MESENCHYMAL STEM CELL-DERIVED EXTRACELLULAR VESICLES AND THEIR MEDICAL USE
20190328792 · 2019-10-31
Inventors
Cpc classification
C07K14/51
CHEMISTRY; METALLURGY
C12N2502/1358
CHEMISTRY; METALLURGY
A61K47/6903
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
A61K2039/55555
HUMAN NECESSITIES
C12N15/111
CHEMISTRY; METALLURGY
C12N2502/00
CHEMISTRY; METALLURGY
C12N5/0663
CHEMISTRY; METALLURGY
C12N15/1136
CHEMISTRY; METALLURGY
A61K35/28
HUMAN NECESSITIES
A61P19/04
HUMAN NECESSITIES
International classification
A61K35/28
HUMAN NECESSITIES
C07K14/51
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a method for obtaining extracellular vesicles derived from mesenchymal stromal cells (MSCs), wherein the extracellular-vesicles are essentially free of vesicles not derived from the MSCs, and wherein the extracellular-vesicles are essentially free of heparin. The invention further relates to the extracellular vesicles derived from mesenchymal stromal cells (MSCs), to a pharmaceutical composition comprising such extracellular-vesicles and to the use of the pharmaceutical composition in the treatment of (a) bone defect(s), tendon defect(s) and/or spinal cord injury. The pharmaceutical composition of the invention can further comprise one or more Bone Morphogenic Proteins (BMPs), such as BMP2.
Claims
1-53. (canceled)
54. Pharmaceutical composition comprising extracellular-vesicles derived from mesenchymal stromal cells (MSCs), wherein the extracellular-vesicles are essentially free of vesicles not derived from the MSCs, and wherein the extracellular-vesicles are essentially free of heparin.
55. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises one or more Bone Morphogenic Proteins and/or PDGF, wherein the one or more Bone Morphogenic Protein(s) (BMP(s)) are selected from the group consisting of Bone Morphogenic Protein 2 (BMP2), Bone Morphogenic Protein 3 (BMP3), Bone Morphogenic Protein 4 (BMP4), Bone Morphogenic Protein 5 (BMP5), Bone Morphogenic Protein 6 (BMP6), Bone Morphogenic Protein 7 (BMP7), Bone Morphogenic Protein 8a (BMP8a), Bone Morphogenic Protein 8b (BMP8b), Bone Morphogenic Protein 12 (BMP12), Bone Morphogenic Protein 13 (BMP13), and Bone Morphogenic Protein 14 (BMP14).
56. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises BMP2.
57. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises BMP4.
58. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises BMP6.
59. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises BMP12.
60. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises BMP13.
61. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises BMP14.
62. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises a dose of the one or more BMP(s).
63. The pharmaceutical composition of claim 54, wherein the dose of the BMP(s) is between about 10 g to about 10 mg.
64. The pharmaceutical composition of 63, wherein the dose of the BMP is about 375 g.
65. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition or the extracellular-vesicles is/are to be administered to the traumatic and/or disordered area.
66. The pharmaceutical composition of claim 54, wherein the vesicles not derived from the MSCs are vesicles that are comprised in a culture medium prior to the culturing of the MSCs, such as vesicles that are comprised in serum, human platelet lysate, or plasma.
67. The pharmaceutical composition of claim 54, wherein the vesicles not derived from the MSCs are vesicles that are comprised in a cell culture supplement, such as serum, human platelet lysate, or plasma.
68. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles comprise at least about 80%, 85%, 90%, 94.4%, 95%, 99% or 99.5% of extracellular-vesicles derived from the MSCs.
69. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles are essentially free of fibrinogen.
70. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles are derived from MSCs cultured in culture medium that is essentially free of vesicles not derived from the MSCs and that is essentially free of heparin, and preferably fibrinogen, or wherein the extracellular-vesicles are derived from MSCs cultured in medium that is essentially free of cell culture supplement, such as serum, human platelet lysate, or plasma.
71. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles are derived from MSCs cultured in culture medium, and wherein the fibrinogen and the vesicles were removed from the culture medium prior to the culturing of the MSCs, and preferably wherein said culture medium was supplemented with a cell culture supplement comprising vesicles.
72. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles comprise hsa-miR-146a-5p, hsa-miR-92a-3p, hsa-miR-21-5p, hsa-miR-148a-3p, hsa-miR-221-3p, hsa-let-7i-5p, and hsa-let-7f-5p.
73. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles are essentially free of hsa-miR-133, hsa-miR-135, hsa-miR-204, hsa-miR-211, hsa-miR-17, and hsa-miR-106.
74. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles comprise: 100% of hsa-miR-146a-5p, at least about 50% of hsa-miR-92a-3p, at least about 15% of hsa-miR-21-5p, at least about 10% of hsa-miR-148a-3p, at least about 15% of hsa-miR-221-3p, at least about 5% of hsa-let-7i-5p, and at least about 5% of hsa-let-7f-5p, wherein the level of hsa-miR-146a-5p is set to 100%.
75. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles comprise: 100% of hsa-miR-146a-5p, less than about 0.1% hsa-miR-133, less than about 0.1% hsa-miR-135, less than about 0.1% hsa-miR-204, less than about 0.1% hsa-miR-211, less than about 0.1% hsa-miR-17, and less than about 0.1% hsa-miR-106, wherein the level of hsa-miR-146a-5p is set to 100%.
76. The pharmaceutical composition of claim 54, wherein the extracellular vesicles have a diameter of about 30 nm to about 150 nm, preferably have a diameter of about 100 nm to about 115 nm.
77. The pharmaceutical composition of claim 54, wherein the extracellular vesicles are positive for CD9, CD81 and TSG101 and are negative for GM130.
78. The pharmaceutical composition of claim 54, wherein the extracellular-vesicles are exosomes.
79. The pharmaceutical composition of claim 54, wherein the MSCs are umbilical cord-derived mesenchymal stromal cells (UC-MSCs) and/or bone marrow-derived mesenchymal stromal cells (BM-MSCs).
80. The pharmaceutical composition of claim 54, wherein the MSCs are umbilical cord-derived mesenchymal stromal cells (UC-MSCs).
81. The pharmaceutical composition of claim 54, wherein the MSCs are positive for the surface markers CD73, CD90 and CD105, and optionally wherein the MSCs are negative for the surface markers CD14, CD19, CD34, CD45 and HLA-DR.
82. The pharmaceutical composition of claim 81, wherein the MSCs are positive for the cell surface markers and are negative for the cell surface markers when the extracellular-vesicles are isolated.
83. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition or the extracellular vesicles is/are in a scaffold.
84. The pharmaceutical composition of claim 83, wherein the scaffold is a matrix.
85. The pharmaceutical composition of claim 84, wherein the matrix is selected of the group consisting of a ceramic implant, a polymer, hydrogel, bone matrix and demineralized bone matrix.
86. A method of treating a bone defect, a tendon defect or a spinal cord injury, wherein the method comprises the administration of a pharmaceutical composition comprising extracellular-vesicles derived from mesenchymal stromal cells (MSCs), wherein the extracellular-vesicles are essentially free of vesicles not derived from the MSCs, and wherein the extracellular-vesicles are essentially free of heparin.
87. The method according to claim 86, wherein the pharmaceutical composition comprises one or more Bone Morphogenic Proteins and/or PDGF, wherein the one or more Bone Morphogenic Protein(s) (BMP(s)) are selected from the group consisting of Bone Morphogenic Protein 2 (BMP2), Bone Morphogenic Protein 3 (BMP3), Bone Morphogenic Protein 4 (BMP4), Bone Morphogenic Protein 5 (BMP5), Bone Morphogenic Protein 6 (BMP6), Bone Morphogenic Protein 7 (BMP7), Bone Morphogenic Protein 8a (BMP8a), Bone Morphogenic Protein 8b (BMP8b), Bone Morphogenic Protein 12 (BMP12), Bone Morphogenic Protein 13 (BMP13), and Bone Morphogenic Protein 14 (BMP14).
88. The method according to claim 86, wherein the pharmaceutical composition comprises BMP2.
89. The method according to claim 86, wherein the pharmaceutical composition comprises BMP4.
90. The method according to claim 86, wherein the pharmaceutical composition comprises BMP6.
91. The method according to claim 86, wherein the pharmaceutical composition comprises BMP12.
92. The method according to claim 86, wherein the pharmaceutical composition comprises BMP13.
93. The method according to claim 86, wherein the pharmaceutical composition comprises BMP14.
94. The method according to claim 86, wherein the pharmaceutical composition comprises a dose of the one or more BMP(s).
95. The method according to claim 94, wherein the dose of the BMP(s) is between about 10 g to about 10 mg.
96. The method according to claim 86, wherein the dose of the BMP is about 375 g.
97. The method according to claim 96, wherein the pharmaceutical composition or the extracellular-vesicles is/are to be administered to the traumatic and/or disordered area.
98. The method according to claim 86, wherein the vesicles not derived from the MSCs are vesicles that are comprised in a culture medium prior to the culturing of the MSCs, such as vesicles that are comprised in serum, human platelet lysate, or plasma.
99. The method according to claim 86, wherein the vesicles not derived from the MSCs are vesicles that are comprised in a cell culture supplement, such as serum, human platelet lysate, or plasma.
100. The method according to claim 86, wherein the extracellular-vesicles comprise at least about 80%, 85%, 90%, 94.4%, 95%, 99% or 99.5% of extracellular-vesicles derived from the MSCs.
101. The method according to claim 86, wherein the extracellular-vesicles are essentially free of fibrinogen.
102. The method according to claim 86, wherein the extracellular-vesicles are derived from MSCs cultured in culture medium that is essentially free of vesicles not derived from the MSCs and that is essentially free of heparin, and preferably fibrinogen, or wherein the extracellular-vesicles are derived from MSCs cultured in medium that is essentially free of cell culture supplement, such as serum, human platelet lysate, or plasma.
103. The method according to claim 86, wherein the extracellular-vesicles are derived from MSCs cultured in culture medium, and wherein the fibrinogen and the vesicles were removed from the culture medium prior to the culturing of the MSCs, and preferably wherein said culture medium was supplemented with a cell culture supplement comprising vesicles.
104. The method according to claim 86, wherein the extracellular-vesicles comprise hsa-miR-146a-5p, hsa-miR-92a-3p, hsa-miR-21-5p, hsa-miR-148a-3p, hsa-miR-221-3p, hsa-let-7i-5p, and hsa-let-7f-5p.
105. The method according to claim 86, wherein the extracellular-vesicles are essentially free of hsa-miR-133, hsa-miR-135, hsa-miR-204, hsa-miR-211, hsa-miR-17, and hsa-miR-106.
106. The method according to claim 86, wherein the extracellular-vesicles comprise: 100% of hsa-miR-146a-5p, at least about 50% of hsa-miR-92a-3p, at least about 15% of hsa-miR-21-5p, at least about 10% of hsa-miR-148a-3p, at least about 15% of hsa-miR-221-3p, at least about 5% of hsa-let-7i-5p, and at least about 5% of hsa-let-7f-5p, wherein the level of hsa-miR-146a-5p is set to 100%.
107. The method according to claim 86, wherein the extracellular-vesicles comprise: 100% of hsa-miR-146a-5p, less than about 0.1% hsa-miR-133, less than about 0.1% hsa-miR-135, less than about 0.1% hsa-miR-204, less than about 0.1% hsa-miR-211, less than about 0.1% hsa-miR-17, and less than about 0.1% hsa-miR-106, wherein the level of hsa-miR-146a-5p is set to 100%.
108. The method according to claim 86, wherein the extracellular vesicles have a diameter of about 30 nm to about 150 nm, preferably have a diameter of about 100 nm to about 115 nm.
109. The method according to claim 86, wherein the extracellular vesicles are positive for CD9, CD81 and TSG101 and are negative for GM130.
110. The method according to claim 86, wherein the extracellular-vesicles are exosomes.
111. The method according to claim 86, wherein the MSCs are umbilical cord-derived mesenchymal stromal cells (UC-MSCs) and/or bone marrow-derived mesenchymal stromal cells (BM-MSCs).
112. The method according to claim 86, wherein the MSCs are umbilical cord-derived mesenchymal stromal cells (UC-MSCs).
113. The method according to claim 86, wherein the MSCs are positive for the surface markers CD73, CD90 and CD105, and optionally wherein the MSCs are negative for the surface markers CD14, CD19, CD34, CD45 and HLA-DR.
114. The method according to claim 113, wherein the MSCs are positive for the cell surface markers and are negative for the cell surface markers when the extracellular-vesicles are isolated.
115. The method according to claim 114, wherein the pharmaceutical composition or the extracellular vesicles is/are in a scaffold.
116. The method according to claim 115, wherein the scaffold is a matrix.
117. The method according to claim 116, wherein the matrix is selected of the group consisting of a ceramic implant, a polymer, hydrogel, bone matrix and demineralized bone matrix.
118. Extracellular-vesicles derived from mesenchymal stromal cells (MSCs), wherein the extracellular-vesicles are essentially free of vesicles not derived from the MSCs, and wherein the extracellular-vesicles are essentially free of heparin.
119. The extracellular-vesicles of claim 118, wherein the pharmaceutical composition or the extracellular-vesicles is/are to be administered to the traumatic and/or disordered area.
120. The extracellular-vesicles of claim 118, wherein the vesicles not derived from the MSCs are vesicles that are comprised in a culture medium prior to the culturing of the MSCs, such as vesicles that are comprised in serum, human platelet lysate, or plasma.
121. The extracellular-vesicles of claim 118, wherein the vesicles not derived from the MSCs are vesicles that are comprised in a cell culture supplement, such as serum, human platelet lysate, or plasma.
122. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles comprise at least about 80%, 85%, 90%, 94.4%, 95%, 99% or 99.5% of extracellular-vesicles derived from the MSCs.
123. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles are essentially free of fibrinogen.
124. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles are derived from MSCs cultured in culture medium that is essentially free of vesicles not derived from the MSCs and that is essentially free of heparin, and preferably fibrinogen, or wherein the extracellular-vesicles are derived from MSCs cultured in medium that is essentially free of cell culture supplement, such as serum, human platelet lysate, or plasma.
125. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles are derived from MSCs cultured in culture medium, and wherein the fibrinogen and the vesicles were removed from the culture medium prior to the culturing of the MSCs, and preferably wherein said culture medium was supplemented with a cell culture supplement comprising vesicles.
126. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles comprise hsa-miR-146a-5p, hsa-miR-92a-3p, hsa-miR-21-5p, hsa-miR-148a-3p, hsa-miR-221-3p, hsa-let-7i-5p, and hsa-let-7f-5p.
127. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles are essentially free of hsa-miR-133, hsa-miR-135, hsa-miR-204, hsa-miR-211, hsa-miR-17, and hsa-miR-106.
128. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles comprise: 100% of hsa-miR-146a-5p, at least about 50% of hsa-miR-92a-3p, at least about 15% of hsa-miR-21-5p, at least about 10% of hsa-miR-148a-3p, at least about 15% of hsa-miR-221-3p, at least about 5% of hsa-let-7i-5p, and at least about 5% of hsa-let-7f-5p, wherein the level of hsa-miR-146a-5p is set to 100%.
129. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles comprise: 100% of hsa-miR-146a-5p, less than about 0.1% hsa-miR-133, less than about 0.1% hsa-miR-135, less than about 0.1% hsa-miR-204, less than about 0.1% hsa-miR-211, less than about 0.1% hsa-miR-17, and less than about 0.1% hsa-miR-106, wherein the level of hsa-miR-146a-5p is set to 100%.
130. The extracellular-vesicles of claim 118, wherein the extracellular vesicles have a diameter of about 30 nm to about 150 nm, preferably have a diameter of about 100 nm to about 115 nm.
131. The extracellular-vesicles of claim 118, wherein the extracellular vesicles are positive for CD9, CD81 and TSG101 and are negative for GM130.
132. The extracellular-vesicles of claim 118, wherein the extracellular-vesicles are exosomes.
133. The extracellular-vesicles of claim 118, wherein the MSCs are umbilical cord-derived mesenchymal stromal cells (UC-MSCs) and/or bone marrow-derived mesenchymal stromal cells (BM-MSCs).
134. The extracellular-vesicles of claim 118, wherein the MSCs are umbilical cord-derived mesenchymal stromal cells (UC-MSCs).
135. The extracellular-vesicles of claim 118, wherein the MSCs are positive for the surface markers CD73, CD90 and CD105, and optionally wherein the MSCs are negative for the surface markers CD14, CD19, CD34, CD45 and HLA-DR.
136. The extracellular-vesicles of claim 135, wherein the MSCs are positive for the cell surface markers and are negative for the cell surface markers when the extracellular-vesicles are isolated.
137. The extracellular-vesicles of claim 118, wherein the pharmaceutical composition or the extracellular vesicles is/are in a scaffold.
138. The extracellular-vesicles of claim 137, wherein the scaffold is a matrix.
139. The extracellular-vesicles of claim 138, wherein the matrix is selected of the group consisting of a ceramic implant, a polymer, hydrogel, bone matrix and demineralized bone matrix.
140. A method for obtaining extracellular-vesicles derived from mesenchymal stromal cells (MSCs) that are essentially free of vesicles not derived from the MSCs, wherein the method comprises: culturing the MSCs in a culture medium that is essentially free of vesicles not derived from the MSCs and that is essentially free of heparin and preferably fibrinogen; and (ii) isolating the extracellular-vesicles derived from the MSCs from the culture medium.
141. The method of claim 140, wherein the vesicles not derived from the MSCs are vesicles that are comprised in the culture medium prior to the culturing step (i).
142. The method of claim 140, wherein the vesicles not derived from the MSCs are vesicles that are comprised in the serum, human platelet lysate, or plasma.
143. The method of claim 140, wherein the culture medium is medium that does not comprise a cell culture supplement, such as serum, human platelet lysate, or plasma.
144. A method for obtaining extracellular-vesicles derived from mesenchymal stromal cells (MSCs), wherein the method comprises: (i) removing fibrinogen from a culture medium, wherein no heparin is added to the culture medium, and removing vesicles from the culture medium, (ii) culturing the MSCs in the culture medium of step (i), and (iii) isolating the extracellular-vesicles derived from the MSCs from the medium.
145. The method of claim 144, wherein the MSCs are cultured in the medium for at least about 48 hours.
146. Extracellular-vesicles obtained according to claim 144.
147. The extracellular-vesicles of claim 146, wherein the extracellular-vesicles are derived from mesenchymal stromal cells (MSCs), wherein the extracellular-vesicles are essentially free of vesicles not derived from the MSCs, and wherein the extracellular-vesicles are essentially free of heparin.
148. The extracellular-vesicles of claim 146, wherein the extracellular-vesicles comprise hsa-miR-146a-5p, hsa-miR-92a-3p, hsa-miR-21-5p, hsa-miR-148a-3p, hsa-miR-221-3p, hsa-let-7i-5p, and hsa-let-7f-5p.
149. The extracellular-vesicle of claim 146, wherein the extracellular-vesicles are derived from mesenchymal stromal cells (MSCs), wherein the extracellular-vesicles comprise at least about 80%, 85%, 90%, 94.4%, 95%, 99% or 99.5% of extracellular-vesicles derived from the MSCs.
150. Mesenchymal stromal cells (MSCs) comprised and/or cultured in a culture medium that is essentially free of vesicles not derived from the MSCs, and preferably wherein the culture medium is essentially free of heparin and preferably fibrinogen.
151. The mesenchymal stromal cells of claim 150, wherein the fibrinogen and the vesicles comprised in the culture medium prior to the culturing of the MSCs were removed, and preferably wherein the culture medium was supplemented with a cell culture supplement comprising vesicles.
152. A culture medium, wherein the culture medium comprises extracellular-vesicles derived from mesenchymal stromal cells (MSCs), and wherein the extracellular-vesicles are essentially free of vesicles not derived from the MSCs, and preferably wherein the culture medium is essentially free of heparin and preferably fibrinogen.
153. The culture medium of claim 152, wherein the fibrinogen and the vesicles comprised in the culture medium prior to culturing the MSCs were removed, and preferably wherein the culture medium was supplemented with a cell culture supplement comprising vesicles.
Description
[0289] The present invention is further described by reference to the following non-limiting figures and examples. The Figures show:
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[0295] Black bars indicate site of osteotomy; WB . . . immature, woven bone; B . . . compact bone; BM . . . bone marrow.
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[0312] The present invention is additionally described by way of the following illustrative non-limiting examples that provide a better understanding of the present invention and of its many advantages.
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EXAMPLE 1: MSC-DERIVED EXOSOMES ENHANCE BONE REGENERATION WITH OR WITHOUT LOW DOSE RHBMP2 IN A RAT FEMUR DEFECT MODEL
1. Methods
[0314] 2.1 Medium Preparation
[0315] Heparin-free and fibrinogen-depleted medium was prepared as follows: Alpha-modified Minimal Essential Medium Eagle (alpha-MEM, M4526, Sigma-Aldrich) was supplemented with 5 mM (N2)-L-Alanyl-L-Glutamine (Dipeptiven, 11051014, Fresenius Kabi, Austria) and 10% pHPL. pHPL was produced as described earlier (Schallmoser, 2007). The mixture was immediately divided into 14 aliquots (40 mL in 50 mL centrifuge tubes), maintained for 4-6 h at 20-24 C. until biogel formation was completed, and stored at 4 C. for 12-18 h. To remove fibrin, the clotted medium was brought to 37 C. The fibrin gel was then physically collapsed by exceeding the shear forces to the matrix, and the precipitated fibrin was pelleted at 2,500g at 20 C. for 12 minutes (Laner-Plamberger, 2015). Finally, the supernatant was filtered (0.22 m). Heparin-free, fibrinogen-depleted and EV-depleted medium was produced from the above by a single centrifugation step at 120,000g for 3 h (employing a Sorvall MX-120 or WX-80 ultracentrifuge, fixed angle rotor, Thermo Scientific). The resulting supernatant was filtered through a 0.22 m filter, to yield EV-depleted medium. HPL-free medium was generated by adding 5 mM Dipeptiven to alpha-MEM, followed by filtration (0.22 m).
[0316] 2.2 Cell Culture
[0317] Cells were cultured at 37 C. and 5% CO.sub.2. Experiments were performed with two different primary human BM-MSC and UC-MSC preparations, respectively. One BM aspirate was directly obtained from the University Clinic for Blood Group Serology and Transfusion Medicine, Paracelsus Medical University, Salzburg (Austria). The second BM aspirate was purchased from AllCells (California, USA). Both donors had signed an informed consent. BM-MSC were isolated from bone marrow as described and were tested negative for mycoplasma. For all experiments, cells were kept in 30 mL medium per T225 flask.
[0318] 2.3 Isolation of pHPL- and MSC-Derived Extracellular Vesicles
[0319] EVs released by MSCs may contribute to biological processes like tissue regeneration, immunomodulation, and neuroprotection. Evaluation of the therapeutic potential of MSC-derived EV and their application in future clinical trials demand thorough characterisation of their content as well as production under defined medium conditions, devoid of both xenogenic substances and serum-derived vesicles. We have addressed the apparent need for such a growth medium and have developed a medium formulation based on pooled human platelet lysate (pHPL) that is free from animal-derived xenogenic additives and depleted from EVs, for the generation of exclusively MSC-derived EV. Depletion of EVs from complete growth medium was achieved by centrifugation at 120,000g for three hours, which eliminated 94.4% (1.4%) of RNA-containing pHPL EVs. MSCs propagated in this medium retain the characteristic surface marker expression, cell morphology, viability, and in vitro osteogenic and adipogenic differentiation potential. The proliferation rates are decreased by 4% after 48 hours and 13% after 96 hours. EVs collected from MSCs cultured in the EV-depleted medium reveal a similar RNA pattern compared to EVs generated in standard pHPL EV-containing medium, but displayed a more clearly defined pattern of proteins characteristic for EVs. Specifically, the EV marker CD9 is present in large amounts in EVs collected from unconditioned medium (see
Heparin-Free and Fibrinogen-Depleted Medium is Prepared as Follows:
[0320] Alpha-modified Minimal Essential Medium Eagle (alpha-MEM, M4526, Sigma-Aldrich) supplemented with 5 mM (N2)-L-Alanyl-L-Glutamine (Dipeptiven, 11051014, Fresenius Kabi, Austria) and 10% pHPL is mixed and immediately divided into 14 aliquots (40 mL in 50 mL centrifuge tubes), maintained for 4-6 h at 20-24 C. until biogel formation is completed, and stored at 4 C. for 12-18 h. To remove fibrin, the clotted medium is brought to 37 C. The fibrin gel is then physically collapsed by exceeding the shear forces to the matrix, and the precipitated fibrin was pelleted at 2,500g at 20 C. for 12 minutes. Finally, the supernatant is filtered (0.22 m). Heparin-free, fibrinogen-depleted and EV-depleted medium is produced by single centrifugation step at 120,000g for 3 h (employing a Sorvall MX120 or WX80 ultracentrifuge, and a fixed angle rotor, Thermo Scientific). The resulting supernatant is filtered through a 0.22 m filter.
Enrichment, Purification and Characterization of EVs
[0321] To obtain EVs released by BM- or UC-MSC, cells are first maintained in heparin-free medium until they reach 70% confluence, whereafter cells are washed with PBS and medium is changed to fresh Heparin-free, fibrinogen-depleted and EV-depleted medium. Conditioned media (CM) are harvested after 48 hours. The CM is first filtered through a 0.22 m filter to remove cell debris and large vesicles, followed by centrifugation at 30,000g for 20 minutes to pellet larger microvesicles. The supernatants are subjected to centrifugation at 120,000g for 3 hours to pellet the EV. The resulting pellets are either resuspended in PBS or Ringer-Lactate solution (for nanoparticle tracking analysis) or in alpha-MEM without supplements (for proteinase and RNase protection assay), or directly lysed in RNA lysis buffer (for RNA isolation, buffer from mirVANA miRNA Isolation Kit, AM1561, Ambion) or RIPA buffer (for Western Blot analysis; R0278, Sigma-Aldrich). EVs are stored at 80 C. prior to nanoparticle tracking analyses (NTA).
[0322] After collection of CM, MSCs are removed from the culture vessel by the addition of TrypLE Select CTS (A12859-01, Gibco), stained with trypan blue and counted using a hemocytometer, and analyzed by flow cytometry to obtain an accurate cell count at the time of harvest. This enables the definition of a cell equivalent (CE) dose for further investigation of the therapeutic activity.
[0323] 210.sup.6 cells are used to obtain an immunophenotype of the cells by flow cytometry (FACS). In order to indicate the MSC phenotype approved for administration to humans in clinical trials, the cells should demonstrate an MSC phenotype to meet modified and improved criteria based upon the minimal criteria for MSCs set forward by the International Society for Cell Therapy (ISCT; Dominici et al., 2006) and approved by international authorities.
Purification StrategyTFF Vs UC
[0324] In order to obtain therapeutically active EVs from large volumes of conditioned media various techniques are available (see for example Nordin et al., 2015 comparing both biochemical and biophysical purification aspects and demonstrating (by NTA, WB and LC/MS/MS) that ultrafiltration techniques generated EVs with the same proteome as those vesicles purified by ultracentrifugation, albeit with even higher yield and purity, as determined by the vesicle-to-protein ratio).
[0325] We confirm in our own studies that the purification process by ultracentrifugation is suitable for obtaining therapeutically active EVs. Tangential flow filtration (TFF) has been evaluated as an alternative, scalable method. Our data demonstrate, that TFF can be used to obtain MSC-derived EVs from large volumes (2-10 L) of MSC conditioned medium and that the resulting RNA profile is not substantially affected and deviating from the criteria set above.
[0326] 2.4 Immunophenotyping and Determination of Cell Viability by Flow Cytometry
[0327] Immunophenotype and viability analysis of MSCs cultured is carried out according to the minimal criteria for defining MSC identity (Dominici et al., 2006). In brief, collected cells are centrifuged (300g for 6 min), resuspended in 5% v/v sheep serum-containing blocking buffer to reach a concentration of 1.510.sup.7 cells/mL and incubated for 20 minutes at +4 C. in dark. 310.sup.5 cells are stained with mouse anti-human monoclonal antibodies against CD90 (IM1839U, Beckman Coulter, France), CD105 (MCHD10505, Life Technologies, Austria), CD14, CD19, CD34, CD45, CD73, HLA-II (DR) (345785, 345802, 345808, 550257, 347400 Becton Dickinson, Austria), or with corresponding isotype controls (345815, 345818, 555743, 345816, 349051, Becton Dickinson, Austria) for 25 minutes at +4 C. in dark. Thereafter samples are washed with cold PBS and resuspended in 100 L 7AAD-containing PBS (1:10 dilution, 0.0005 w/v % final concentration) and stained for 10 minutes at room temperature protected from light. Finally 400 L cold PBS is added and the samples are measured immediately using FACSCanto II flow cytometer (Becton Dickinson) until 1000 events were recorded per staining. Blue (488 nm) and red (633 nm) laser excited fluorescence signals are detected with the following standard light filters: FITC: 530/30 nm; PE: 585/42 nm; APC: 660/20 nm; 7AAD: 670LP. Results are analyzed with WinList software 8.0 (Verity Software House, Topsham, USA). [FSCA, SSCA] dot plot analyses are applied for debris exclusion, and a doublet discrimination panel is set on the FSC channel for the detection of height and width of the fluorescence signals. The ratio of the viable cells is determined on [SSC, 7AAD] dot plot.
[0328] 2.5 RNA Isolation and Detection
[0329] RNA was isolated from EV pellets using mirVana miRNA Isolation Kit (AM1561, Ambion) according to the manufacturer's instructions for total RNA isolation. One microliter of isolated RNA was analyzed for quality, size distribution and yield with Agilent RNA 6000 Pico chips (5067-1513) on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif.) according to the manufacturer's protocol.
[0330] 2.6 Proliferation Assays
[0331] Proliferative capacity of MSC is monitored in real-time employing the impedance-based growth measurement system xCELLigence RTCA DP (Roche/ACEA) and E-Plates 16 (05469830001, Roche/ACEA). Experiments are performed according to the manufacturer's instructions. For each data set 500 or 1000 cells per well are seeded in quadruplicates. The impedance value of each well is automatically monitored by the xCELLigence system every 5 minutes over a period of 96 hours and expressed as a CI (cell index) value. The rate of cell growth is determined by comparing the percent difference in the mean normalized CI values between the cells.
[0332] 2.7 Differentiation Assays
[0333] MSC were grown until confluence. Osteogenic differentiation was induced in medium supplemented with 0.1 M dexamethasone (D4902, Sigma-Aldrich), 10 mM beta-glycerophosphate (G9422, Sigma-Aldrich) and 0.05 mM L-ascorbic acid (A4403, Sigma-Aldrich). Adipogenic differentiation was initiated in medium supplemented with 10 g/ml insulin (10516, Sigma-Aldrich), 1 M dexamethasone (D4902, Sigma-Aldrich), 0.1 mM indomethacin (Landesapotheke Salzburg, Austria), and 0.5 mM 3-isobutyl-1-methylxanthine (15879, Sigma-Aldrich). Media were changed every 3 days, and 14 days after differentiation induction cells were fixed with 4% paraformaldehyde (0335.3, Roth). Osteogenic differentiation was detected by staining with 0.5% Alizarin Red S (A5533, Sigma-Aldrich), adipogenic differentiation by staining with 1% Sudan III (S4136, Sigma-Aldrich).
[0334] 2.8 Proteinase and RNase Protection Assay
[0335] EV pellets isolated from BM-MSC conditioned medium A were resuspended in 4 mL of alpha-MEM (without Dipeptiven and pHPL), filtered through a 0.22 m filter to remove persistent precipitates and divided into four aliquots. One sample was incubated with proteinase K (0.05 g/l final concentration, Sigma, P6556) at 37 C. for 30 minutes. Proteinase K activity was inhibited by incubation at 90 C. for 5 minutes. Subsequently, the sample was incubated with RNase A (0.5 g/l final concentration, Sigma, R4642) for 20 minutes at 37 C. As controls, samples were treated identically but adding PBS instead of proteinase K and/or RNase A. Samples were centrifuged at 120,000g for 3 hours (S55-A2 fixed angle rotor, k-factor: 39.8), and finally RNA was extracted and analysed as described above. To evaluate effectiveness of the proteinase K and RNase A digestion, purified vesicular RNA was incubated with proteinase K with or without subsequent RNase A treatment.
[0336] 2.9 Western Blot Analysis
[0337] Pelleted EV derived from media and conditioned media or cells as control were lysed in 1RIPA buffer (R0278, Sigma-Aldrich) supplemented with proteinase inhibitor cocktail (P8340, Sigma-Aldrich) and incubated at 4 C. for 15 minutes with occasional mixing, followed by centrifugation at 12,000g and 4 C. for 10 minutes. Protein concentration was determined with Agilent Protein chip (5067-1575) on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif.). One hundred g of protein lysates were separated on 4-15% polyacrylamide gradient gels (456-1084, Bio-Rad Laboratories) and transferred onto nitrocellulose membranes (170-4158, Bio-Rad Laboratories). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for one hour at room temperature and probed with primary antibodies against CD9 (sc-13118, Santa Cruz Biotechnology), CD81 (sc-7637, Santa Cruz Biotechnology), TSG101 (sc-7964, Santa Cruz Biotechnology), and GM130 (610823, BD Transduction Laboratories), all diluted 1:250 in TBS-T containing 0.5% non-fat dry milk, with incubation at room temperature for 4 hours. After extensive washing with TBS-T, the secondary antibody (goat anti-mouse HRP-conjugated, K4004, DAKO), diluted 1:200 in TBS-T containing 0.5% non-fat dry milk, was applied for one hour at room temperature. The proteins were detected with ECL Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare) and ChemiDoc MP System (Bio-Rad).
[0338] 2.10 Animal Study Design
[0339] All animal experiments and procedures were conducted in accordance with Austrian laws on animal experimentation and were approved by Austrian regulatory authorities (permit number BMWF-66.012/0035-WF/V/3b/2016). 26 male Fischer-344 rats (age 2 months; weight approx. 200 grams; Charles River Laboratories, Germany) were randomly assigned to seven groups. A 5 mm, critical-sized, mid-femoral defect was created (see section 2.4) and bone defects were treated with a scaffold only (control I; n=4), received a collagen sponge with a low dose of rhBMP2 (1 g control II; n=2), or received a scaffold loaded with EVs derived from bone marrow-derived mesenchymal stromal cells (BM-MSCs; treatment I; n=5), or umbilical cord-derived mesenchymal stromal cells (UC-MSCs; treatment II; n=5). In addition, one group each received BMSC-EVs (treatment III; n=5) or UC-MSC-EVs (treatment IV; n=5) combined with 1 g of rhBMP2 respectively. Assessment of bone healing was performed by radiography (2, 4, and 6 weeks post-surgery), by histology (6 weeks post-surgery; n=2 per treatment), and CT analysis (6 weeks post-surgery).
[0340] 2.11 Implant Preparation
[0341] Cylinders of equine collagen sponges (6 mm, Biopad Collagen, Euroresearch s.r.l, Italy) were prepared using dermal punches (pfm medical, Germany). BMSC-EVs and UCMSC-EVs resuspended in a final volume of 50 l lactated Ringer's solution (Fresenius Kabi, Austria) were combined with 30 l 2% NOVATACH MVG GRGDSP alginate solution (FMC Biopolymer, Norway) and loaded onto a collagen sponge. Gelation at room temperature was induced by the addition of 10 l of a CaCl.sub.2 solution (0.7 g/L in sterile water). For the treatment groups III and IV 1 g of rhBMP2 (Peprotech, Austria) was added to the alginate in addition to 50 l of EV preparation. For control groups, scaffolds were either loaded with an alginate/NaCl solution only (control I) or with 1 g rhBMP2 in alginate (control II) in a final volume of 80 l. Scaffold were implanted immediately after preparation.
[0342] 2.12 Surgical Procedure
[0343] A 5 mm critical-sized femoral defect was created according to a modified version of a previously published procedure (Kunkel et al., 2015, Betz et al., 2010, Einhorn et al., 1984). Briefly, surgeries were performed under isoflurane gas anesthesia (4% for induction, 2% for maintenance; SomnoSuite, Kent Scientific, U.S.A.) in oxygen (450 ml/min) and were warmed using an electric heating pad to prevent hypothermia (Harvard Apparatus; Holliston, Mass.). After aseptic preparation for surgery a 3-4 cm skin incision was made and the shaft of the femur was carefully exposed. After creating four drill holes using a 0.9 mm drill bit (Gebrder Brasseler, Lemgo, Germany) two custom-made fixation plates were secured to the femur using 4 threaded Kirschner wires (MEDE Technik, Emmingen, Germany). A 5 mm segmental defect was created using a Gigli wire saw (0.9 mm; RISystem, Davos, Switzerland) and subsequently the site was thoroughly rinsed with sterile 0.9% saline solution. A collagen sponge (control or loaded with MSC-EVs and/or rhBMP2; see section 1.2) was press-fitted into the defect area and the wound was subsequently closed in layers. After surgery, each animal was given an antibiotic (clindamycin, 45 mg/kg) and the analgesic meloxicam (1 mg/kg) and 3 ml 0.9% NaCl solution. After recovery from anesthesia animals were allowed free movement and analgesia was provided by subcutaneous injection of buprenorphine (0.01 mg/kg; 2 daily) and meloxicam (1 mg/kg; 1 daily) for three days after surgery. Animals had free access to food and water and were frequently monitored daily for any complications or abnormal behavior.
[0344] 2.13 Radiographic Assessment
[0345] Bone-healing was evaluated with serial radiography 4, 6, and 8 weeks post-surgery. While under general anesthesia, craniocaudal and mediolateral radiographs of the operated femur were obtained.
[0346] 2.14 Microcomputed Tomography
[0347] Blinded micro-CT scans of the explanted femurs were conducted using a SCANCO CT 50 system (Scanco Medical, Basserdorf, Switzerland). All samples were scanned nominally to the diaphyseal axis of the femora at 70 kVp and 200 A using a voxel size of 34.4 m. For 3D reconstruction slices of 34.4 m were recorded and subsequently a VOI including the entire osteotomy gap and 1 mm proximally and distally was chosen for analysis. Bone volume (BV) is expressed as meanSD.
[0348] 2.15 Descriptive Histology
[0349] Explanted femurs were fixed in 4% paraformaldehyde in PBS- for 48 hours, and decalcified samples were embedded in paraffin according to routine histological procedures. Sections of 7 m were stained with Masson's Goldner trichrome stain following standard protocols (Mulisch et al., 2010). All images were acquired by bright-field microscopy and were processed with Adobe Photoshop CS6. Only global corrections were performed (i.e. unsharp masking) and no specific feature within an image was manipulated.
Results:
[0350] 3.1 EV miRNA Profile
[0351] Mesenchymal stromal cells (MSCs) of various tissue origin display differential therapeutic effects despite their overall similar surface marker expression. Similarly, extracellular vesicles (EVs) that largely mediate the paracrine effects of MSC differ with respect to their protein and RNA content. EVs are purified from naive MSCs by differential ultracentrifugation and characterized by NTA and Western blotting. Agilent Bioanalyzer-based total RNA profiling and miRNA Next Generation Sequencing (NGS) were employed to identify differences in the EVs RNA content.
[0352] RNA profiling revealed that EVs from UC-MSC contained significantly larger amounts of total RNA and miRNAs than EV from BM-MSC. miRNA sequencing results confirmed the increased content of miRNAs found in UC-MSC-derived EVs. However, the most abundant miRNA in EV from all three naive tissue sources was miR-146a, which is involved in immunosuppressive processes (see
[0353] Immunosuppressive miRNAs packaged into EV from naive MSC may reflect their immunomodulatory potential. miRNA profiling can thus be employed to define the targeted therapeutic activity in MSC-derived EV to promote future preclinical and clinical trials. miRNA 146a-5p was used as the reference miRNA since this miRNA consistently showed the high values (see
[0354] Despite some overlapping miRNAs, the profiles for bone marrow and adipose tissue derived MSC exosomes reported in Baglio et al (2015) differ from the miRNA profiles in extracellular vesicles obtained by the herein described methods (see above and below). Therefore, the purified extracellular-vesicles of the present invention are different compared to the vesicles purified in Baglio et al.
[0355] Biomolecular signature profiles are required to determine the identity of the therapeutic agent. This present profile is well suited to support the animal data in vivo as well as the in vitro cell differentiation and proliferation data.
[0356] miRNA sequencing of EVs from BM-MSCs and UC-MSCs derived from different donors revealed a consistent profile that supports the observed pro-osteogenic activity observed in vitro and in vivo:
hsa-miR-146a-5p
hsa-miR-92a-3p
hsa-miR-21-5p
hsa-miR-30d-5p
hsa-miR-148a-3p
hsa-miR-320a
hsa-let-7i-5p
hsa-miR-221-3p
hsa-let-7f-5p
[0357] Further, hsa-miR-22-3p/hsa-miR-30d-5p could individually be determined as occurring in amounts less than 2% in relation to 100% of hsa-miR-146a-5p.
[0358] On the basis of the above findings, further characterization of the miRNA profiling can be carried out. For example, the quantification may be ranked according to miRNA abundance and/or levels. The purification of the extracellular vesicles was herein described above and below. Therefore, the miRNA profile in extracellular vesicles and a ranking according to the level of the miRNA abundance was also determined for extracellular-vesicles derived from UC-MSCs (see Table 1). Comparison of the miRNA content of UCMSC-derived vesicles obtained by either ultracentrifugation or tangential flow filtration (TFF) and grown in either HPL-containing standard medium (herein referred to as culture medium) revealed the sensitivity of the biological process of extracellular vesicle production.
[0359] The data revealed that the levels of particular miRNAs among the 20 most abundant miRNAs remained constant in the extracellular vesicle preparations compared to extracellular vesicles obtained from cells that had been maintained in serum free medium devoid of growth factor containing serum. For example, the hsa-miR-146a-5p, hsa-miR-92a-3p, hsa-miR-21-5p, hsa-miR-148a-3p, hsa-miR-221-3p, hsa-let-7i-5p, and hsa-let-7f-5p was constantly determined among the 20 most abundant miRNAs (see Table 1), whereas miR-22-3p, miR-222-3p, miR-486-5p and miR-191-5p were not determined among the 15 most abundant miRNAs.
[0360] In particular, miRNA 486 was not significantly not detected among the 20 most abundant miRNAs in extracellular-vesicles obtained by the herein described methods compared to the extracellular-vesicles purified by other strategies and in the presence of heparin.
[0361] The miRNA ranking differed from that reported of other cell types and lineages provided with the methodology of the prior art.
TABLE-US-00006 TABLE 1 Comparison of miRNA content of MSC-derived vesicles obtained by ultracentrifugation or tangential flow filtration (TFF) and grown in HPL-containing culture medium. The miRNA levels were similar in the extracellular vesicles isolated by either ultracentrifugation or TFF. Standard med./Ultracentr. Standard med./TFF miRNA TPM miRNA TPM hsa-miR-148a-3p 97730 hsa-miR-92a-3p 89803 hsa-miR-21-5p 95111 hsa-miR-21-5p 85704 hsa-let-7f-5p 82777 hsa-miR-146a-5p 79871 hsa-miR-146a-5p 75778 hsa-let-7f-5p 52473 hsa-let-7i-5p 68000 hsa-miR-100-5p 48681 hsa-let-7g-5p 43773 hsa-miR-148a-3p 46263 hsa-miR-92a-3p 40092 hsa-let-7i-5p 41879 hsa-miR-100-5p 37871 hsa-miR-30d-5p 41366 hsa-miR-151a-3p 33527 hsa-miR-151a-3p 30775 hsa-miR-103a-3p 31518 hsa-let-7a-5p 28170 hsa-let-7a-5p 29677 hsa-let-7g-5p 25289 hsa-miR-26a-5p 28120 hsa-miR-221-3p 23922 hsa-miR-584-5p 19316 hsa-miR-26a-5p 23248 hsa-miR-221-3p 18175 hsa-miR-10a-5p 22793 hsa-miR-10a-5p 13547 hsa-miR-584-5p 22637 hsa-miR-30d-5p 12635 hsa-miR-103a-3p 17568 hsa-miR-320a 11538 hsa-miR-486-5p 17433 hsa-miR-126-3p 11388 hsa-miR-320a 13582 hsa-miR-143-3p 10742 hsa-miR-25-3p 11917 hsa-miR-191-5p 10406 hsa-miR-143-3p 11013
[0362] Upon serum starvation an overrepresentation of miRNA 100 was observed. Comparative studies indicated that the origin of miR-100 dates back to the bilaterian ancestor. In human cancers, miR-100 has been reported to function as either a oncogenic miRNA or a tumor suppressive miRNA, which depends on tumor types and microenvironment. miR-100 functions in numerous important biological processes such as metabolism, cell cycle, migration, epithelial-mesenchymal transition, differentiation and cell survival. Moreover, miR-100 re-sensitizes tumor cells to chemotherapeutic drugs.
[0363] Accordingly, the umbilical cord MSC derived (and bone marrow derived) extracellular vesicles obtained by the herein described methods contain a unique miRNA profile that distinguish them from bone marrow or adipose tissue MSC derived EV preparations.
[0364] 3.2 Immunomodulation in MLR Assay
[0365] A robust immunosuppression potency assay has been established (Ketterl et al., 2015) using CFSE pre-labeled pooled and cryopreserved PBMCs, which can be tested off-the-shelf for mitogenesis-driven lymphocyte proliferation and MLR. The inhibitory potential of individual MSCs is compared to pooled MSCs as a reference normalizing donor variation in this combined assay format. We have modified and utilized this assay to evaluate the immunomodulatory potential of EVs. Notably, EVs from UC-MSCs perform comparable to parental UC-MSCs. This is a unique modification of a cell-oriented potency assay that aids in the in vitro determination of the immunomodulatory potential of purified EVs.
[0366] 3.3 NTA (Nanoparticle Tracking Analysis)
[0367] EV suspensions were diluted in PBS to result in a concentration of 4-710.sup.7 particles/mL. Amount and size of particles were measured using a ZetaView Nanoparticle Tracking Analyzer (ParticleMetrix). Minimum brightness was set at 20 arbitrary units (AU), sensitivity at 85 AU and temperature at 20 C. Five exposures at 11 measurement positions were recorded for each sample. Particle size was calculated according to the Stokes-Einstein equation by the ZetaView software. Data on particle numbers are presented as arithmetic meanstandard deviation (see
[0368] 3.4 General Animal Health
[0369] One animal from treatment group II (UC-MSC-EV) died 2 days after surgery due to unknown reasons. One animal each from treatment group II and treatment group III (BMSC-EVs plus 1 g rhBMP2) had to be excluded from the study due to fixator plate loosening. All other animals showed normal behavior with non-restricted weight bearing 48 hours after surgery and no obvious signs of adverse reactions were observed after implantation of the collagen scaffolds.
[0370] 3.5 Radiographic Evaluation, CT, and Descriptive Histology
[0371] For the vast majority of the animals from the treatment groups calcified tissue had formed within the defect region 6 weeks post-osteotomy (
[0372] Comparisons of the bone volume (BV) formed within the defect area 6 weeks after the surgery revealed no significant differences (p>0.05) between the various treatment groups (
TABLE-US-00007 TABLE 2 Bone Volumes (BV) in mm.sup.3 determined from CT scans Treat. III Treat. IV Treat. I Treat. II (BM-MSC- (UC-MSC- Ctrl. I Ctrl. II (BM-MSC- (UC-MSC- EV + 1 g EV + 1 g (scaffold (1 g BV EV; EV; rhBMP; rhBMP; only; rhBMP2; (mm.sup.3) n = 5) n = 3) n = 4) n = 5) n = 4) n = 2) Median 11.13 14.64 24.11 27.47 2.944 15.78 Minimum 5.495 14.06 19.86 18.27 0.0223 2.326 Maximum 21.29 21.74 27.86 35.09 7.339 29.24 Mean 12.32 16.82 23.98 25.87 3.313 15.78 Std. Deviation 6.317 4.277 4.305 6.818 3.404 19.03 Lower 95% CI 4.473 6.189 17.13 17.4 2.103 155.2 of mean Upper 95% CI 20.16 27.44 30.84 34.33 8.728 186.8 of mean
[0373] 4.2 Bone Formation Assessed by Rat Femur Segmental Defect Model
[0374] Animals of the rat femur segmental defect model were treated with BMSC-EV, UCMSC-EV and BMSC-EV plus BMPs2, and UCMSC-EV plus BMP2. For the vast majority of the animals from the treatment groups calcified tissue had formed within the defect region 6 weeks post-osteotomy. However, no bony union was achieved, despite the significant formation of immature bone tissue. Treatment with vesicles with or without a low dose of BMP2 resulted in a significantly higher bone volume (BV) when compared to the scaffold only group. For most cases a bony closure of the medullary canal with a variable amount of appositional bone formation was evident. In contrast, for those animals that had received the scaffold without EVs or rhBMPs (control I), only minimal amounts of calcified tissue was evident. Interestingly, for one femur which had been treated with 1 g rhBMP-2 only (control II) a significant amount of calcified tissue had formed 6 weeks post-surgery, whereas for the second animal no significant amount of new bone was seen, as evidenced by X-ray and CT analysis.
[0375] In summary, treatment of a critical-size defect with EVs isolated from MSCs resulted in the formation of significant amounts of calcified tissue. The treatment with UC-MSC-EVs induced moderately more tissue regeneration when compared to the treatment with BM-MSC-EVs. Further, the co-application of 1 g rhBMP2 further enhanced bone formation. A dose of 1 g rhBMP2 per defect may represent a low dose. By downscaling the clinical dose based on body weight (12 mg of rhBMP2 for patients with an average body weight of 75 kg), a dose of 33.3 g/200 g would be administered in a preclinical study in rats. Interestingly, Yasko et al., compared rhBMP2 dosages in a 5 mm rat osteotomy defect and found that 11 g rhBMP2 resulted in endochondral bone formation and gap bridging, while a reduction to 1.4 g rhBMP2 could not induce healing in this critical size defect. Therefore, the co-administration of -EVs derived from MSCs further enhanced the osteoinductivity of rhBMP2.
EXAMPLE 2: A GMP-GRADE STANDARD PROTOCOL FOR EXCLUSIVELY HUMAN MESENCHYMAL STROMAL CELL-DERIVED EXTRACELLULAR VESICLES
[0376] As an alternative to serum-free cell culture conditions, serum/HPL depleted of its EV can be used for investigation and production of cell-derived EV. Several protocols for the depletion of FBS-derived EV have been reported and applied for EV research in the past, but the use of EV-depleted HPL for MSC propagation has not been reported thus far. A protocol for producing a medium is described that is free of xenogenic substances and based on supplementation with EV-depleted pHPL. The effect of this medium was evaluated on bone marrow (BM)-MSC morphology, immunophenotype, proliferation and differentiation properties as well as on RNA and protein composition in the purified EV. The data reveal that media produced under GMP compliant conditions and supplemented with 10% EV-depleted pHPL are suitable for the production and characterization of BM-MSC-derived EV.
Materials and Methods
Medium Preparation
[0377] Heparin-free and fibrinogen-depleted medium (medium A) was prepared as follows: Alpha-modified Minimal Essential Medium Eagle (alpha-MEM, M4526, Sigma-Aldrich) was supplemented with 5 mM (N2)-L-Alanyl-L-Glutamine (Dipeptiven, 11051014, Fresenius Kabi, Austria) and 10% pHPL. pHPL was produced as described earlier (Schallmoser et al., 2007). The mixture was immediately divided into 14 aliquots (40 mL in 50 mL centrifuge tubes), maintained for 4-6 h at 20-24 C. until biogel formation was completed, and stored at 4 C. for 12-18 h. To remove fibrin, the clotted medium was brought to 37 C. The fibrin gel was then physically collapsed, and the precipitated fibrin was pelleted at 2,500g at 20 C. for 12 minutes (Laner Plamberger et al., 2015). Finally, the supernatant was filtered (0.22 m) to yield medium A. Heparin-free, fibrinogen-depleted and EV-depleted medium (medium B) was produced from medium A by a single ultracentrifugation step at 120,000g for 3 h (employing a Sorvall MX120 ultracentrifuge, fixed angle rotor S50-A, k-factor 60.7, Thermo Scientific). The resulting supernatant was filtered through a 0.22 m filter, to yield medium B (EV-depleted medium). FBS-containing medium was prepared by adding 10% FBS (CC-4101A, Lonza) and 5 mM Dipeptiven to alpha-MEM, and immediate filtration (0.22 m). Serum-/pHPL-free medium (medium C) was generated by adding 5 mM Dipeptiven to alpha-MEM, followed by filtration (0.22 m). pHPL-reduced medium (medium D) was obtained by fivefold dilution of medium A with medium C; pHPL-reduced and EV-depleted medium (medium E) was prepared by fivefold dilution of medium B with medium C (see Table 3).
TABLE-US-00008 TABLE 3 Overview of media used in this study. Medium name Medium content Medium A (10% pHPL) alpha-MEM + 5 mM Dipeptiven + 10% pHPL Medium B (10% HPL/ alpha-MEM + 5 mM Dipeptiven + 10% pHPL, EV-depleted) EV-depleted Medium C (serum-/ alpha-MEM + 5 mM Dipeptiven pHPL-free) Medium D (2% pHPL) alpha-MEM + 5 mM Dipeptiven + 2% pHPL Medium E (2% pHPL/ alpha-MEM + 5 mM Dipeptiven + 2% pHPL, EV-depleted) EV-depleted FBS Medium alpha-MEM + 5 mM Dipeptiven + 10% FBS
[0378] All pHPL-based media are heparin-free and fibrinogen-depleted.
[0379] Abbreviations: pHPL, pooled human platelet lysate; EV, extracellular vesicles; FBS, fetal bovine serum.
Cell Culture
[0380] Cells were cultured at 37 C. and 5% CO.sub.2. Experiments were performed with two different primary human BM-MSC preparations. One BM aspirate was directly obtained from the University Clinic for Blood Group Serology and Transfusion Medicine, Paracelsus Medical University, Salzburg (Austria). The second BM aspirate was purchased from AllCells (California, USA). Both donors had signed an informed consent. BM-MSC were isolated from bone marrow as described (Lotvall, 2014) and tested negative for mycoplasma. Following primary cell isolation, medium A was used as the basic cell culture medium. For all experiments, cells were kept in 30 mL medium per T225 flask.
Isolation of pHPL-, FBS- and BM-MSC-Derived Extracellular Vesicles
[0381] EV present in pHPL were obtained by ultracentrifugation at 120,000g for 3 hours. EV from FBS were isolated from alpha-MEM containing 10% FBS in the same manner. To obtain EV released by BM-MSC, cells were first maintained in medium A. At 70% confluence, cells were washed with PBS and medium was changed to fresh medium. Conditioned media (CM) were harvested after 48 hours. The CM were first filtered through a 0.22 m filter to remove cell debris and large vesicles, followed by ultracentrifugation at 30,000g for 20 minutes to pellet larger microvesicles. The supernatants were subjected to ultracentrifugation at 120,000g for 3 hours to sediment the EV. The resulting pellets were either resuspended in PBS (for nanoparticle tracking analysis) or in alpha-MEM without supplements (for proteinase and RNase protection assay), or directly lysed in RNA lysis buffer from mirVANA miRNA Isolation Kit, AM1561, Ambion (for RNA isolation) or RIPA buffer (for Western Blot analysis; R0278, Sigma-Aldrich). Ultracentrifugation was performed in an S50-A fixed angle rotor (k-factor: 60.7, Thermo Scientific). EV were stored at 80 C. prior to nanoparticle tracking analyses (NTA). After collection of CM, cells were removed from the culture vessel by the addition of TrypLE Select CTS (A12859-01, Gibco), stained with trypan blue and counted using a hemocytometer for immunophenotyping and determining the live-dead ratio of the harvested cells.
Immunophenotyping and Determination of Cell Viability by Flow Cytometry
[0382] Immunophenotype and viability analysis of BM-MSC was carried out according to the minimal criteria for defining MSC identity (Dominici et al., 2006). In brief, collected cells were centrifuged (300g for 6 min), resuspended in 5% v/v sheep serum-containing blocking buffer to reach a concentration of 1.510.sup.7 cells/mL and incubated for 20 minutes at +4 C. in dark. 310.sup.5 cells were stained with mouse anti-human monoclonal antibodies against CD90 (IM1839U, Beckman Coulter, France), CD105 (MCHD10505, Life Technologies, Austria), CD14, CD34, CD45, CD73, HLA-II (DR) (345785, 345802, 345808, 550257, 347400 Becton Dickinson, Austria), or with corresponding isotype controls (345815, 345818, 555743, 345816, 349051, Becton Dickinson, Austria) for 25 minutes at +4 C. in dark. Thereafter samples were washed with cold PBS and resuspended in 100 L 7AAD-containing PBS (1:10 dilution, 0.0005 w/v % final concentration) and stained for 10 minutes at room temperature protected from light. Finally, 400 L cold PBS was added and the samples were measured immediately using a FACSCanto II flow cytometer (Becton Dickinson). For each measurement 10.000 events were recorded per staining. Blue (488 nm) and red (633 nm) laser excited fluorescence signals were detected with the following standard light filters: FITC: 530/30 nm; PE: 585/42 nm; APC: 660/20 nm; 7AAD: 670LP. Results were analyzed with WinList software 8.0 (Verity Software House, Topsham, USA). [FSCA, SSCA] dot plot analyses were applied for debris exclusion, and a doublet discrimination panel was set on the FSC channel for the detection of height and width of the fluorescence signals. The ratio of the viable cells was determined on [SSC, 7AAD] dot plot.
RNA Isolation and Detection
[0383] RNA was isolated from EV pellets using mirVana miRNA Isolation Kit (AM1561, Ambion) according to the manufacturer's instructions for total RNA isolation. One microliter of isolated RNA was analyzed for quality, size distribution and yield with Agilent RNA 6000 Pico chips (5067-1513). The profile (based on size discrimination/nucleotide length determination) of the miRNA was determined by Agilent Small RNA chips (5067-1548) on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif.) according to the manufacturer's protocol.
Nanoparticle Tracking Analysis
[0384] EV suspensions were diluted in PBS to obtain 4-710.sup.7 particles/mL. Amount and size of particles were measured using a ZetaView Nanoparticle Tracking Analyzer (ParticleMetrix). Minimum brightness was set to 20 arbitrary units (AU), sensitivity to 85 AU and temperature to 20 C. Five exposures at 11 measurement positions were recorded for each sample. Particle size was calculated according to the Stokes-Einstein equation by the ZetaView software.
Proliferation Assays
[0385] The proliferative capacity of BM-MSC was monitored in real-time employing the impedance-based growth measurement system xCELLigence RTCA DP (Roche/ACEA) and E-Plates 16 (05469830001, Roche/ACEA). Experiments were performed according to the manufacturer's instructions. For each data set 500 or 1000 cells per well were seeded in quadruplicates. The impedance value of each well was automatically monitored by the xCELLigence system every 5 minutes over a period of 96 hours and expressed as a CI (cell index) value. The rate of cell growth was determined by comparing the difference in the mean normalized CI values between the cells.
Differentiation Assays
[0386] BM-MSC were grown in medium A until confluence. Osteogenic differentiation was induced using medium supplemented with 0.1 M dexamethasone (D4902, Sigma-Aldrich), 10 mM beta-glycerophosphate (G9422, Sigma-Aldrich) and 0.05 mM L-ascorbic acid (A4403, Sigma-Aldrich). Adipogenic differentiation was initiated using medium supplemented with 10 g/ml insulin (10516, Sigma-Aldrich), 1 M dexamethasone (D4902, Sigma-Aldrich), 0.1 mM indomethacin (Landesapotheke Salzburg, Austria), and 0.5 mM 3-isobutyl-1-methylxanthine (15879, Sigma-Aldrich). Media were changed every 3 days, and 14 days after induction of differentiation cells were fixed with 4% paraformaldehyde (0335.3, Roth). Osteogenic differentiation was detected by staining with 0.5% Alizarin Red S (A5533, Sigma-Aldrich), adipogenic differentiation by staining with 1% Sudan III (S4136, Sigma-Aldrich).
Proteinase and RNase Protection Assay
[0387] EV pellets isolated from BM-MSC conditioned medium A were resuspended in 4 mL of alpha-MEM (without Dipeptiven and pHPL), filtered through a 0.22 m filter to remove undisssolved precipitates and divided into four aliquots. One sample was incubated with proteinase K (0.05 g/L final concentration, Sigma, P6556) at 37 C. for 30 minutes. Proteinase K activity was inhibited by incubation at 90 C. for 5 minutes. Subsequently, the sample was incubated with RNase A (0.5 g/L final concentration, Sigma, R4642) for 20 minutes at 37 C. As controls, samples were treated identically but adding PBS instead of proteinase K and/or RNase A. Samples were centrifuged at 120,000g for 3 hours (S55-A2 fixed angle rotor, k-factor: 39.8), and finally RNA was extracted and analysed as described above. To evaluate effectiveness of the proteinase K and RNase A digestion, purified vesicular RNA was incubated with proteinase K with or without subsequent RNase A treatment.
Western Blot Analysis
[0388] EV derived from growth media or conditioned media, or cells (as control) were lysed in 1RIPA buffer (R0278, Sigma-Aldrich) supplemented with proteinase inhibitor cocktail (P8340, Sigma-Aldrich) and incubated at 4 C. for 15 minutes with occasional mixing, followed by centrifugation at 12,000g at 4 C. for 10 minutes. Protein concentration was determined using an Agilent Protein chip (5067-1575) on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif.). One hundred g of protein lysates were separated on 4-15% gradient polyacrylamide gels (456-1084, Bio-Rad Laboratories) and transferred onto nitrocellulose membranes (170-4158, Bio-Rad Laboratories). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for one hour at room temperature and probed with primary antibodies against CD9 (sc-13118, Santa Cruz Biotechnology), CD81 (sc-7637, Santa Cruz Biotechnology), TSG101 (sc-7964, Santa Cruz Biotechnology), and GM130 (610823, BD Transduction Laboratories), all diluted 1:250 in TBS-T containing 0.5% non-fat dry milk, with incubation at room temperature for 4 hours. After extensive washing with TBS-T, the secondary antibody (goat anti-mouse HRP-conjugated, K4004, DAKO), diluted 1:200 in TBS-T containing 0.5% non-fat dry milk, was applied for one hour at room temperature. Proteins were detected with ECL Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare) and ChemiDoc MP System (Bio-Rad).
Statistical Methods
[0389] Data are presented as arithmetic meanstandard error of the mean (SEM). Data were tested for Gaussian distribution using the Kolmogorov-Smirnov normality test and for outliers by Grubb's test. Data were compared using two-tailed unpaired t-test (*, p<0.05; **, p<0.01; ***, p<0.001). The significance level was 0.05 (95% confidence intervals). Analysis was achieved using GraphPad Prism 5 software and Microsoft Office EXCEL 2010.
Results
[0390] A Single Ultracentrifugation Step Eliminates RNA-Containing EV Derived from pHPL Below Detection Limit
[0391] The standard medium A used in this study is entirely free from xenogenic substances and is supplemented with 10% pHPL. For depletion of pHPL-derived EV, medium A was centrifuged at 120,000g for three hours. The resulting supernatant is referred to as medium B (EV-depleted medium), the resulting pellet was used to analyze the pHPL-derived EV in medium A. To assess the efficacy of EV depletion, medium B was subjected to another 3-hour centrifugation step at 120,000g, and the EV yield from medium A and B was compared (
BM-MSC Propagated in EV-Depleted Medium Retain their Characteristic Morphology, Phenotype, Viability and Differentiation Potential, but Exhibit Slightly Decreased Proliferation Potential
[0392] We examined whether the EV-depleted medium is suitable for propagation and ex vivo expansion of MSC. BM-MSC isolated from two different donors and cultured in EV-depleted medium were tested for surface marker expression, proliferation and differentiation potential. No morphological changes could be observed between BM-MSC grown in medium A versus medium B (see
[0393] The proliferation rate of BM-MSC propagated in medium B did not change significantly after 48 hours but was decreased by 13% after 96 hours, compared to cells in medium A (
EV-Depleted Medium Conditions Result in Enrichment of BM-MSC-Derived EV
[0394] Next we analyzed the EV released from cells expanded in EV-depleted medium. BM-MSC were first grown in medium A to reach a confluence of 70%, followed by a medium change to either fresh medium A or medium B. EV were purified from conditioned media after 48 hours. CM B contained on average 70% fewer particles than CM A, when calculated either per cell or per mL (
[0395] EV RNA profiles from identical volumes of CM demonstrated that CM A contained more vesicular RNA than CM B, but that the size distribution of isolated RNA was similar (
[0396] To confirm that the RNA purified from BM-MSC-derived EV pellets is present in vesicles and not in extracellular protein-RNA complexes, EV pellets derived from cells cultured in medium A were treated with RNase, with or without pre-treatment with proteinase K. Proteinase K digestion was optimized using cell lysates (see
pHPL-Reduced Culture Conditions Strongly Affect MSC Proliferation and Differentiation Capacities as Well as EV RNA Profiles
[0397] Media formulations using reduced pHPL levels (e.g. 2% versus 10%) seem advantageous for large scale expansions and clinical scale EV purification, as smaller amounts of fibrinogen-depleted and EV-depleted medium (medium B) have to be produced. To assess whether such media are equally suitable, we compared BM-MSC characteristics and RNA profiles of EV released in 10% pHPL (medium A), 10% pHPL/EV-depleted (medium B), 0% pHPL (serum-/pHPL-free, medium C), 2% pHPL (medium D), and 2% pHPL/EV-depleted (medium E) alpha-MEM (see Table 3). Flow cytometric analyses indicated no change in the MSC immunophenotype after two passages in pHPL-reduced media D and E (
[0398] Notably, cells showed no signs of increased cell death in either medium, as investigated by microscopy (see
[0399] When BM-MSC were transferred from medium A to medium D (2% pHPL) or medium E (2% pHPL/EV-depleted), we detected a reduction in the proliferative capacity to 81% and 72.5%, respectively, after 48 hours. After 96 hours, proliferation in media D and E were reduced to 56% (donor AC 1-001)/62% (donor DA001) and 49% (donor AC 1-001)/59% (donor DA001), respectively, compared to cells in medium A (
[0400] The most striking differences between cells cultured in 10% pHPL versus 2% pHPL or 0% pHPL were observed for their differentiation potential. With serum-/pHPL-free or pHPL-reduced media as basal differentiation medium, BM-MSC exhibited a significantly increased propensity for matrix mineralization following chemical induction of osteogenic differentiation (
[0401] To further characterize EV released by BM-MSC cultured in pHPL-reduced or serum-/pHPL-free media, cells were first maintained in standard medium A until 70% confluence, followed by a period of 48 hours in media C, D, or E and subsequent EV isolation. CM D contained 17.61.9% (donor AC 1-001)/22.50.5% (donor DA001), CM E 8.80.4% (donor AC 1-001)/11.80.4% (donor DA001) of the amount of nanosized EV particles detected in CM A per cell. In CM C, only 40.4% (donor AC 1-001)/6.30.5% (donor DA001) of the number of EV determined for CM A per cell were detected (
[0402] Thus, BM-MSC propagated in pHPL-reduced or entirely serum-/pHPL-free media display variations in their proliferation and differentiation potential and release EV with altered RNA contents in comparison to cells cultured in standard medium.
[0403] In this study, a protocol was developed for the production of a pHPL-supplemented medium that is fibrinogen-depleted, free of non-human components and depleted of pHPL-derived EV, and investigated its suitability for the collection and characterization of human BM-MSC-derived EV.
[0404] This pHPL EV depletion protocol demonstrated considerable effectiveness in terms of eliminating vesicle-derived RNA. After centrifugation, no RNA-containing pHPL EV were detected. The overall nanoparticle content in the EV-depleted medium was reduced to around 19% of the starting content Evidence was provided that pHPL EV-depleted medium was suitable for propagation and large scale expansion of BM-MSC. Cells displayed high viability, exhibited characteristic MSC immunophenotype and were able to differentiate into osteogenic and adipogenic cells. The proliferation rate was decreased to 87% of the rate in standard pHPL medium after 96 hours. However, standard pHPL medium can be used for cell propagation until the desired cell confluence is reached, and changed for EV-depleted medium only prior to EV collection, thereby circumventing a longer EV production time due to decreased proliferative capacity of the parental cells. Application of EV-depleted medium for the production of BM-MSC-derived EV showed that the overall EV RNA pattern remained unchanged. The RNA profiles reflected the EV RNA content and composition. RNA was protected from proteinase K and RNase degradation indicating that the RNA purified from the EV pellets is present inside the membrane-encircled compartment of the vesicles that cannot be penetrated by proteinase K, and is not part of larger, RNA protecting structures such as AGO-2 or HDL.
[0405] BM-MSC EV harvested from cells propagated in standard pHPL medium may represent a mixture of BM-MSC-derived EV and pHPL-derived EV. In standard pHPL medium, the amount of nanoparticles prior to cell propagation was comparable to that in the conditioned medium after 48 hours. Thus, uptake and/or degradation of EV and release of EV by BM-MSC seemed to be balanced under this medium condition. In contrast, the conditioned pHPL EV-depleted medium contained twice the amount of nanoparticles determined in the pHPL EV-depleted starting medium, suggesting that in BM-MSC cultured in a medium, which was reduced by 81.2% in pHPL-derived EV, the release of EV can exceed their uptake/degradation. Significant amounts of RNA in EV-depleted medium were not detected. Thus, the RNA present in EV from pHPL EV-depleted conditioned medium originated from BM-MSC.
[0406] Removal of serum-/HPL-derived EV from a culture medium prior to collection of CM for EV enrichment from any cell type is recommended in order to yield an exclusively cell-derived EV population. Cell-derived EV fractions of highest achievable purity and a minimum of co-purifying substances are required to determine their therapeutic effect and to exclude that any measurable therapeutic effect arises from HPL-derived EV. HPL contains a large amount of growth factors and cytokines that contribute to tissue regeneration. A reduction of pHPL from 10% to 2% with or without prior EV-depletion strongly promoted the osteogenic differentiation potential of BM-MSC, but decreased the differentiation towards adipocytes. Also in serum-/pHPL-free alpha-MEM, BM-MSC displayed elevated osteogenic differentiation, but showed no sign of adipogenic differentiation.
EXAMPLE 3
Cytokine Profiling of Extracellular Vesicle Preparations Obtained by the Methods Provided Herein
[0407] Comparative analysis of cytokine content in extracellular-vesicle preparations from cells expanded in the presence of HPL or in synthetic media was performed. The extracellular-vesicles were analyzed by the ProcartaPlex multiplex immunoassays for the Luminex platform (Invitrogen; ProcartaPlex Mix&Match Human 20-plex (affymetrix eBioscience), Cat.no.: EPX200-19407-801, Lot no.: 140982000). ProcartaPlex multiplex immunoassays are bead-based assays for protein quantification based on the principles of a sandwich ELISA with the use of Luminex xMAP (multianalyte profiling) technology. ProcartaPlex assays are suitable for use with serum, plasma, cell and tissue lysates, and cell culture supernatants. Assays are provided in multiple formats across six species (including human, mouse, rat, nonhuman primate, porcine, and canine).
[0408] The use of multiplex immunoassays for multiple analyte detection is a valuable tool for the comprehensive study of biological systems. As these systems are comprised of networks of secreted proteins including cytokines, chemokines, growth factors, and other proteins, multiplex immunoassays are an efficient method for biomarker profiling of a large set of proteins from a small sample; see INVITROGEN's procartaplex multiplex immunoassays brochure for more details.
[0409] The cytokine profiles of UC-MSC EVs obtained from cells either grown in standard HPL containing medium and serum-free synthetic medium and enriched by either ultracentrifugation (Ultracentrif.) or tangential flow filtration (TFF) were compared.
[0410] For each assay 50 microliter of EV solution were used, corresponding to a cell equivalent of 2010.sup.6 cells.
[0411] Any cytokine detectable in HPL but not in synthetic medium indicated the contribution of the platelet lysate. Only cytokines that were detected in both preparations were considered for analysis and evaluation. Of note, cytokine profiling of EV-containing therapeutic preparations was a novelty, owing to the stringent focus on vesicles.
TABLE-US-00009 TABLE 4 Cytokine profiling of UCMSC-derived extracellular vesicle preparations. Condition Condition Standard Syn. Condition med./ Condition medium/ Syn. Ultracentri- Standard Ultracentri- medium/ NAME fugation med./TFF fugation TFF SDF-1 1277.7 470.9 636.5 367.5 183.7 80.4 209.3 63.0 alpha IL-27 42.9 0.4 70.9 3.3 11.5 0.1 11.5 0.1 IL-1 beta 3.6 0.1 3.7 0.3 2.5 0.1 2.5 0.0 IL-2 201.6 2.1 259.7 3.4 0.0 0.0 IL-5 123.0 10.3 163.8 35.4 0.0 0.0 IL-6 390.4 6.3 1167.5 158.0 16.4 16.4 144.2 32.7 IL-8 302.3 21.0 432.2 85.3 49.5 10.3 82.6 2.6 IL-10 7.9 0.2 9.5 3.6 0.0 0.0 Eotaxin 33.9 0.3 44.1 1.3 0.0 4.4 1.0 IL-1RA 844.1 70.1 754.2 221.3 0.0 0.0 RANTES 104.1 0.1 109.4 10.5 48.8 10.5 75.8 5.3 IFN- 5.6 0.6 10.8 1.1 0.0 0.0 gamma GM-CSF 58.0 0.2 80.3 1.7 25.8 0.3 27.0 0.7 TNF-alpha 86.9 6.6 108.6 0.0 0.0 0.0 VEGF-D 10.9 0.9 30.6 3.6 0.0 0.0 bNGF 145.1 8.5 160.2 34.9 0.0 0.0 EGF 37.5 2.7 64.8 3.9 0.0 0.0 BDNF 125.6 24.7 208.6 65.7 119.8 93.7 230.6 90.1 IL-15 59.1 0.7 102.8 8.0 0.0 0.0 PDGF-BB 35.2 2.5 68.1 0.5 0.0 0.0
Median Values:
[0412]
TABLE-US-00010 SDF1 600 pg/mL IL-6 800 pg/mL IL8 350 pg/mL GM-CSF 70 pg/mL BDNF 170 pg/mL
BDNF 329 aa
[0413] Differential analysis of extracellular-vesicle-associated cytokines revealed a specific enrichment in the neuroprotective cytokine Brain Derived Neurotrophic Factor (BDNF). Enrichment and purification strategies that take advantage of the Tangential Flow System versus conventional ultracentrifugation increase the BDNF content by 70%. BDNF is specifically produced by the therapeutically-active UC-MSCs and packaged into or onto vesicles and is not serum-derived since identical values of BDNF were retained in extracellular-vesicle preparations derived from both HPL containing and serum-free synthetic culture media. The HPL-containing medium was prepared as described above in Example 1 (e.g. section 2.1) and Example 2.
[0414] Synthetic medium was prepared by combining the basal TheraPEAK Medium (Lonza) and the supplement (Cat. No.: MSCGM-CD Bulletkit 00190632) according to manufacturer's guidelines. Medium was sterile filtered by 0.22 micron filtration. Purification of MSC-derived extracellular vesicles from cells grown in synthetic serum-free medium was conducted as for the standard medium derived extracellular vesicles.
Demonstrated Immunomodulatory Activity
[0415] The regenerative and immunomodulatory activity of MSCs is partially mediated by secreted vesicular factors. EVs exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies.
[0416] We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reported the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs prepared by the above described methods in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. These results supported an immunomodulatory potential of EVs obtained by the methods of the invention.
TSG-6 Expression and Therapeutic Activity
[0417] TSG-6 (277 aa) is a biomarker to predict efficacy of human MSCs in modulating inflammation in vivo. Comparison of hMSCs from a small cohort suggested that hMSCs from female donors compared with male donors were more effective in suppressing inflammation in the cornea model, expressed higher levels of TSG-6. The use of TSG-6 expression as a biomarker could improve selection of the most effective MSCs for clinical trials and overcome some of the variations in patient responses. The clinical trials with human MSCs from bone marrow and other tissues are proceeding even though cultures of the cells are heterogeneous and there is large variability among preparations of hMSCs due to differences among donors, culture conditions, and inconsistent tissue sampling. However, there was currently no in vitro bioassay for the evaluation of hMSC efficacy in vivo. Therefore, the value of the data obtained from current clinical trials may well be compromised by variations in the quality of the hMSCs used. This study provides the first biomarker that can predict the efficacy of hMSCs in suppressing sterile inflammation in vivo (Lee et al., 2014).
[0418] We have confirmed the specific enrichment of TSG-6 in umbilical cord MSC-derived extracellular vesicles obtained by the above described methods.
Patellar Tendon Enthesis Defect Model:
[0419] The regenerative capacity of the UC-MSC-derived extracellular-vesicles obtained by the methods described above was demonstrated by the treatment of tendon defects (enthesopathies). The UC-MSC-derived extracellular-vesicles were analyzed in to a patellar tendon enthesis defect model in rats. A total of 57 skeletally mature rats (age 3 months; weight approx. 300 grams; Janvier Labs, France) were randomly assigned to 5 groups. Under general anaesthesia a window defect in the patellar tendon (right hind limb) was created using a 2 mm sterile dermal punch (pfm medical, Germany) and the cartilaginous enthesis was thoroughly removed using the punch in order to thoroughly destroy the tendon insertion site. Subsequently, the defect were either left untreated (empty control; n=9), treated with a fibrin clot only (25 l, TISSEEL; Baxter, Germany), or received fibrin clots loaded with either UC-MSC-derived extracellular-vesicles only (10E6 cell equivalents), 0.5 g rhuBMP2 only (Peprotech, UK), or a combination of UC-MSC-derived extracellular-vesicles (10E6 cell equivalents) and 0.5 g rhuBMP2. Subsequently the wound was closed and the animals were allowed to move freely and to immediately load the treated limb. 2 weeks post-surgery the animals were sacrificed, X-Ray analysis was conducted to exclude heterotopic ossification and tissue samples (tibia-patellar tendon-quadriceps muscle) were harvested for biomechanical evaluation using a uniaxial tensile testing regimen (Zwick/Roell, Germany).
[0420] For maximum tensile loads no significant differences between the test samples were observed. To the contrary, the stiffness of the samples did differ by trend. Pairwise comparisons between the control group and the treatment groups revealed:
[0421] First the defects treated with a combination of UC-MSC-derived extracellular-vesicles and 0.5 g BMP2 were significantly less stiffer when compared to the control group (p=0.0315; Mann-Whitney test); see
[0422] Second, the median values of the defects treated with UC-MSC-derived extracellular-vesicles alone were lower by trend when compared to the control group.
TABLE-US-00011 TABLE 5 Stiffness of specimens 2 weeks post-surgery Stiffness (N/mm) control EVs rhuBMP2 rhuBMP + EVs n 9.0 8.0 9.0 9.0 Minimum 77.2 52.6 68.4 50.7 25% Percentile 90.6 61.6 76.6 52.7 Median 95.3 78.5 90.0 74.5 75% Percentile 128.1 118.3 101.6 95.6 Maximum 160.0 139.4 126.6 98.0
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