22.7 24.4

CD34 41.4 24.3 28.5

CD133 35.9 37.6 36.2

CD34+CD133 32.8 21.9 25.3

CD117 56.4 45.6 42.1

CD36 2.9 2.1 0.6

GPA 1.1 0.4 0.3

CD3 0.9 0.1 0

CD14 1.8 0.4 0

CDl9 0 0.3 0.3

HLA-DR 10.4 9.6 11.2

Table 3B. Analysis of the phenotype of MSCs derived from CB or placenta, frozen in DMSO or in Glycerol, thawed and re-cul^¬ tured with HMF-SC, added up with HMF-GF mix.

CB CB Placenta

(DMSO) (DMSO) (DMSO)

CD105 42 43 51

HLA-ABC 71.4 68.4 70.9

CD105 + HLA-ABC 40.3 42.3 50.8

HLA-DR + HLA-ABC 11.1 3.8 12.5

CD105 + HLA-DR 9.3 1.5 2.1

CD44 98.6 99.7 96.2

CD29 62.9 66.4 56.2

CD44+CD29 55.2 53.9 43.1

HLA-DR+CD29 3.9 1.8 2.9

HLA-DR+CD44 1.2 3.7 2.5 HLA-DR 13 4.8 3.6

CD3 0.4 2.8 0

CDl 9 10.6 12.7 2.3

CD14 1.7 3.7 1.9

Placenta CB CB

(Glycerol) (Glycerol) (DMSO)

CD105 51 31 29

HLA-ABC 57.3 61.9 55.4

CD105 + HLA-ABC 44.3 26.8 18.2

HLA-DR + HLA-ABC 1.9 10.7 20.6

CD105 + HLA-DR 3.7 1.4 11.3

CD44 91.4 99.2 98.6

CD29 71.9 53.4 62.8

CD44+CD29 70 51.3 60.4

HLA-DR+CD29 3.9 1.8 2.9

HLA-DR+CD44 0.4 1.3 1.7

HLA-DR 4.9 7.8 17.6

CD3 1.4 0 0

CDl 9 4.3 2.1 0.9

CD14 3.8 3.7 5.4

The results of the phenotypical analysis by FACS provided similar results both regarding the comparison between the use of DMSO 10% or Glycerol 10% and the comparison between a single freezing/thawing cycle and the repetition of it twice or thrice (Table 3). Accordingly, the culture following the procedure described herein allows the generation of stable SC of different origin and type while being fully compatible with different freezing methods and usages.

SCs expansion after 10 days culture from fresh cells, isolated from cord-blood, in presence of different media and GF mixtures

The results regarding the expansion of HSCs derived from human term CB in presence of HMF-SC and HMF-GF mix were compared with those obtained in presence of StemSpan and a standard GF mix. In addition different combination between the standard human SC medium and relative standard GF mix and those described herein were tried as depicted in Fig. 1.

Accordingly, the following conclusion could be reached: • Cells cultured with HMF-SC and HMF-GF mix proliferated much better than

• Cells expanded in StemSpam + standard GF mix, a condition in which the cells proliferated slower and the apoptosis increased

• Cells grown in StemSpam + HMF-GF mix, a condition in which the cells proliferated slower, but formed many clusters and showed reduced levels of apoptosis in comparison with culture in which HMF-GF mix was substituted with a standard GF mix

• Cells grown in HMF-SC + standard GF mix, a condition in which the apoptosis levels were mildly increased in comparison with culture in which HMF-SC was substituted with the standard medium StemSpan.

SCs expansion after ten days culture from fresh cells, isolated from cord-blood in presence of variants of HMF-SC

During the development of the HMF-SC medium, several combinations of factors were tried, in order to determine the optimal conditions for the expansion of different types SCs according to their source, especially regarding increased cell proliferation and reduced cell apoptosis. In addition, the cytokine cocktail under investigation was designed and optimized to be highly selective against contaminants derived from already differentiated cells, known to be massively present in the tissues constituting the sources of the SCs generated and expanded during the experiments, such as T-cells, B-cells, granulocytes, monocytes, erythrocytes and platelets. The developing of HMF-SC was carried out in a series of phases. In the following paragraphs, some of the most important steps of its development are reported. In all the experiments, the following GF mix was used:

The original composition of HMF-SC includes the following ingredients :

In this case, SCs could be expanded for ten days before the levels of apoptosis became massive in correletion with the detection of high levels of spontaneous differentiation towards fully mature hematopoietic cells. Upon completion of the culture, it was possible to harvest about l*10⁷ HSCs starting from 10OmL of CB. In this case, the lymphocyte contamination constituted 50% of the total amount of cells. In any case, the harvested HSCs were able to transdifferentiate into erythroid precursors, which can survive for further ten days without undergoing spontaneous differentiation towards erythrocytes or showing high levels of apoptosis. After this period, the number of harvested erythroid precursors was 2.5*10⁷. Upon right stimulation, they were able completely differentiate into enucleated erythrocytes without showing major apoptotic events. The medium added up with the GF mix was stable for 4 days at 4°C. After a cycle of freezing/thawing (in FBS 90% and DMSO 10%) the viability of the cells was equal to 70% of the initial amount but their phenotype was not stable upon reculturing.

After analysing such results, the original composition of the medium was changed according to the following indications:

In this case, the SCs could be expanded for two weeks without observing significant cell apoptosis and/or spontaneous differentiation. The maximum amount of SCs, expanded according to this modified protocol comprised 3*10⁷ cells, while the lymphocyte contamination was kept below 30% of the total amount of cells. In this case too, it was possible to transdifferentiate the harvested HSCs into erythroid precursors, which could be kept in culture for 30 days without showing significant levels of apoptosis or spontaneous differentiation. The maximum amount of erythroid precursor obtained at harvesting was about 7*10⁷ cells. Upon addition of the right maturation mix, they could be completely differentiated into enucleated erythrocytes without showing major apoptotic events. The medium added up with the GF mix was stable for 1 week at 4°C. After a cycle of freezing/thawing (in FBS 90% and DMSO 10%) cell viability reached 78% of the original value measured at harvesting and at least 60% of the cells showed a stable phenotype upon recultur- ing. According to this modified protocol, the lymphocyte contamination was kept below the 15% of the final amount of cells.

Finally the medium was modified according to the following indications :

According to this newly modified protocol, the SCs could be expanded for ten days without the setting of major apoptotic or spontaneous differentiation events. At harvesting, the maximum amount of SCs was 5-7*10⁷, while the lymphocyte contamination was kept below 25% of the total amount of cells. In this case too, it was possible to transdifferentiate the harvested HSCs into erythroid precursors, which could be further expanded for additional 50 days without showing spontaneous differentiation and/or cell apoptosis. The maximum expansion shown by such erythroid precursors was about l*10⁸ and they could completely differentiate into enucleated erythrocytes without showing major apoptotic events upon maturation with the right cocktail. The medium added up with the GF mix was stable for 1 week at 4⁰C. Upon a cycle of freezing/thawing (in FBS 90% and DMSO 10%) the cell viability was equal to 80% of the original value measured at harvesting and the SC phenotype was maintained by more than 80% of the cells upon reculturing. Upon reculturing, the lymphocyte contamination decreased to 10% of the total amount of cells .

For an overview of the results obtained in this series of experiments, refer to Table 4

Table 4. Analysis of the phenotype of fresh-cultured HSCs and MSCs, derived from CB and immediately cultured (Rl-4), or frozen in DMSO (Rl-4 after R), cultured in 4 different (1-4) developing versions of HMF-SCs, in presence of HMF-GF mix.

Rl after R2 after R3 after RF after

Rl R2 R3 RF F F F F

CD117 12.7 42.3 33.7 56.7 4.7 23.7 37.9 55.9

ABC-G2+CD117 0.4 0.7 2.6 2.3 0.4 0.1 2.8 2

CD34 31.2 42.8 61.2 67.4 11.4 34.6 55.8 69.8

ABC-G2+CD34 0.2 0.1 0.1 2.1 0.2 0.3 0.3 1.8

CD34+CD117 5.9 22.4 11.7 52.3 3.2 11.3 4.8 53.5

SSEA-4+CD34 0.2 0.4 0.2 1.9 0.1 0.1 0.1 1.6

CD133 0.7 2.1 0.2 53.2 0.3 1.7 0.3 49.8

CD117+CD133 0.2 2 0.1 50.7 0.1 0.8 0.3 48.2

CD34+CD133 0.2 2.1 0.2 32.7 0.3 0.3 0.1 34.9

CD34+HLA-DR 21.5 17.6 10.3 15.4 10.4 25.7 11.2 18.7

CD34+HLA-ABC 24.3 37.6 39.7 65.3 5.9 21.1 43.2 67.9

SSEA-4+ABC-G2 0.1 0.2 0.1 2.3 0.1 0.1 0.2 2.9

CD133+ABC-G2 0.2 0.2 0.2 0.9 0.3 0.1 0.1 1.4

CD117+SSEA-4 0.2 0.1 0.1 0.7 0.1 0.1 0.1 1.2

CD9+CD34 11.4 14.3 23.4 36.8 4.3 3.5 11.1 28.7

CD34+CD166 9.3 8.7 15.4 39.8 3.8 2.9 4.3 31.8

CD10+CD34 2.8 4.5 2.9 21.3 0.8 2.9 0.4 18.7

CD34+CD105 3.9 4.9 22 9 39.7 0.9 2.1 13.2 29.8

CD29+CD34 1.4 0.9 19.8 41.2 0.3 0.1 10.8 33.9

CD34+CD73 1.3 0.1 17.6 40.7 0.2 0.4 21.8 43.9

CD13+CD34 1.9 0.3 2.1 38.2 0.1 0.4 0.9 38.6

SSEA-4 0.5 0.8 0.4 2.4 0.1 0.8 0.9 1.3

ABC-G2 0.8 1.6 2.3 2.9 0.1 0.4 1.1 2.1

HLA-DR 39.8 29.4 31.2 18.9 42.7 35.5 39.7 16.4

CD44 92.3 97.3 96.3 91.4 95.9 91.8 89.9 93.8

CD29 24.3 39.8 43.7 69.8 10.5 21.1 54.3 56.9

CD44+CD29 21.6 31.2 42.1 28.9 8.4 11.7 29.8 15.9

CD90 11.2 56.2 78.3 75.7 10.3 32.9 61.2 68.1

CD90+CD29 18.1 33.2 41.1 47.9 2.3 21 33.8 38.6

CD44+CD90 3.9 50.3 70.9 41.2 2.1 22.9 60.8 47.8

CD105 10.6 16.8 32.3 37.2 5.9 9.7 24.1 33.9

HLA-ABC 23.2 45.9 89.6 96.5 18.7 30.1 71.2 91.7

CD105+HLA-ABC 9.3 11.3 31 31.4 1.1 8.1 20.6 31.8

HLA-DR+HLA-ABC 20.3 15.3 29.4 18.9 10.2 11.1 33.9 15.3

HLA-DR+CD105 10.1 9.3 2,1 3.9 5.4 5.2 20.1 3.8

CDl66 10.2 42.9 69.8 54.3 4.8 67.4 55.9 55.1 CD166+CD9 2.5 8.9 31.2 6.5 1.1 3.4 2.7 7.9

CD9 15.7 23.4 46.8 41.9 7.9 10.8 20.5 54.2

CDlO 9.3 39.8 2.1 34.7 5.5 48.9 1.1 32.9

CD9+CD10 1.2 21.4 1.1 3.2 1.3 2.3 0.7 1.1

CD10+CD166 5.9 34.9 1.9 23.6 1.1 23.5 0.3 15.4

CD73 4.5 89.4 28.8 87.6 6 91.2 43.2 73.2

CD73+CD29 4.1 33.1 19.2 28.1 1.1 19 10.4 36.8

CD13 3.9 54.8 21.2 55.2 4.2 19.2 13.2 54.1

CD13+CD29 1.1 30.1 2.9 64.3 0.6 10.8 1.1 73.9

CD13+CD73 0.9 29.9 2.8 63.2 0.1 12.3 1.9 65.4

CD45 78.9 89.4 89.7 43.9 81.4 54.8 94.3 53.6

CDl9 34.8 2.1 4.5 3.4 11.5 3.4 5.1 2.1

CD3 54.3 22.1 17.9 6.9 34.6 20.9 24.6 3.4

CD14 68.9 27.7 21.3 18.9 76.3 30.5 13.2 23.5

SCs differentiation into erythroid cells

In order to determine the transdifferentiation potentials of HSCs towards the red blood cell lineage, HSCs expanded in HMF-SC were further differentiated into fully enucleated erythrocytes upon addition of:

1. 10 U/mL Epo, 10 ng/mL insulin, 3xlO^"6 M ZK112.993, and 1 mg/ mL iron-saturated human transferrin (GFl) or

2.100 ng/mL IL-3, 200 U/mL GM-CSF and 5U/mL Epo (GF2).

The rate of differntiation was estimated through the analysis of hemoglobin accumulation. Within seven days and with a partial medium change every second day, HSCs fully differentiated into enucleated erythrocytes and produced high amounts of hemoglobin (Fig. 2) . These results were even slightly better than those observed during the analysis of erythroid cells expanded and fully differentiated in StemSpam and GFl (Control) .

Summary of the example :

The present example provides a medium to expand SCs from cord-blood, placenta or amniotic fluid.

It could be shown that in this medium both HSCs and MSCs as well as fibroid-like cells (amniotic cells) could be expanded to obtain a minimum of 5*10⁷ cells without the need of enrichment steps . It could also be shown that the HSCs maintained their pheno- type upon a freezing/thawing step when cryostored in DMSO 10% or Glycerol 10%. During expansion before or after a freezing/thawing cycle, the potential contaminations by T-cells, B-cells and monocytes were kept always below 30% of the total amount of cells .

In addition, HSCs have been shown of being capable to trans- differentiate into erythroid precursor that in turn could be further expanded and finally fully differentiate into enucleated erythrocyte in in vitro conditions.

Claims

Claims :

1. A plain growth medium for in vitro stem cell expansion comprising:

Selenium 5ng/ml to O.lmg/mL,

Transferrin 5mg/ml to lOOmg/mL,

Insulin 2.5μg/ml to lmg/mL,

Pyruvate, preferably sodium pyruvate, 0.05 to 1 mM, L-glutamine 0.5 to 10 mM,

Nucleosides 1 to 100 μg/ml at least one amino acid, preferably non-essential amino acid

5 to 1000 μg/ml, and Iscove's modified Dulbecco ' s medium (IMDM).

2. Medium according to claim 1, characterised in that the medium comprises further LDL, preferably human LDL, in an amount of 1 to 100 μg/ml.

3. Medium according to claim 1 or 2, characterised in that the medium comprises further an iron salt, preferably ferrous sulphate, in an amount of 1 to 100 nM.

4. Medium according to any one of claims 1 to 3, characterised in that the medium comprises:

Selenium 70μg/mL,

Transferrin 50mg/mL,

Insulin 0.5mg/mL,

Sodium pyruvate 0. ImM,

L-glutamine 2mM,

Nucleosides lOμg/ml,

Non-essential amino acid lOOμg/ml, and Iscove's modified Dulbecco ' s medium (IMDM) .

5. Medium according to any one of claims 1 to 4, characterised in that the medium comprises:

Selenium 70μg/mL,

Transferrin 50mg/mL,

Insulin 0.5mg/mL,

Sodium pyruvate 0. ImM,

L-glutamine 2mM, Nucleosides 10μg/ml,

Non-essential amino acid lOOμg/ml,

Iron Sulphate 40 nM, human LDL 40μg/mL and

Iscove's modified Dulbecco ' s medium (IMDM) .

6. Use of a medium according to any one of claims 1 to 5 for cultivating hematopoietic, amniotic and/or mesenchymal stem cells .

7. Method for cultivating hematopoietic, amniotic and/or mesenchymal stem cells comprising the steps:

- providing a stem cell source, and

- incubating said source with medium according to any one of claims 1 to 5.