Cardiomyocyte-like cell clusters derived from hBS cells

A cardiomyocyte, cell technology, applied in the field of cardiomyocyte-like cell clusters, can solve problems such as influence

Inactive Publication Date: 2010-12-15
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on the variation in strategy used and the efficiency with which hBS cells differentiate into cardiomyocytes, it appears that hBS cell lines behave quite differently, suggesting that the actual hBS cell line used may affect the final results

Method used

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  • Cardiomyocyte-like cell clusters derived from hBS cells
  • Cardiomyocyte-like cell clusters derived from hBS cells
  • Cardiomyocyte-like cell clusters derived from hBS cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0277] Example 1: Differentiation of hBS cells into cardiomyocyte-like cell clusters

[0278] Spontaneously contracting cells were derived from undifferentiated hBS cells cultured on MEF cells (Heins et al 2004 Stem Cells). The cell line that is used for this test can be hBS cell line SA002, SA121, SA001, SA002.5, SA461 (Cellartis AB, Goteborg, Sweden, http: / / www.cellartis.com), and they can be according to Heins et al. Proliferation described in 2004. These cell lines are listed in the NIH stem cell registry and the UK StemCell bank. hBS cells were detached from the feeder layer by incubation with collagenase IV (200 U / ml) at 37°C for 10-15 minutes. This cell suspension was transferred to a 15 ml tube, and after the cells were pelleted, the supernatant was removed. Clones were resuspended in Knock OutDMEM supplemented with 20% FBS, 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol / l β-mercaptoethanol and 1% non-essential amino acids (all from Invitrogen, Carlsbad, Californ...

Embodiment 2

[0281] Example 2. Microarray analysis of cardiomyocyte-like cell clusters (CMLCs) derived from hBS cells

[0282] Cell Culture and Differentiation

[0283] The hBS cell line SA002 (Cellartis AB, Sweden, http: / / www.cellartis.com )proliferation. The cell line is listed in NIH stem cell registry and UK Stem Cell bank. Differentiation of hBS cells was performed as described in Example 1 above. When hBS cells were differentiated by this strategy, clusters of spontaneously contracting cardiomyocyte-like cell clusters (CMLCs) were frequently observed using light microscopy. For microarray experiments, 3 separate samples (biological replicates) of CMLC were collected. For comparison purposes, two independent samples from undifferentiated hBS cells and one sample from a mixed population of differentiated cells (ie, absence of contracting CMLCs) were also collected for analysis. Following selection and removal of spontaneously contracting CMLC in culture differentiated as descri...

Embodiment 3

[0334] Example 3. CMLC real-time QPCR derived from hBS cells

[0335] Cell culture and RNA extraction

[0336] Spontaneously contracting CMLCs were derived from undifferentiated hBS cells (SA002) (see Example 1). hBS cells were detached from MEF feeder layers by incubation with collagenase IV (200 U / ml) at 37°C for 10-15 minutes. This suspension was transferred to a 15ml tube, and after the cells had pelleted, the supernatant was removed. Clones were resuspended in culture medium and mechanically dissociated into small aggregates of undifferentiated cells using a pipette. The cell suspension was dispensed (200 μl / well) into 96-well plates (Costar, Corning, untreated, v-bottom) and centrifuged at 400×g for 5 min. The plate was then incubated at 37°C for 6-8 days. The formed 3D structures were then transferred to gelatin-coated dishes containing medium. After two days, spontaneous contractile clusters of cells (CMLCs) were observed. Some of these clusters were harvested 0-...

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Abstract

A cluster comprising cardiomyocyte-like cells, wherein the cluster has i) contracting cells, ii) cells that are electrically connected, and expresses iii) cardiac markers including Nkx.2.5, troponin and myosin, iv) markers for functional adrenergic receptors, v) markers for functional muscarinic receptors, vi) markers for functional ion-channels including hERG, Na+, Ca2+ and K+ channels, vii) one or more endodermal markers selected from the group consisting of AFP, TF, APOA2, AHSG, SERPINA1, APOA1, APOC3, TTR1 APOB, and RBP4. A method for preparing the clusters and the use of the clusters in drug discovery and toxicity screenings are described.

Description

technical field [0001] The present invention relates to novel cardiomyocyte-like cell clusters (CMLCs) derived from hBS cells, and to the potential use of such cardiomyocyte-like cell clusters in eg drug discovery and development, toxicity testing, cell therapy and medicine. The clusters contained cells expressing endoderm, mesoderm, and cardiac markers. The invention also relates to methods for the preparation of said clusters, compositions comprising one or more CMLCs for use in therapy and toxicity testing. The composition is stable after storage of the composition at a temperature of at least -80°C for at least 2 years, ie the properties and viability of the clusters do not substantially change during this storage period. Background technique [0002] Mature cardiomyocytes are considered terminally differentiated cells, and thus they have no or very low proliferative capacity. Human cardiomyocytes can be isolated from cardiac biopsies, but the process is complicated an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/077
CPCC12N2501/70C12N2501/115C12N2506/02C12N2503/02C12N2501/235C12N2501/16C12N5/0657C12N2501/999A61P9/00
Inventor P·萨蒂皮K·奥克松C·阿梅恩J·辛纳格伦K·达莱伯格D·斯蒂尔
Owner CELLECTIS SA
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