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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-9005897-B2
Publication Date
2015-04-14
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 discloses a general method to derive lineage‑restricted, self‑renewing progenitor cell lines from embryonic stem cells by selecting for self‑renewal and applying culturing conditions that discourage embryonic stem propagation and favor progenitor expansion, preferably feeder‑free. Specific experimental steps and formulations are omitted here as [procedural detail omitted for safety]. The resulting progenitor cell lines are lineage‑restricted compared to the parental embryonic stem cell and are described as non‑pluripotent and non‑tumorigenic.
The disclosure describes derived lines exemplified by mouse E‑RoSH and human huES9.E MSC‑like lines that show loss of pluripotency markers OCT4 and alkaline phosphatase, telomerase/OCT4 profiling changes, and upregulation of endothelial markers flk‑1, Tie‑2, c‑kit, runx‑1 and PDGFRα. The lines exhibit an MSC surface profile CD29+, CD44+, CD105+, CD166+, CD34−, CD45−, consistent with lineage restriction relative to the parental embryonic stem cell.
The progenitor lines demonstrate functional differentiation potential and growth properties, including endothelial features such as tubule formation, acetylated LDL uptake, and vWF/PECAM‑1 immunoreactivity, and differentiation into adipocyte and osteocyte lineages with PPARγ and Alp1 expression. They exhibit rapid population doubling, long‑term maintenance, genetic stability across passages, a stable karyotype, and lack of teratoma formation, supporting suitability for downstream screening or differentiation assays and therapeutic applications.
Claims Coverage
The patent includes two independent claims and the summary below extracts six main inventive features across those claims.
Use of a parental human embryonic stem cell or descendants
Providing a parental embryonic stem cell or descendants of a parental embryonic stem cell as the starting material, wherein the parental embryonic stem cell is a human embryonic stem cell; the specification also notes dispersal of an embryonic stem cell colony ([procedural detail omitted for safety]) as a preparative step.
Feeder-free culture in rich media lacking embryonic stem growth regulators
Culturing the parental embryonic stem cell in the absence of feeder cells in rich media comprising essential nutrients and serum or serum replacement, wherein the rich media does not comprise additional growth regulators or hormones that promote growth of embryonic stem cells, and wherein the culturing produces mesenchymal progenitor cells which self‑renew.
Establishment of a lineage-restricted, self-renewing mesenchymal progenitor cell line maintainable >20 generations
Establishing, in the absence of transformation, a mesenchymal progenitor cell line from the mesenchymal progenitor cells which self‑renew, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 20 generations and is lineage restricted compared to the parental embryonic stem cell.
Screening candidate molecules on mesenchymal progenitor cells
Culturing cells from the mesenchymal progenitor cell line in the presence of a candidate molecule and determining the effect of the candidate molecule on treated cells compared with untreated cells or cells treated with an inert compound.
Derivation of differentiated cells from the mesenchymal progenitor cell line for screening
A differentiated cell derived from the mesenchymal progenitor cell line for downstream analysis. [procedural detail omitted for safety]
Screening candidate molecules on differentiated cells
Screening candidate molecules using the differentiated cell derived from the mesenchymal progenitor cell line, with effects determined relative to controls. [procedural detail omitted for safety]
The independent claims center on starting from a human embryonic stem cell source, using feeder‑free rich‑media culture conditions that avoid embryonic stem growth regulators to produce self‑renewing mesenchymal progenitor cells, establishing a lineage‑restricted progenitor cell line maintainable for more than 20 generations, and using either the progenitor cells or differentiated derivatives as substrates for candidate molecule screening.
Stated Advantages
Generation of lineage‑restricted, non‑pluripotent progenitor cell lines.
Progenitor cell lines that are non‑tumorigenic.
Genetic stability of derived progenitor lines across passages.
Long‑term maintainability in cell culture for many generations (examples and claims state more than 20 generations, with dependent claims and examples noting higher thresholds).
Suitability of derived progenitor lines for derivation of differentiated cells and use in screening and therapeutic applications.
Demonstrated functional differentiation potential (endothelial, adipocyte, osteocyte) enabling use in functional assays.
Documented Applications
Screening candidate molecules for effects on human mesenchymal progenitor cells or on cells differentiated from a human mesenchymal progenitor cell line. [procedural detail omitted for safety]
Use in downstream screening or differentiation assays, supported by the derived lines' long‑term cultureability and lineage restriction.
Derivation of differentiated cells including endothelial and mesenchymal lineages and further differentiated cell types such as adipocytes and osteocytes.
Functional assays demonstrating endothelial features, including tubule formation, acetylated LDL uptake, and vWF/PECAM‑1 immunoreactivity, and differentiation into adipocyte and osteocyte lineages with marker expression (PPARγ and Alp1) as documented characterizations.
Regenerative and therapeutic uses.
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