Perinatal tissue derived mesenchymal stem cells: method of preparation and uses thereof

Abstract

The present invention discloses the preparation of placenta tissue derived CD106.sup.high CD151+Nestin+ mesenchymal stem cells (MSCs). In a first aspect, the invention relates to a particular method to prepare these cells at industrial scale and the cell population generated thereby. In a second aspect, the invention relates to a cell culture obtained by said particular method, containing placental CD106.sup.high CD151+Nestin+ MSCs expressing the vascular cell adhesion molecule 1 (VCAM-1) marker. The present application shows that said placental CD106.sup.high CD151+Nestin+ MSCs are capable of inducing angiogenesis in vitro and in vivo. The herein presented results also show that administering said placental CD106.sup.high CD151+Nestin+ MSCs to individuals suffering from an ischemic disease or from a disorder of the circulatory system results in a detectable improvement of one or more symptoms of said disease or disorder. Therefore, in a third aspect, the invention relates to placental CD 106.sup.high CD151+Nestin+ MSCs for use as a medicament for treating subjects suffering from an ischemic disease, a disorder of the circulatory system, an immune disease, an organ injury or an organ function failure.

Claims

1. A method for obtaining CD106+CD151+Nestin+mesenchymal stem cells (MSCs), said method comprising: culturing a population of undifferentiated MSCs in a first culture medium devoid of any exogenous growth factor until said undifferentiated MSCs reach 85-90% confluence, removing the first culture medium, introducing a second culture medium to the culture of undifferentiated MSCs comprising between 10 and 20 ng/ml of added Interleukin 1? and between 10 and 20 ng/ml of added Interleukin 4, culturing said undifferentiated MSCs in the second culture medium for at least 48 hours to obtain CD106+CD151+Nestin+MSCs, and characterizing the CD106+CD151+Nestin+MSCs based on marker expression, wherein over 60% of the CD106+CD151+Nestin+MSCs express CD106 at a detectible level, wherein over 98% of the CD106+CD151+Nestin+MSCs express CD151 at a detectible level, wherein over 98% of the CD106+CD151+Nestin+MSCs express Nestin at a detectible level wherein over 95% of the CD106+CD151+Nestin+MSCs express CD73, CD90, CD105, and CD166 at a detectible level, and wherein less than 2% of the CD106+CD151+Nestin+MSCs express CD45, CD34, and HLA-DR at a detectible level.

2. The method of claim 1, wherein said undifferentiated MSCs are obtained by cell isolation from an explant of umbilical cord fragment or by cell isolation from a placenta tissue fragment.

3. The method of claim 1, wherein over 98% of the MSCs do not express markers CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, and CD133 at a detectable level.

4. A method for enhancing the CD106 expression level of undifferentiated CD106+CD151+Nestin+mesenchymal stem cells (MSCs), said method comprising: culturing a population of undifferentiated MSCs in a first culture medium devoid of any exogenous growth factors until said undifferentiated MSCs reach 85-90% confluence, removing the first culture medium, introducing a second culture medium to the culture of undifferentiated MSCs comprising between 10 and 20 ng/ml of added Interleukin 1? and between 10 and 20 ng/ml of added Interleukin 4, culturing said undifferentiated MSCs in the second culture medium for at least 48 hours to produce undifferentiated CD106+CD151+Nestin+MSCs, and characterizing the CD106+CD151+Nestin+MSCs based on marker expression, wherein the CD106 expression is enhanced such that over 60% of the undifferentiated CD106+CD151+Nestin+MSCs express CD106 at a detectible level, wherein over 98% of the undifferentiated CD106+CD151+Nestin+MSCs express CD151 at a detectible level, wherein over 98% of the undifferentiated CD106+CD151+Nestin+MSCs express Nestin at a detectible level, wherein over 95% of the undifferentiated CD106+CD151+Nestin+MSCs express CD73, CD90, CD105, and CD166 at a detectible level, and wherein less than 2% of the undifferentiated CD106+CD151+Nestin+MSCs express CD45, CD34, and HLA-DR at a detectible level.

5. The method of claim 4, wherein said undifferentiated MSCs are obtained by cell isolation from a placenta tissue fragment.

6. The method of claim 4, wherein over 98% of the MSCs do not express markers CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, and CD133 at a detectable level.

7. A method for obtaining CD106+CD151+Nestin+mesenchymal stem cells (MSCs), comprising the steps of: a) collecting mononuclear cells contained in a perinatal biological tissue or fluid, b) culturing the mononuclear cells in a first culture medium devoid of any exogenous growth factor, wherein said mononuclear cells are passaged when they reach 85-90% confluence, c) characterizing the mononuclear cells based on marker expression to obtain a population of undifferentiated MSCs, wherein 95% of the undifferentiated MSCs express the markers CD73, CD90, CD105 and CD166, and less than 2% express the markers CD45, CD34 and HLA-DR, d) seeding the population of undifferentiated MSCs obtained from step c) at a density of 1000 to 5000 MSCs per cm2 into a second culture medium, e) once the population of undifferentiated MSCs in step d) reaches 40-50% confluency, adding between 10 and 20 ng/ml of Interleukin 1? and between 10 and 20 ng/ml of Interleukin 4, and culturing said population of undifferentiated MSCs for at least 48 hours, f) collecting the population of undifferentiated MSCs from step e) when they reach 90-95% confluence, g) characterizing the population of undifferentiated MSCs obtained from step f) based on expression of markers comprising CD106, CD151, and Nestin to obtain a population of CD106+CD151+Nestin+MSCs, wherein the CD106 expression is enhanced such that over 60% of the undifferentiated CD106+CD151+Nestin+MSCs express CD106 at a detectible level, wherein over 98% of the undifferentiated CD106+CD151+Nestin+MSCs express CD151 at a detectible level, wherein over 98% of the undifferentiated CD106+CD151+Nestin+MSCs express Nestin at a detectible level.

8. The method of claim 7, wherein over 98% of the MSCs do not express markers CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, and CD133 at a detectable level.

Description

DESCRIPTION OF THE FIGURES

(1) In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments and illustrated figures. The figures together with a detailed description below serve to further illustrate the embodiments and explain various principles and advantages in accordance with the present disclosure.

(2) FIG. 1 discloses a diagram showing how the CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs subpopulation of the invention can be prepared.

(3) FIG. 2 shows the morphology of the MSCs attached to culture flasks in placenta, according to Example 1.

(4) FIG. 3 shows the adipogenic and osteogenic cell differentiation of CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs. The results show that CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs subpopulation are capable of adipogenic and osteogenic differentiation in appropriate medium. The white arrows highlight lipid droplets and strongly mineralized area.

(5) FIG. 4 shows a pathological examination by angiography: Representative angiograms obtained on postoperative day 21.

(6) FIG. 5 shows a pathological examination by LDPI: Representative LDPI images obtained on postoperative day 0 and day 21. On the above panel, the area is still affected 3 weeks after the transplant, as shown by the white arrow. On the lower panels, black arrows highlight perfused area, 3 weeks after the transplant.

(7) FIG. 6 shows the results of placenta derived MSCs infusion for patients with diabetes. A total of 15 patients with diabetes (11 men and 4 women) were included in this clinical trial. (A) shows a reduction in mean insulin requirement after 3 times of treatment with perinatal tissue derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs in this group of 15 patients, which was statistically significant (P<0.001). (B) shows that the treatment of perinatal derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs also significantly improved the level of glycosylated hemoglobin of patients with diabetes (P<0.001). These results show that perinatal derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs may be a good choice for patients with diabetes.

(8) FIG. 7. (A) show the ascites level using ultrasonic examination before CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs infusion in a patient suffering from decompensated cirrhosis. And (B) show the ascites level using ultrasonic examination one month after the CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs were administered. (C) show that the liver functions of said patient improved significantly. CA125 expression decreased to normal level. In addition, total protein, albumin, globulin in serum also increased significantly.

(9) FIG. 8 shows the evaluation of the perfusion rate in a NOD/SCID mouse hindlimb ischemia model after arterial ligation and injection of various doses of umbilical cord MSCs of the invention, or saline solution or VEGF.

EXAMPLES

(10) For simplicity and illustrative purposes, the present invention is described by referring to exemplary embodiments thereof. It will be apparent, however, to one of ordinary skill in the art that the present invention may be practiced without limitation to these specific details. In other instances, well known methods have not been described in detail so as not to unnecessarily obscure the present invention.

(11) 1. Method to Obtain the MSCs of the Invention from Placental Tissue

(12) The method of the invention has been realized by: a) separately collecting human placenta tissue from multiple mother donors; b) washing the placenta tissue three times using 1?PBS, dissected in 1 mm.sup.3 cubes and washing the cubes tissue again to remove most of the blood from the tissue. c) digesting the placenta tissue of each donor separately with collagenase, centrifuging the digested tissue and collecting the mononuclear cells, d) seeding the mononuclear cells into a culture medium; e) trypsinizing and passage the cells once they reach 85-90% confluence; f) characterizing the cells based on the percentage of cells which express positive markers CD73, CD90, CD105 and CD166, and negative markers CD45, CD34 and HLA-DR; g) seeding the cells in a culture medium containing 90% Dulbecco's Modified Eagle's Medium/F12-Knockout (DMEM/F12-KO) and 10% FBS and growth factors at a seeding density of 1000 to 5000 MSCs per cm.sup.2 when they comprise at least 95% of the positive markers and at most 2% of the negative markers, h) obtaining CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs comprising over 80% cells which express CD106, 98% for CD151, 98% for Nestin, and 95% cells which express positive markers CD73, CD90, CD 105 and CD 166, and less than 2% cells which express negative markers CD45, CD34 and HLA-DR (for transplantation use) and adding 20 ng/mL of IL1? (provided by Peprotech) and 20 ng/mL of IL4 (provided by Peprotech) when they are 40-50% confluent; i) trypsinizing and collecting the cells once they reach 90-95% confluence; j) characterizing the cells based on the percentage of cells which express positive markers CD73, CD90, CD105 and CD166, and negative markers CD45, CD34 and HLA-DR.

(13) This method enabled to generate a subpopulation of placental-derived MSCs with a yield of at least 1?10.sup.5 cells/cm.sup.2 MSCs (5?10.sup.6 MSCs in T75 cm.sup.2 flasks when they are 90% confluent) that can be used in allogenic administrations. These cell cultures comprised over 95% cells which express positive markers CD73, CD90, CD105 and CD166, and less than 2% cells which express negative markers CD45, CD34 and HLA-DR.

(14) The human perinatal tissue derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs were fibroblast-like and grow very well on plastic flask (cf. FIG. 2).

(15) Three independent experiments have been performed (experiment 1, experiment 2 and experiment 3). 20 ng/mL of both Interleukin 1 (?) and 4 have been added when the cells are 40-50% confluent (see FIG. 1). At that point, MSCs comprise over 95% cells which express the positive markers CD73, CD90, CD105 and CD166, and less than 2% cells which express the negative markers CD45, CD34 and HLA-DR. After this, MSCs keep growing till 90% cell confluent (see Tables 1 and 2 below), in practice during about 2 days.

(16) Of note, the expression of MSCs cell surface protein CD106, CD151 and Nestin increased significantly after the interleukin 1 and 4 were added.

(17) TABLE 1 shows the expression of CD11b, CD19, CD29, CD31, CD34, CD45, CD73, CD90, CD 105, CD106, CD151, Nestin and HLA-DR on placenta tissue derived MSCs before adding interleukin 1 and 4 obtained in placenta Example 1, 2, and 3. These results show that placenta derived MSCs have classical cellular surface markers just like bone marrow and adipose tissue derived MSCs.

(18) TABLE-US-00001 TABLE 1 Surface markers Experiment 1 Experiment 2 Experiment 3 IgG-PE 0.4% 0.9% 0.1% IgG-FITC 0.5% 0.1% 0.4% CD11b-PE 0.2% 1.4% 0.3% CD19-FITC 0.7% 0.2% 0.6% CD34-FITC 0.9% 0.1% 0.3% CD45-PE 0.2% 1.1% 0.3% CD31-FITC 0.3% 0.1% 0.4% CD73-PE 99.2% 99.1% 99.8% CD90-PE 99.7% 100% 98.9% CD105-FITC 96.7% 98.1% 98.8% CD106-PE 31.5% 23.2% 30.6% CD151-PE 95.6% 96.1% 93.8% CD29-PE 99.4% 100% 99.9% CD44-FITC 99.6% 99.4% 99.2% Nestin-PE 82.5% 78.7% 84.1% HLA-DR-PE 0.0% 0.9% 0.0%

(19) TABLE 2 shows the expression of CD11b, CD19, CD29, CD31, CD34, CD45, CD73, CD90, CD 105, CD106, CD151, Nestin and HLA-DR on the placenta tissue derived MSCs after adding interleukin 1 and 4 in Experiments 1, 2 and 3. The results show that CD106 and Nestin protein marker increased significantly 48 hours after adding the IL-1 and IL-4 in complete medium. Meanwhile, the CD106.sup.high CD151.sup.+Nestin.sup.+ cells still have classical MSCs cellular surface phenotypic marker.

(20) TABLE-US-00002 TABLE 2 Surface markers Experiment 1 Experiment 2 Experiment 3 IgG-PE 0.1% 0.6% 0.2% IgG-FITC 0.2% 0.4% 0.4% CD11b-PE 0.2% 1.0% 0.1% CD19-FITC 0.4% 0.8% 0.8% CD34-FITC 0.4% 0.2% 0.5% CD45-PE 0.5% 0.8% 0.7% CD31-FITC 0.7% 0.5% 0.8% CD73-PE 99.9% 99.9% 99.7% CD90-PE 99.3% 100% 99.7% CD105-FITC 99.8% 99.8% 95.5% CD106-PE 82.7% 81.9% 84.3% CD151-PE 98.2% 99.4% 98.3% CD29-PE 100% 100% 99.6% CD44-FITC 99.8% 99.8% 99.6% Nestin-PE 93.7% 92.9% 90.6% HLA-DR-PE 0.0% 1.3% 0.5%

(21) These subpopulations can secrete more cellular growth factors, cytokines, immunomodulation factors and inflammation factors (see Table 3). These CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs will therefore have a better potential of immunomodulation and angiogenesis.

(22) Table 3 shows the related expression levels of growth factors secreted in the spent media by q-PCR. The results show that placenta tissue derived MSCs have more protein expression or secretion of IL-6, IL-8, IL-10, HGF, ANG, MMP2, VEGF-A and TGF-? at 48 hours after adding IL-1 and IL-4 in complete medium.

(23) TABLE-US-00003 TABLE 3 Growth factors Before IL-1 and il-4 Post IL-1 and IL-4 T-test IL-6 1.03 ? 0.29 15.06 ? 3.24 P < 0.05 IL-8 1.07 ? 0.31 13.74 ? 4.51 P < 0.05 IL-10 0.82 ? 0.31 7.84 ? 2.32 P < 0.05 HGF 0.85 ? 0.42 4.19 ? 1.63 P < 0.05 ANG 1.13 ? 0.28 3.58 ? 1.25 P < 0.05 MMP 2 0.95 ? 0.42 3.92 ? 1.29 P < 0.05 VEGF-A 1.18 ? 0.24 2.83 ? 0.76 P < 0.05 TGF-? 0.96 ? 0.29 2.07 ? 0.37 P < 0.05 bFGF 1.05 ? 0.35 1.47 ? 0.44 P > 0.05 SDF 1.13 ? 0.27 1.92 ? 0.66 P > 0.05
2. Method to Obtain the MSCs of the Invention from Umbilical Cord Tissues

(24) The following reagents have been used in the present example:

(25) TABLE-US-00004 Cellgenix 001011-050 IL-1b GMP Cellgenix 001003-050 IL-4 GMP (250 ?g/mL) Aliquots 15 ?L Peprotech 200-01B Recombinant Human IL-1b 100 ?g Peprotech 200-04 Recombinant Human IL-4 100 ?g BD 555749 PE Mouse IgG1, Isotype Control BD 555647 PE Mouse Anti-Human CD106 51-10C9 RUO BD 550257 PE Mouse Anti-Human CD73 AD2 RUO BD 556057 PE Mouse Anti-Human CD151 14A2.H1 RUO Miltenyi 130-081-002 CD34-PE, human Miltenyi 130-092-654 CD31-FITC, human Miltenyi 130-099-295 CD90-PE-Vio770, human Miltenyi 130-111-788 Anti-HLA-DR-FITC, human Miltenyi 130-094-941 CD105-PE, human Miltenyi 130-110-631 CD45-FITC, human Miltenyi 130-091-835 Mouse IgG2a-PE Miltenyi 130-092-213 Mouse IgG1-FITC Miltenyi 130-096-654 Mouse IgG1-PE-Vio770 Miltenyi 130-104-693 MACS Comp Bead Kit anti-REA Miltenyi 130-097-900 MACS Comp Bead Kit anti-Mouse Igk
2.1. Cell Isolation by Explant Method

(26) The umbilical cord was removed from the transport solution and cut in 2-3 cm long sections. To avoid contamination by adherent blood cells, each cord segment containing a blood clot that cannot be removed was discarded. The sections were then disinfected in a bath of antibiotics and antifungal agents composed of ?MEM+Vancomycin 1 g/L+Amoxicillin 1 g/L+Amikacin 500 mg/L+Amphotericin B 50 mg/L for 30 min at room temperature (RT). Antibiotics were extemporaneously dissolved in sterile water for injection.

(27) The sections of umbilical cord were removed from the bath and quickly rinsed in 1?PBS at RT. The epithelial membrane was slightly sectioned without touching the vessels. Each section was then detailed in slices of 0.5 cm thickness and disposed at the bottom of a 150 cm.sup.2 plastic flask with lid. 6 to 10 slices per flask were disposed with at least a 1 cm radius circle of free space around each slice, and left to adhere for 15 min without medium at RT.

(28) After adhesion, complete medium (?MEM+5% CPL+2 U/mL heparin) was added carefully, to keep the explants adherent to the bottom of the flask. The flasks were then incubated at 37? C., 90% humidity and 5% CO2.

(29) The culture medium was changed after 5 to 7 days.

(30) At day 10 after isolation, the migration of the cells out of the explants was controlled by inverted microscopy. If a circle of adherent cells was visible around most of the explants, they were carefully removed, by picking them out of the flask, through the lid, with a sterile, disposable, single-use pair of tweezers.

(31) From this step, the confluency of the cells was visually checked every other day and, if needed, a medium change was performed at day 17.

(32) When the cells reached 70-90% confluency or at D20, the medium was removed and cells were washed with 30 mL of 1?PBS per flask. Cells were then removed with Trypzean? and collected with the old medium and centrifuged 10 min at 2500 rpm. Supernatant was discarded and cells were then suspended in a cryopreservation solution consisting in ?MEM+100 mg/mL HSA+10% DMSO and cryopreserved.

(33) 2.2. Cell Isolation by Enzymatic Method

(34) The umbilical cord was removed from the transport solution and cut in 2-3 cm long sections. To avoid contamination by adherent blood cells, each segment containing a blood clot that cannot be removed was discarded. The sections were then disinfected in a bath of antibiotics and antifungal agents composed of ?DMEM+Vancomycin 1 g/L+Amoxicillin 1 g/L+Amikacin 500 mg/L+Amphotericin B 50 mg/L for 30 min at room temperature (RD. Antibiotics were extemporaneously dissolved in sterile water for injection.

(35) The cord was then cut in small pieces and immersed in an enzymatic cocktail comprising 2.7 mg/mL collagenase type I and 0.7 mg/mL hyaluronidase, incubated for 3 h at 37? C. with gentle agitation, followed by the addition of 2.5% trypsin and a further incubation for 30 min.

(36) The digested suspension was diluted 1:2 with medium to reduce the viscosity of the suspension and passed through a nylon mesh to obtain single suspension. Cells were centrifuged at 300?g for 20 min, and seeded at 10,000 cells/cm.sup.2 with fresh medium.

(37) The culture medium was changed after 5 to 7 days and every 7 days after that.

(38) When the cells reached 70-90% confluency or at D20, the medium was removed and cells were washed with 30 mL of 1?PBS per flask. Cells were then removed with Trypzean? and collected with the old medium and centrifuged 10 min at 2500 rpm. Supernatant was discarded and cells were then suspended in a cryopreservation solution consisting in ?MEM+100 mg/mL HSA+10% DMSO and cryopreserved.

(39) 2.3. Cell Thawing and Culture

(40) Cells were thawed following a classical protocol. Briefly, cryotubes were removed for liquid nitrogen and quickly plunge into a 37? C. water bath. As soon as there was no ice left in the tube, cells were diluted in preheated (37? C.) complete medium (?MEM+0.5% (v/v) ciprofloxacine+2 U/mL heparin+5% (v/v) LP) and quickly centrifuged (300 g, RT, 5 min).

(41) After centrifugation, the cells were suspended in preheated complete medium, and assessed for number and viability (blue trypan/Mallassez hemocytometer).

(42) The cells were seeded in two 75 cm.sup.2 plastic culture flasks in complete medium, and incubated (90% humidity, 5% CO.sub.2, 37? C.).

(43) 2.4. Cell Stimulation

(44) After a few days of expansion, the cells were checked for confluency. When confluency reached 30 to 50%, the old medium was discarded and replaced either by fresh complete medium for unstimulated condition, or by fresh medium completed with 10 ng/mL of IL-1? and 10 ng/mL of IL-4.

(45) Cells were then incubated at least 2 days before the flow cytometry experiments.

(46) 2.5. Cell Harvesting and Flow Cytometry Analysis

(47) After 2 to 3 days of expansion/stimulation, the cells were checked for confluency. If confluency was up to 80%, the cells were harvested. Briefly, the old medium was discarded and the cells were washed with 1?DPBS. Trypsin EDTA was added and the cells were incubated 5 min at 37? C. Trypsin was neutralized with at least 2? the volume of medium, and the cell suspension was harvested and assessed for number and viability.

(48) The flow cytometry experiment required 1?10.sup.6 cells, which were centrifuged and resuspended in 1?DPBS+0.4% HSA.

(49) The cells were labelled for CD73, CD90 CD105, CD106, CD151 and CD31, CD34, CD45, HLA-DR according to the following protocol: 1. The cells were assessed for viability and number. 2. The volume of suspension necessary to obtain 20 000 cells/labelling tube was placed in a 15 mL propylene plastic tube. 3. The tube was centrifuged 5 min at 300 g and 4? C. 4. The cells were resuspended with 1?DPBS+0.4% HSA (dilute 10? 4 mg/mL HSA in 1?DPBS). The volume necessary was 500 ?L per labelling tube.

(50) Extracellular staining was performed for CD106 and CD151 markers as recommended by the FACS manufacturer and antibodies providers. Intracellular staining was performed for Nestin. For intracellular staining, the Fixation/Permeabilization solution called BD Cytofix/Cytoperm kit was used.

(51) If the cells were not analyzed immediately after the staining, a fixation step was performed, by contacting the cells with a 1?DPBS 0.5% formaldehyde solution.

(52) After labelling, the cells were washed with 1?DPBS+0.4% HSA and permeabilized for Nestin labelling.

(53) The labelled cells were analyzed with the Accuri C6+ BD Biosciences cytometer, and results were analyzed with the BD Accuri C6 Plus software.

(54) 2.6. Results

(55) Several batches of umbilical cord-derived MSCs (HB-COR001 to COR005-MSC) have been obtained by cultivating cord-MSCs isolated by means of the explant methods disclosed above (2.1.) in the conditions exposed in point 2.4. above. For Cord 3 and 5 (COR003 and COR005), two MSC populations (MSC1 and MSC2) have been obtained concomitantly, by repeating the steps of the invention in a separate manner.

(56) The expression of CD106 at the surface of said cells was measured by flow cytometry.

(57) All tested batches of MSC exhibited a drastic increase (>60%) in the CD106 expression levels (see table 4).

(58) TABLE-US-00005 TABLE 4 CD106 expression of Umbilical Cord-derived MSCs after stimulation with IL1? and IL4 CD106 levels before and after full stimulation (10 ng/mL of BOTH IL-1b and IL-4) Cord % of CD106 % of CD106 Umbilical Cord-derived MSC before stimulation after stimulation HB1-COR001-MSC1 15.71% 97.34% HB1-COR002-MSC1 9.58% 70.62% HB1-COR003-MSC1 8.25% 69.42% HB1-COR003-MSC2 8.25% 59.47% HB1-COR005-MSC1 9.89% 71.26% HB1-COR005-MSC2 9.89% 75.48%
3. Impact of the Isolation Protocol on CD106 Expression of Umbilical Cord-Derived MSCs

(59) Mesenchymal stem cells were isolated from umbilical cord using the two different methods exposed above (see 2.1. and 2.2.): by explant isolation and by enzymatic digestion.

(60) All cells were cultivated in the same culture medium (?MEM+5% platelet lysate (LP)), stimulated at passage 3 according to the method of the invention and collected 2 days after stimulation. Cells viability is measured and cells were counted.

(61) The expression of several phenotypic markers, including CD106, was then measured by flow cytometry.

(62) All MSCs, whatever the isolation method or the stimulation condition, expressed phenotypic markers in a classical way: CD73, CD90, CD105 are over 95% positive, while CD31, CD34, CD45 and HLA-DR were negative.

(63) CD151+ and Nestin were also both over 95% positive, independently of the stimulation or the isolation method.

(64) Without stimulation, the MSCs isolated by enzymatic digestion expressed higher levels of CD106 before stimulation (53%) than the MSCs isolated with the explants methods (20%) They were however less sensitive to stimulation with the inflammatory cocktail: a smaller increase in CD106 has been observed (+4% increase vs +60% increase).

(65) The explants method is therefore the preferred method of the invention for umbilical cord-derived MSCs.

(66) TABLE-US-00006 TABLE 5 viability and cell count of the MSCs obtained after the two experimental protocols exposed in 2.1. and 2.2. COR27Enz P3 + 1 COR88Ex P3 + 1 MSCs isolated by MSCs isolated by the Enzymatic digestion explants method Condition Non stimulated Stimulated Non stimulated Stimulated Viability 97.8% 95.8% 99.2% 97.1% Cell count 6.0 ? 10.sup.6 4.65 ? 10.sup.6 6.2 ? 10.sup.6 8.26 ? 10.sup.6

(67) TABLE-US-00007 TABLE 6 molecular markers of umbilical cord-derived MSCs obtained by the two experimental conditions exposed in 2.1. and 2.2. COR27 COR27 COR88 COR88 Enz (?) Enz (+) Ex (?) Ex (+) CD73+ 100.00% 99.88% 99.99% 99.99% CD90+ 99.98% 99.87% 99.95% 98.56% CD105+ 99.99% 100.00% 99.99% 99.99% CD31? 99.97% 99.59% 99.85% 99.92% CD34? 93.57% 93.70% 99.35% 97.91% CD45? 99.92% 99.94% 99.96% 99.98% HLADR? 100.00% 100.00% 99.99% 99.99% CD106+ 53.41% 57.21% 20.68% 80.31% CD151+ 99.99% 100.00% 99.99% 100.00% Nestin+ 100.00% 100.00% 100.00% 99.98%
4. Effects of Stimulation with an IL1-IL4 Combination Versus IL1 or IL4 Alone Stimulation on CD106 Levels of Umbilical Cord Mesenchymal Stem Cells

(68) Mesenchymal stem cells were isolated from two umbilical cord by explant isolation (see 2.1.).

(69) All cells were cultivated in the same culture medium (?MEM+5% platelet lysate (LP)), stimulated at passage 4 according to the method of the invention with a mix of 10 ng/mL of IL-1b and 10 ng/mL of IL-4, or by each interleukin separately (10 ng/mL each) and collected 2 days after stimulation. Cells viability is measured and cells were counted.

(70) The expression of several phenotypic markers, including CD106, was then measured by flow cytometry.

(71) All MSCs, regardless of the stimulation condition, expressed phenotypic markers in a classical way: CD73, CD90, CD105 are over 95% positive, while CD31, CD34, CD45 and HLA-DR were negative. CD151+ was also over 95% positive, independently of the stimulation.

(72) Regarding the CD106 marker, the increase of expression observed with the stimulation with a combination of IL1 and IL4 is 3 to 5-fold higher than the increase of expression observed with a single interleukin stimulation (see Table 7.).

(73) TABLE-US-00008 TABLE 7 CD106 expression levels obtained after stimulation of MSCs with a combination of IL1-IL4 or with a single IL. % of CD106 CORD 1 CORD 2 Unstimulated cells 11.60% 5.00% IL-1b + IL-4 stimulation 75.88% 54.29% IL-1b stimulation 32.19% 13.41% IL-4 stimulation 22.88% 15.77%
5. Neovascularization Effect of Placental Derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs in Diabetic Rats

(74) This example focuses on the therapeutic neovascularization effect of placental derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs. It provides insights into their potential for clinical use as a cell-based therapy combined with insulin injection for treating critical hind limb ischemia in diabetes. Our results showed that Placenta derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs participate in angiogenesis and therapeutic vascularisation in order to improve ischemia and restore blood flow perfusion by directly differentiating into vascular cells. In addition, placenta derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs improved ischemia damage and functional recovery in diabetic rats.

(75) Immunodeficient male nude rats of six weeks of age were purchased from Vital River Laboratories (Charles River Laboratories suppler in China). Diabetes was induced with a single intraperitoneal injection of streptozotocin (70 mg/kg in Citrate buffer solution, only prepared immediately prior to injection) after overnight fasting. Fasting plasma glucose levels were measured every week and rats with Plasma glucose between 11 mM and 15 mM were considered to be diabetic. Age- and weight-matched nude rats receiving an intraperitoneal citrate buffer injection were used as non-diabetic controls (glycaemia between 5.5 and 8 mM).

(76) Two weeks later, diabetic nude rats were anesthetized (60 mg/kg pentobarbital intraperitoneally) and the left femoral artery were occluded by ligating it with 3-0 silk. The ligature was applied 0.5 cm proximally to the bifurcation of the saphenous and popliteal arteries. Lipiodol (1.5 ml/kg) was used to induce an embolism intravascularly. A sham ligature was applied to the left femoral artery with the left hind limb remaining non ischemic.

(77) After the ischemia model was established, the surgical limb deficiency was obvious as the limb could no longer support any weight and the paw was swollen and red. Over time the ischemic damage improved to different degrees in all the groups. The recovery of limb function was significantly increased in CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs subpopulation (FIG. 4).

(78) More precisely, the histological data on FIG. 4 show that the capillary density was increased in the cell transplant groups compared to the PBS group. In addition, more capillary numbers were observed in the CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs subpopulation cell transplant groups in diabetic rats and non-diabetic rats in comparison with the PBS group.

(79) FIG. 5 showed the functional evidence of ischemia-induced changes using vascularisation LDPI. The image shows that blood flow was completely blocked in the left hind limb on the surgical day; this is indicated by the deep dark colour. Three weeks later, the blood flow perfusion was restored to some degree in all the groups but did not return to normal in the PBS group where the ischemia/non-ischemia perfusion ratio only achieved 52%. The perfusion recovery in the cell transplant groups was significantly higher. The ratio was 81% in the P-MSCs group of the prior art (P<0.05 vs PBS group) and 86% for the CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs subpopulation group (P<0.01 vs PBS group).

(80) The perfusion recovery in both MSCs subpopulation was significantly higher. Therefore, the results revealed that MSCs improve blood flow perfusion and that the MSCs of the invention are more effective in doing so. In fact, the MSCs of the invention showed a significant improvement in the restoration of blood flow in both diabetic and non-diabetic rats.

(81) The results revealed that the MSCs of the invention improve blood flow perfusion more efficiently than the MSCs of the prior art. This example indicates that administration of the MSCs of the invention is a promising new approach for diabetic critical limb ischemia.

(82) 6. Therapeutic Effect of Placenta Derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs of the Invention to Patients with Diabetes

(83) This example focuses on the therapeutic effect of placenta derived CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs of the invention to patients suffering from diabetes. It provides insights into their potential for a clinical use as a cell-based therapy diabetes.

(84) A total of 15 patients with diabetes (11 men and 4 women) were included in this clinical trial. The inclusion criteria were patients with type 2 diabetes diagnosed from November 2013 to November 2014 in Tianjin General Hospital. The patients were between 30 and 85 years of age, duration of diabetes?3 years, requiring insulin for optimal glycemic control in a dose of ?0.7 U/kg/day at least for 1.5 year, having insulin dysfunction, poorly controlled blood glucose fluctuation with insulin-based treatment, and willingness to participate in the study. These 15 patients were between 42 and 67 years of age, with a median age of 59 years old; duration of diabetes from 3 years to 17 years, with an median of 8 years; daily insulin requirement from 38 units to 90 units, with an average of 58.7 units. Simultaneous glucose tolerance test, insulin release test, C peptide stimulation test and the determination of glycosylated hemoglobin was examined every three months. The cardiac, liver and kidney function tests were performed. The adverse events and side effects were observed during treatment. It was considered it to be effective if daily insulin requirement reduced by ?50% after treatment and lasted more than 3 months.

(85) The patients received three intravenous infusions of the placenta derived MSCs (P-MSCs) of the invention, with one month interval of infusion. The total number of P-MSCs administered for each patient was of (1.0-1.5)?10.sup.6/kg, with an average of 1.25?10.sup.6/kg. At the same time, patients continued to apply insulin, and adjusted the insulin dose according to blood glucose levels. For complications, the original general treatments were maintained. All of the patients were followed up after therapy for at least 6 months.

(86) This clinical trial showed a reduction in mean insulin requirement after 3 administrations of placenta derived MSCs of the invention in this group of 15 patients, which was statistically significant (P<0.001). The insulin injection dosage for patients with diabetes almost decreased by half after Placenta-derived MSCs of the invention were administered (as compared with the mean insulin dosage taken during the 6 months prior to MSCs administration) (FIG. 6 A).

(87) This clinical trial showed that the treatment with placenta derived MSCs of the invention significantly decreased the level of glycosylated hemoglobin of patients with diabetes (P<0.001) (FIG. 6B).

(88) These data support that the MSCs of the invention can be used for treatment of patients with diabetes.

(89) 7. Therapeutic Effect of the Placenta Derived MSCs of the Invention to Patients with Aplastic Anemia

(90) This example focuses on the therapeutic effect of placenta derived MSCs of the invention to patients with aplastic anemia and provides insights into their potential for clinical use as a cell-based therapy for aplastic anemia.

(91) Aplastic anemia is mostly considered as an immune-mediated bone marrow (BM) failure syndrome, characterized by hypoplasia and pancytopenia with fatty BM and reduced angiogenesis. Previous investigations have demonstrated that acquired aplastic anemia is manifested by abnormalities of HSCs/HPCs and hematopoietic microenvironment. Lots of evidences have hinted that aplastic anemia might be a syndrome characterized by stem/progenitor-cell disorders including HSCs/HPCs and MSCs. MSCs support hematopoiesis and regulate almost overall immune cells function to maintain the hematopoietic and immune homeostasis. MSCs can modulate the functions of the main immune cell including T cells, B cells, monocytes, DCs, NKTs and neutrophils [5]. MSCs possess remarkable immunosuppressive properties on Th1, Th17 and CTLs. MSCs inhibit the proliferation of T cells, IFN-? and TNF-? secretion by Th1 cells while promoting IL-10 production by Th2 cells and the expansion of Treg cells. However, recent researches showed that MSCs from aplastic anemia patients had poor proliferation and deficient immune suppression of MLR, PHA-induced T cell activation and IFN-? release [6, 7]. Our recent study showed that MSCs from aplastic anemia patients were reduced in suppressing the proliferation and clonogenic potential of CD4+ T cells while promoting Treg cells expansion. MSCs were also found defective in suppressing the production of TNF-? and IFN-? by CD4+ cells. However, there was no significant difference in regulating the production of IL-4, IL-10 and IL-17 [8]. In addition, our research also showed that MSCs from aplastic anemia patients showed aberrant morphology, decreased proliferation and clonogenic potential, and increased apoptosis as compared with BM-MSCs from healthy controls. MSCs from aplastic anemia patients were susceptible to be induced to differentiate into adipocytes but more difficult to differentiate into osteoblasts. Consistent with abnormal biological features, a large number of genes implicated in cell cycle, cell division, proliferation, chemotaxis and hematopoietic cell lineage showed markedly decreased expression in MSCs from these aplastic anemia patients. Conversely, more genes related with apoptosis, adipogenesis and immune response showed increased expression in MSCs from aplastic anemia patients. The gene expression profile of MSCs further confirmed the abnormal biological properties and provided significant evidence for the possible mechanism of the destruction of the BM microenvironment in aplastic anemia [9].

(92) MSCs is a promising therapeutic candidate for treating aplastic anemia due to the following two important facts: i) the hematopoiesis supportive and potent immunosuppressive capability of MSCs in general and ii) the biological characteristic difference and functional deficiency observed in MSCs derived from patients with aplastic anemia.

(93) In a clinical trial, a 6-year-old girl with intermittent fever continued for more than a month was treated with MSCs. Aplastic anemia was confirmed after two bone marrow biopsies revealed hypocellularity. There was no improvement in the peripheral blood phenotype after treatment of cyclosporine and stanozolol for almost 6 months. Hematopoietic stem cell transplantation was not good choice because it was very difficult to find a matched donor. Therefore, the patient was administered by intravenous infusion with 1?10.sup.7 of the placenta derived MSCs of the invention. The peripheral blood phenotype improved significantly after the 1.sup.st MSCs transplantation. However, the patient was still dependent on blood products transfusion including red blood cell and platelet transfusion. 6 months later, the patient accepted for the 2.sup.nd time a 1?10.sup.7 intravenous injection of the MSCs of the invention. The phenotype of peripheral blood improved significantly and reached almost normal levels 12 months after the 2.sup.nd injection. In addition, the patient was completely independent on blood products transfusion 12 months after the 2.sup.nd injection (Table 8). These data support that the placenta derived MSCs of the invention can be used for clinical treatment of patients with aplastic anemia.

(94) TABLE-US-00009 TABLE 8 The phenotype of peripheral blood during the treatment of placenta derived MSCs. 6 months post 12 months post Phenotype of PB Pre-treatment 1.sup.st MSCT 2.sup.nd MSCT WBC 0.64 ? 10.sup.9/l 4 ? 10.sup.9/l 5 ? 10.sup.9/l ANC 0.03 ? 10.sup.9/l 1 ? 10.sup.9/l 1.5 ? 10.sup.9/l HGB 48 g/l 80 g/l 110 g/l PLT 3 ? 10.sup.9/l 28 ? 10.sup.9/l 53 ? 10.sup.9/l
8. Therapeutic Effect of the Placenta Derived MSCs of the Invention to Patients with Liver Diseases

(95) This example focuses on the therapeutic effect of placenta derived MSCs obtained according to the method of the invention (i.e., with IL1 and IL4 treatment) administered to patients suffering from liver diseases. It provides insights into their potential for clinical use as a cell-based therapy for liver diseases.

(96) In this clinical trial, a 40-year-old man was confirmed suffering from alcoholic hepatitis for more than ten years. The patient was confirmed with decompensated cirrhosis two years ago although he accepted conventional treatment. Because there is no good choice for treatment of decompensated cirrhosis, the patient accepted voluntarily an intravenous injection of 4?10.sup.7 MSCs of the invention.

(97) In this clinical trial, the ultrasonic examination results showed an ascite level of 9.7 cm before MSCs were administered (see FIG. 7A). However, the ascite level decreased to 3.6 cm one month after the administration of the MSCs (FIG. 7 B). Furthermore, the results showed that the liver functions of patient with decompensated cirrhosis improved significantly after the administration of the MSCs. Moreover, CA125 expression in serum decreased to normal level according to the clinical references.

(98) These results demonstrate the excellent clinical reaction in decompensated cirrhosis patients treated with the MSCs of the invention.

(99) In addition, total protein, albumin, globulin in serum also increased significantly to reach the normal level according the clinical references (FIG. 7 C). These results showed that patient's liver synthetic and metabolic function improved one month after the intravenous injection of MSCs.

(100) Therefore, the placenta derived MSCs of the invention is a very good cytotherapeutic choice for liver diseases.

(101) 9. Angiogenic Potential of the Cells of the Invention, Obtained from Umbilical Cord

(102) The pro-angiogenic potential of the MSC of the invention has been further tested in a hindlimb ischemia murine model.

Material & Methods

(103) Twelve 8-week-old NOD/SCID mice (Laboratoire Janvier) were anesthetized with a mix of ketamin and xylazine. The left proximal and distal parts of the femoral artery of the left leg was then ligated (6.0 silk suture, Ethicon, Issy-Les-Moulineaux, France) and the part between ligations excised. Mice that exhibit more than 80% of ischemia after surgery were intramuscularly injected in the gastrocnemius muscle of the ischemic limb with 2 doses of MSCs of the invention as obtained according to the protocols exposed in point 2.4. above (0.5?10.sup.6 cells per animal in Group 2, 0.05. 10.sup.6 cells per animal in Group 3). A saline solution injection was used in the Control Group 1. A dose of 20 ng/mL of VEGF/mice was also injected in the Control Group 4.

(104) The injection scheme as well as the dose was derived for the intended clinical use of the cells and the dose administered to animals was >10 times the proposed dose for a phase I/II clinical trial in humans (which is of about 25?10.sup.6 cells/kg, that means about 0.5?10.sup.6 cells/mouse (average mouse weight=20 gr)).

(105) Hind limb blood flow was measured using a scanner-laser Doppler (Laser Doppler Perfusion system, Perimed PeriScan PIM III). The average perfusion of ischemic and non-ischemic limbs was determined before and after ischemia, and every 7 days until 21 days after ischemia. Blood flow-dependent changes in laser frequency were imaged using different colored pixels. Images were analyzed to quantify blood flow by using a blood perfusion analysis software (LPDIwin). Percentage of perfusion was expressed as the ratio of the ischemic to the non-ischemic hind limb.

(106) Hematoxylin-Eosin Staining of Hind Limb Muscle Sections

(107) Mice were euthanized at the end of the experiment (D21). Hind-limb gastrocnemius muscles were dissected and stained to visualize the distribution of the cells nuclei in the muscular fiber. Muscles isolated from the ischemic hind limb were fixed in paraffin and stained with hematoxylin/eosin.

Results

(108) FIG. 8 shows the mean reperfusion rate after hind limb ischemia in mice treated with different injections: NaCl 0.9% (negative controln=8), VEGF 20 ng/ml (positive controln=6), 50.Math.10.sup.3 MS cells (n=9) and 500.Math.10.sup.3 MS cells (n=7).

(109) These results demonstrated that administration of high doses of the MSCs of the invention in mice after ligation of the femoral artery resulted in higher perfusion rates when compared to vehicle (NaCl) of VEGF administered alone. A 100% perfusion rate was observed as early as 7 days post cell injection for mice receiving the highest dose of cells (0.5?10.sup.6 cells). There was a statistically significant difference with the other groups.

(110) Also, a dose response effect is likely since in this preliminary experiment the highest dose of cells (0.5?10.sup.6 cells) seems to achieve better perfusion rate than a 10 folds lower dose.

(111) These results confirm the actual angiogenic potency of the MSCs of the invention to improve blood perfusion in this mouse model of CLI.

(112) Results Observed in the Hematoxylin-Eosin Staining (Data Not Shown):

(113) In the normal, non-ischemic muscles, cells nuclei are distributed in the periphery of the muscular fiber.

(114) In the ischemic muscles of mice from Groups 1 (NaCl 0.9%) and 4 (VEGF 20 ng/mL), 21 days after ischemia induction, cells exhibit very few nuclei in the periphery of the muscular fiber, since they are still regenerating.

(115) In the ischemic muscles of mice from Group 3 (0.05.Math.10.sup.6 MSC cells or 50 k), 21 days after ischemia induction, 50% of muscular fibers are regenerated and exhibit nuclei in the periphery of the fiber. The regeneration happens faster than in Groups 1 and 4.

(116) In the ischemic muscles of mice from Group 2 (0.5?10.sup.6 MSC cells or 500 k), 21 days after ischemia induction, 100% of muscular fibers are regenerated and exhibit nuclei in the periphery of the fiber. The staining profile of the muscle is identical to the normal, non-ischemic, muscle. In this experiment, the 500 k cells dose allows a complete regeneration of the muscle.

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