Neuron-like cell sourced from humanized adipose-derived stem cells, preparation method and application thereof

A technology of neuron-like cells and adipose-derived stem cells, which is applied in the field of stem cells and biomedicine, and can solve problems such as little consideration of clinical transformation applications, toxicity to the human body, and low maturity of neuron-like cells.

Active Publication Date: 2014-11-05
SHANGHAI EAST HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, previous studies were more concerned with the evaluation and mechanism of neuronal differentiation of hADSC or hBMSC in vitro, and less considered the clinical translation application
However, due to the strong proliferation and differentiation potential of embryonic stem cells, the methods currently applied to embryonic stem cells cannot be applied to adipose-derived stem cells.
In addition, the inducers used for hADSC induction in the prior a

Method used

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  • Neuron-like cell sourced from humanized adipose-derived stem cells, preparation method and application thereof
  • Neuron-like cell sourced from humanized adipose-derived stem cells, preparation method and application thereof
  • Neuron-like cell sourced from humanized adipose-derived stem cells, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1. Characterization of hADSCs by cell morphology and CD surface markers

[0100] The tissue cells obtained from liposuction were diluted with 20% fetal bovine serum (JRH Bioscience, USA) basal medium DMEM / F-12 (Invitrogen, Japan) and seeded in T-75flasks (Falcon, Becton Dickinson, Japan). ), so that its concentration is 6×10 7 cells / cm 2 . The medium was changed every two weeks. After 15 days of culture, hADSCs adhered to the wall, showed a typical spindle shape, and expanded in a vortex manner. Hematoxylin and eosin (HE) staining showed that hADSCs were mononuclear cells, such as figure 1 Shown in B. Identify hADSCs by immunohistochemical staining for CD surface markers, such as figure 1 a. Quantification of the positive rate of CD surface markers as figure 1 c.

[0101] These results indicated that more than 85% of hADSCs highly expressed the mesenchymal stem cell markers CD29, CD44, and CD105, whereas less than 5% of hADSCs expressed the hematopoieti...

Embodiment 2

[0102] Example 2. Stem cell transcription factors and differentiation to three lineages indicate pluripotency of hADSCs

[0103] Classic stem cell transcriptional markers such as Sox2, Oct4, c-Myc, Nanog, and KLF4 have been widely used to characterize induced pluripotent stem cells (iPSCs). To characterize the stemness of hADSCs, this study applied immunohistochemical staining and designed primers for transcription factor markers and semi-quantitative RT-PCR to detect the expression of these factors, compared with ESCs. The results showed that: with immunohistochemical staining, hADSC expressed Sox2, Oct4, c-Myc and Nanog at the protein level ( figure 2 A). Human embryonic stem cell (hESC) line H9 was used as a positive control, and Sox2, Oct4, c-Myc, Nanog and KLF4 were expressed by semi-quantitative RT-PCR, and Oct4, Nanog and KLF4 were significantly highly expressed in hESC. The number of positively expressed stem cell transcriptional markers indicated that more than 80%...

Embodiment 3

[0104] Example 3. Differentiation of hADSCs to neurons in plastic culture dishes into hADSC-NLCs

[0105] The inventors induced hADSCs to differentiate into neurons into hADSC-NLCs in plastic culture dishes. The steps of neuron induction included: first incubating hADSCs with DKK1 for 24 hours, and then using Safford, K.M, etc. (Biochem Biophys Res Commun294, 371, 2002) described in the mixed medium culture for 3 days. During induction, hADSC morphology changes, the cytoplasm retracts toward the nucleus, and the cell body becomes spherical, as in image 3 A and B are shown.

[0106] In order to confirm that the expression of neuronal markers is really due to the induction of the mixed induction solution, hADSCs were first immunohistochemically stained with Tuj1, MAP2, NeuN, Synapsin1 / 2 and vGAT without any treatment. hADSCs basally express Tuj1 and MAP2 but not NeuN, Synapsin1 / 2 and vGAT, as image 3 C shown. Basal expression of some neuronal markers in hADSCs, such as Tuj...

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Abstract

The invention provides a method for preparing neuron-like cells sourced from humanized adipose-derived stem cells. The method includes following steps: pre-incubating the humanized adipose-derived stem cells through a wnt pathway inhibitor, such as DKK1; inducing the pre-incubated humanized adipose-derived stem cells to differentiate into the neuron-like cells; and promoting the obtained neuron-like cells to mature by means of neurotrophic factors, such as BDNF, NGF and NT3. The neuron-like cells sourced from humanized adipose-derived stem cells are highly matured, can express mature nerve cell markers and ion channels and can enable a complex synapse structure to be observed so that the neuron-like cells can be used for treating neurodegenerative diseases and nervous-injury diseases and preparing corresponding medicines.

Description

technical field [0001] The invention relates to the fields of stem cells and biomedicine. Specifically, the present invention relates to human-derived adipose stem cell (Human-Adipose Derived Stem Cell, hADSC)-derived neuron-like cell (neuron-like cell, NLC) and its preparation method and in the prevention or treatment of neurodegenerative diseases application. Background technique [0002] Embryonic stem cells (ESCs) are a sufficient source of cells for the treatment of various degenerative diseases because of their unique self-renewal and differentiation pluripotency. But at the same time, these characteristics of ESC have also become the main difficulties in its clinical treatment. Tumor formation has been reported despite transplantation of pre-differentiated or pre-sorted ESC-derived cells. This raises safety concerns for the application of ESC-derived cells in humans. In addition, the use of embryonic stem cells is also highly controversial ethically. [0003] At ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/0793A61K35/12A61P25/00A61P25/28A61P25/16A61P25/14
Inventor 徐俊高山峨
Owner SHANGHAI EAST HOSPITAL
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