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A novel population of multipotent cardiac precursor cells derived from human blastocysts derived stem cells

A technology of cells and populations, applied in the field of pluripotent cardiac precursor cell populations

Inactive Publication Date: 2009-08-26
塞拉帝思股份公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Numerous reports have demonstrated the ability of hESCs to differentiate into contractile myocytes, but so far there have been no reports on the identification and isolation of individual intermediate cardiac progenitor populations from hESCs

Method used

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  • A novel population of multipotent cardiac precursor cells derived from human blastocysts derived stem cells
  • A novel population of multipotent cardiac precursor cells derived from human blastocysts derived stem cells
  • A novel population of multipotent cardiac precursor cells derived from human blastocysts derived stem cells

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0273] Preparation of feeder cell layer

[0274] Before plating the foreign-free human fibroblast feeder cells, the tissue culture wells were coated with 0.1% recombinant human gelatin (Fibrogen) at room temperature for at least 1 hour. The confluent monolayers of exogenous hFF003 (passages 5 to 8) cells grown in IMDM, 10% human serum and 1% penicillin-streptomycin were then treated with mitomycin C (Sigma) for 2.5 hours. Place the feeder cells treated with mitomycin C on the IVF wells (Becton Dickinson), every 2.89cm in the medium 2 200,000 cells, the medium is based on DMEM (as above), supplemented with 10% (v / v) human serum, 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol / l β-mercaptoethanol and 1% non-essential amino acids ( Gibco Invitrogen Corporation). Before placing the blastocyst with inner cell cluster cells and cells derived from it or hBS cells, change the medium to DMEM (as above), now instead of supplementing with 20% (v / v) human serum, 10ng / mL Human recombinant bF...

Embodiment 1

[0310] Basal cells derived from undifferentiated hBS cells

[0311] Suspension culture

[0312] The undifferentiated hBS cells are maintained and propagated in vitro. On the 4-5 days after the passage, use a stem cell cutting tool to manually separate the undifferentiated hBS cell colonies and place them in the differentiation medium (KO-DMEM, supplemented with 1mM Glutamax, 0.1mM β-MeOH, 1% NEAA, 1% PeST and 20% FBS) in suspension culture. The cells are maintained in suspension for 1-6 days, and then transferred to a gelatin-coated tissue culture dish, resulting in the adhesion of differentiated cell clusters and the continued proliferation and differentiation of cells. Four days after plating, the adherent clusters of differentiated hBS cells appeared as a monolayer of cells. These cells grow in a characteristic dense network that resembles a maze, making the area of ​​the base cell easy to distinguish when using an optical microscope. The morphology of basal cells is similar to...

Embodiment 2

[0316] Characterization of Basal Cells

[0317] Basal cells were derived from undifferentiated hBS cells as described above. They were cultured in differentiation medium (KO-DMEM, supplemented with 1 mM Glutamax, 0.1 mM β-MeOH, 1% NEAA, 1% PeST and 20% FBS) for up to 37 days. Cells were fixed in PFA at different time points and assessed by immunohistochemistry (Noaksson et al. Stem Cells 2005). The antigenic markers used are vimentin, desmin, α-striated muscle actin and short tail protein. Basal cells express α-striated muscle actin and short tail protein ( Picture 9 ) But does not express vimentin and desmin. The cell lines used are SA002, LOTAL002 (passage 26 times); SA002.5, LOTBE002.5 (passage 32 times) and SA461, LOTBF461 (passage 33 times).

[0318] Table I summarizes the results obtained by IHC analysis of basal cells.

[0319] Table I

[0320] Antigenic marker

Days of differentiation ＊

The result

Antibody information

Vimentin

25

Negati...

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Abstract

The present invention discloses a novel population of multipotent cardiac precursor (MCP) cells derived from human blastocysts derived stem cells, methods for the preparation thereof and use of the cells for in vitro testing. Basement cells derived from hBS cells are also disclosed and method for the preparation of MCP cells from basement cells. The MCP cells have the following characteristics i) at least 1 % of the cells exhibit no antigen expression of one or more markers for undifferentiated cell, the marker being selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and Oct-4, ii) at least 1% of the cells exhibit no protein expression of one or more of a neural marker including nestin or GFAP iii) at least 1% of the cells exhibit protein and / or gene expression of one or more of a mesodermal marker including brachyury, vimentin or desmin iv) at least 1% of the cells exhibit protein and / or gene expression of Flk-1 (KDR). Furthermore, the MCP cells have a characteristic morphology. They grow as clusters of small, round and phase-bright cells; individual cells are 5-20 [mu]m in diameter and each cluster is composed of 2-500 cells. They form clusters of round or elongated shape, that appear as loosely adherent cell clumps that as illustrated in figure 2 panel a, b and c. Furthermore, they have a relatively high nucleus-to-cytoplasma ratio, e.g. 1 :2-1 :64 of the total volume of the cell and / or appear as balloons on a string, as illustrated in figure 18, schematic sketch. Moreover, the MCP cells are non-contracting.

Description

Invention field [0001] The present invention relates to a new population of pluripotent cardiac precursor (MCP) cells derived from hBS cells, and the potential applications of such MCP cells in, for example, medical treatment, drug development, cell therapy, and toxicity detection. [0002] In addition, MCP cells have the ability to differentiate into spontaneous contractile cardiomyocyte-like cells, which makes them attractive for in vitro studies of cardiogenesis such as early cardiomyogenesis processes or myocardial regenerative diseases or malformations. [0003] MCP cells are obtained after culturing so-called basement cells which are also derived from hBS cells. Therefore, the present invention also relates to these basal cells that have been shown to provide MCP cells. Background of the invention [0004] Cardiovascular disease is the cause of a large number of deaths in the world and a major burden on the healthcare system. For end-stage heart disease, heart transplantati...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N5/071C12N5/077
CPCC12N5/0672C12N2506/02C12N2500/44C12N5/0606C12N5/0657C12N2533/54
Inventor 彼得·萨蒂皮卡罗利妮·阿克松卡罗琳·阿梅
Owner 塞拉帝思股份公司
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