Method of deriving mesenchymal stem cells from ES cells using FGF2
Inventors
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Assignees
Paracrine Therapeutics Pte Ltd
MemberParacrineParacrineParacrine develops regenerative therapies for chronic wound care, leveraging autologous cell-based treatments and real-time AI platforms to enhance healing precision, predictability, and efficiency. The company operates globally, focusing on scalable and cost-effective biological solutions that integrate advanced machine learning for improved patient outcomes.
Paracrine develops regenerative therapies for chronic wound care, leveraging autologous cell-based treatments and real-time AI platforms to enhance healing precision, predictability, and efficiency. The company operates globally, focusing on scalable and cost-effective biological solutions that integrate advanced machine learning for improved patient outcomes.
Publication Number
US-8962318-B2
Publication Date
2015-02-24
Expiration Date
Abstract
We disclose a method comprising: (a) providing an embryonic stem (ES) cell; and (b) establishing a progenitor cell line from the embryonic stem cell; in which the progenitor cell line is selected based on its ability to self-renew. Preferably, the method selects against somatic cells based on their inability to self-renew. Preferably, the progenitor cell line is derived or established in the absence of co-culture, preferably in the absence of feeder cells, which preferably selects against embryonic stem cells. Optionally, the method comprises (d) deriving a differentiated cell from the progenitor cell line.
Core Innovation
The invention provides a defined, scalable, clinically oriented method to derive homogeneous mesenchymal stem cell (MSC) cultures and mesenchymal progenitor cell lines from human embryonic stem cells. The method uses feeder-free, serum-free propagation comprising FGF2, provision and dispersion of embryonic stem cell colonies [procedural detail omitted for safety], selection of MSCs by cell surface marker expression including CD105+ / CD24−, and expansion in serum-containing medium to establish lineage-restricted, self-renewing, non-transformed MSC-containing progenitor lines and monoclonal MSC isolates.
The resulting hESC-MSCs are homogeneous, karyotypically stable, long-term expandable, and show absence of teratoma formation. They exhibit very high genome-wide expression similarity among isolates, cluster with adult MSCs (including BM-MSC similarity), and display adipogenic and chondrogenic differentiation capacity with limited osteogenic mineralization. Negative selection against pluripotency markers (OCT4, Nanog, Sox2, Utf1) and high inter-isolate quantitative reproducibility are documented.
Proteomic and computational analysis of MSC-conditioned media identifies a reproducible secretome totaling 201 unique gene products, dominated by cytokines, chemokines and extracellular matrix components. Pathway analysis implicates Jak-STAT, MAPK, TGF-beta, mTOR, Toll-like receptor and FcεRI pathways, predicting roles in metabolism, defense response, cell migration, and tissue differentiation including vascularization, hematopoiesis and skeletal development. The data support a paracrine mechanism and the use of secreted proteins or conditioned media as therapeutics.
Claims Coverage
This coverage addresses two independent claims and eight main inventive features drawn from those claims.
Feeder-free propagation in serum-free medium comprising FGF2
Propagating an embryonic stem cell in the absence of a feeder cell layer, in a serum-free medium comprising FGF2.
Selection of mesenchymal stem cells by surface marker expression
Selecting mesenchymal stem cells from the propagated population based on expression of one or more cell surface markers.
Expansion of selected MSCs in serum-containing medium
Growing the mesenchymal stem cells selected from the propagated population in medium comprising serum to expand the selected cells.
Establishment of a lineage-restricted, self-renewing mesenchymal progenitor cell line without transformation
Establishing a mesenchymal progenitor cell line comprising MSCs that maintains self-renewal without transformation and is lineage restricted compared to the embryonic stem cell.
Provision of an ESC colony
Providing an embryonic stem cell colony as the starting material for derivation of a cell culture.
Dispersion of the ESC colony
[procedural detail omitted for safety]
Propagation of dispersed cells without feeder in serum-free media comprising FGF2 and without transformation
Propagating the dispersed cells in the absence of a feeder layer, in serum-free media in the presence of FGF2, and in the absence of transformation.
Selection and serum expansion to derive a cell culture
Selecting cells from the propagated population based on expression of one or more cell surface markers and growing the selected cells in medium comprising serum to derive a cell culture from the embryonic stem cell.
The independent claims cover methods that combine feeder-free, serum-free propagation comprising FGF2, selection by surface marker expression, and serum-containing expansion to establish lineage-restricted, self-renewing, non-transformed MSC-containing lines or derive cell cultures, with an explicit ESC colony starting material and dispersal step.
Stated Advantages
Scalable, clinically oriented method to derive MSCs or progenitor cell lines from embryonic stem cells.
Renewable human embryonic stem cell source.
Derived mesenchymal progenitor cell lines maintain self-renewal without transformation and are lineage restricted compared to ES cells.
Derived cells are karyotypically stable and long-term expandable.
Produces homogeneous MSC cultures and monoclonal MSC isolates with very high genome-wide expression similarity and quantitative reproducibility among isolates.
MSCs cluster with adult MSCs (including BM-MSC similarity).
Derived MSCs are non-teratogenic in SCID mice.
Generates MSCs that exhibit adipogenic and chondrogenic differentiation with limited osteogenic mineralization.
Batch consistency and reproducibility across independent conditioned media batches.
Conditioned media and secreted proteins explain paracrine therapeutic potential.
Use of serum-free chemically defined media.
Provides cell populations suitable for downstream applications including drug screening and regenerative therapies.
High transcript detection reproducibility (reported 86–88% transcript detection).
Documented Applications
Drug screening
Regenerative therapies
Use of one or more secreted proteins or conditioned media (CM) as therapeutics.
Paracrine-mediated tissue repair, including cardiovascular, hematopoietic and musculoskeletal applications.
Differentiation of derived MSCs to adipocytes, chondrocytes and osteocytes.
A method to identify regulators of MSC formation by testing candidate molecules
Predicted tissue-differentiation effects including vascularization, hematopoiesis and skeletal development.
Molecular/marker characterization of derived mesenchymal-like cells.
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