Medicinal composition for inducing direct conversion of fibroblasts into nerve cells, and use of medicinal composition

A fibroblast, human fibroblast technology, applied in the biological field, can solve problems such as limiting therapeutic applications

Inactive Publication Date: 2017-01-18
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method of exogenously integrating genes limits its current therapeutic applications

Method used

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  • Medicinal composition for inducing direct conversion of fibroblasts into nerve cells, and use of medicinal composition
  • Medicinal composition for inducing direct conversion of fibroblasts into nerve cells, and use of medicinal composition
  • Medicinal composition for inducing direct conversion of fibroblasts into nerve cells, and use of medicinal composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1 Small molecule compounds induce human adult fibroblasts to be neural cells

[0112] The initial human fibroblasts (HAFs, FS090609, from the foreskin of a 28-year-old male) were not found to have neuronal contamination (eg Figure 5 shown in A).

[0113] Then we used VCR in the neuron-inducing medium, and found that: using VCR alone to act on the initial human fibroblasts, no neuron-like cells were detected. That is, it is impossible to induce differentiation into neural cells by simply using VCR to act on naive human fibroblasts. The composition and content of the VCR are: VPA, 0.5mM (Calbiochem, 676380, 1M stock solution, H 2 O); CHIR-99021, 3 μM (Axon, 1386, 10 mM stock, DMSO); RepSox, 1 μM (Biovison, 1894, 10 mM stock, DMSO).

[0114] We then used the VCR plus the compounds shown in Table 1 to form a compound composition to treat HAFs (naive human fibroblasts, FS090609).

[0115] Table 1

[0116]

[0117]

[0118]

[0119]

[0120]

[012...

Embodiment 2

[0132] Example 2 Physiological properties of hcin cells

[0133] To test whether hciN has neurons with fundamental electrophysiological properties, such as firing action potentials and membrane currents, we performed whole-cell patch-clamp recordings on day 14 of chemically treated hcin cells with complex neuronal morphology. Recorded repetitive action potentials ( figure 2 A and Image 6 C). Steps of depolarizing voltage elicit fast sodium inward currents in voltage-clamp mode ( figure 2 B; 93%, n=15). In addition, fast energy was completely blocked by the sodium channel blocker tetrodotoxin (TTX), which is an inward current. To test whether hcin cells have functional glutamate and GABA receptors, we gave hcin cells exogenous glutamate ( figure 2 C, left panel; 50%, n=10) and γ-aminobutyric acid ( figure 2 C, right panel; 67%, n=9) Induced inward currents were found, indicating that functional glutamate and GABA receptors are present in hcin cells. Astrocytes have ...

Embodiment 3

[0136] Example 3 hcin cells have neuronal gene expression characteristics

[0137] To further characterize hcin cell types, we used FluidigmBiomark to quantitatively analyze the expression of 25 neuronal genes and 7 non-neuronal genes at the single-cell level. TF-induced neurons (Ladewig et al., 2012) served as positive controls ( Figure 7 A). FACS sorting of hcin and TF-iN in 21 days of cell culture (FACS purification, image 3 B and 7C). Heatmap analysis showed that hcins main neuron genes, channel genes, and synaptic genes were consistently expressed, but glia-specific and fibroblast-specific genes were not ( image 3 A). The expression pattern of hcin cells is similar to TF-induced neurons but distinct from naive fibroblasts.

[0138] The primers used are shown in Table 4.

[0139]

[0140]

[0141] We further compared global gene expression by microarray analysis in HES-derived neurons (control neurons), HAFS, pre-hcin cells (Vcrfsgy-treated HAFs on days 3 an...

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Abstract

The invention relates to the biotechnical field, and concretely relates to a medicinal composition for inducing direct conversion of fibroblasts into nerve cells, and a use of the medicinal composition. Transdifferentiation between nerve cell non-genealogies is realized through micro-molecular compound combination without exogenous gene. VCRFSGY treated fibroblasts rapidly leave the cell cycle at the third day is found for the first time, which hints that neuron cells formed by chemical induction of human fibroblasts may bypass the proliferation intermediate state, so the medicinal composition has extremely high clinic and market values. Additionally, a situation that Retinoic acid or Parnate can be respectively added on the basis of a VCRFSGY compound combination in order to well promote the induction efficiency is further found.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pharmaceutical composition for inducing the direct transformation of fibroblasts into nerve cells and uses thereof. Background technique [0002] Terminally differentiated somatic cells can be reprogrammed into pluripotent stem cells by forced expression of a specific set of transcription factors, suggesting that cell fate is reversible (Ladewig et al., 2013; Takahashi and Yamanaka, 2006; Yu et al. ., 2007). Moreover, the combination of different lineage-specific transcription factors can directly transdifferentiate human and murine somatic cells into cardiomyocytes, bypassing the pluripotent state (Ieda et al., 2010; Nam et al., 2013), hepatocytes ( Du et al., 2014; Huang et al., 2014) and nerve cells (Pang et al., 2011; Vierbuchenet al., 2010). This provides an alternative approach for the construction of disease models and regenerative medicine. Interestingly, these transdiff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0618C12N2500/30C12N2506/1307
Inventor 裴钢赵简裘斌龙
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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