Methods for differentiating mesenchymal stem cells
Inventors
Pietri, Sandra • Nguyen, Xuan Mai • Bastianelli, Enrico • Ena, Sabrina • Laruelle, Pierre-Yves • Tytgat, Isabelle
Assignees
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Publication Number
US-11447749-B2
Publication Date
2022-09-20
Expiration Date
Abstract
The application discloses a method for obtaining MSC-derived cells with improved transplantation properties from MSC, the method comprising a cell size reduction step, wherein said cell size reduction step is characterized by contacting MSC or MSC-derived cells in vitro or ex vivo with heparin or a derivative or analogue thereof at a concentration of at least 0.01 IU/ml. The application further provides a method for obtaining mesenchymal stem cell-derived cells from mesenchymal stem cells (MSC) comprising contacting MSC in vitro or ex vivo with FGF-2, TGFβ and at least 0.01 IU/ml heparin or a derivative or analogue thereof. The invention also provides the so-obtained cells and cell populations, as well as further products comprising such and uses thereof.
Core Innovation
The invention relates to obtaining progenitor cells committed to the osteoblastic lineage from mesenchymal stem cells (MSC). The method differentiates MSC in vitro or ex vivo by contacting the MSC with fibroblast growth factor-2 (FGF-2), transforming growth factor beta (TGFβ), and heparin or a derivative or analogue thereof at a concentration of at least 0.01 IU/ml.
After contacting, the progenitor cells committed to the osteoblastic lineage are harvested to obtain a substantially pure population. The resulting progenitor cells have the ability to differentiate into cells of the osteoblastic lineage and are described as osteoprogenitors or pre-osteoblasts.
The disclosure further describes MSC-derived bone-forming cell populations generated with heparin, including marker expression consistent with an osteogenic progenitor phenotype, increased osteoblastic marker gene expression, and DKK1 downregulation relative to comparison cells. It also reports reduced cell size with greater homogeneity, in vitro mineralization and chondrogenesis, and characterization by mesenchymal markers positive and CD45 largely negative.
Claims Coverage
The document includes one independent claim with dependent claims. The claim set centers on differentiating MSC into substantially pure osteoblastic-lineage committed progenitor cells using FGF-2, TGFβ, and heparin or a derivative or analogue at a specified minimum concentration, followed by harvesting the cells.
Osteoblastic-lineage progenitor differentiation with FGF-2, TGFβ, and heparin
Differentiating MSC into progenitor cells committed to the osteoblastic lineage by contacting the MSC in vitro or ex vivo with fibroblast growth factor-2 (FGF-2), transforming growth factor beta (TGFβ), and heparin or a derivative or analogue thereof at a concentration of at least 0.01 IU/ml.
Harvesting substantially pure osteoblastic-lineage committed progenitors
Harvesting the progenitor cells committed to the osteoblastic lineage to obtain a substantially pure population of the progenitor cells committed to the osteoblastic lineage, wherein the progenitor cells have the ability to differentiate into cells of the osteoblastic lineage.
Osteoprogenitors or pre-osteoblasts as the osteoblastic-lineage committed progenitors
The progenitor cells committed to the osteoblastic lineage are osteoprogenitors or pre-osteoblasts.
Substantially pure osteoblastic-lineage progenitor population with at least 90% by number
The substantially pure population of progenitor cells committed to the osteoblastic lineage contains at least 90% by number osteoblastic-lineage progenitor cells.
Substantially pure osteoblastic-lineage progenitor population with at least 95% by number
The substantially pure progenitor-cell population committed to the osteoblastic lineage contains at least 95% by number osteoblastic-lineage committed progenitor cells.
Osteoblastic-lineage committed progenitor cell concentration range
Using a substantially pure population of progenitor cells committed to the osteoblastic lineage at a concentration between about 1×10^7/ml and about 1×10^8/ml.
Increased expression of an osteoblastic marker gene versus MSC
The progenitor cells committed to the osteoblastic lineage show increased expression of an osteoblastic marker gene compared with its expression in MSC, where the marker gene is selected from RUNX2, ALPL, BMP2, OPG, POSTN, CADM1, CX43, CD10, and WISP1.
The independent claim covers differentiating MSC into substantially pure osteoblastic-lineage committed progenitor cells by contacting with FGF-2, TGFβ, and heparin or derivative or analogue at at least 0.01 IU/ml, then harvesting such that the progenitors retain the ability to differentiate into osteoblastic-lineage cells. Dependent claims further specify progenitor subtypes, purity thresholds, concentration ranges, and increased expression of osteoblastic marker genes versus MSC.
Stated Advantages
Improved transplantation properties linked to small size of the osteoblastic/osteochondroblastic lineage cells.
Reduced immunogenicity marker HLA-DR (reported as reduced HLA-DR with heparin).
Enhanced osteogenic activity markers, reported as higher ALP with heparin.
Documented Applications
Parenteral/intravascular administration, described in connection with standardized, small, homogeneous MSC-derived cells from the size reduction approach.
Transplantation, referenced as improved transplantation properties of osteoblastic/osteochondroblastic lineage cells produced with FGF-2, TGFβ, and heparin and linked to small size.
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