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Methods for enriching populations of cells

A cell group and cell technology, applied to embryonic cells, animal cells, vertebrate cells, etc., can solve problems such as high cost and low efficiency

Pending Publication Date: 2022-02-08
RUTGERS THE STATE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To isolate MUSE cells, fluorescence-activated cell sorting (FACS) is commonly used, but this method is inefficient and costly (Heneidi, S., et al. PLoS One, 2013.8(6): p.e64752)

Method used

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  • Methods for enriching populations of cells
  • Methods for enriching populations of cells
  • Methods for enriching populations of cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] This example describes the materials and methods used in the subsequent examples.

[0098] Isolation of HUC-MSCs

[0099] HUC is packaged in a bottle filled with shipping medium, which includes KH 2 PO 4 (0.20g / L), Na 2 HPO 4 (anhydrous, 1.15g / L), KCl (0.20g / L) and NaCl (8.00g / L). The bottle was kept at 4°C by surrounding it with ice. All cords were collected with patient consent and in compliance with the requirements of the Rutgers University Ethics Committee. The transport from the patient to the lab took a day. Table I lists the antibodies used in this study.

[0100] Isolation of human umbilical cord (HUC) MSCs followed the protocol described below. First, place HUC in a 10-cm Petri dish. HUC was then cut into 1-cm pieces and dissected longitudinally. Next, the HUC artery and vein were removed, and the HUC tissue was washed, followed by isolation of Wharton's jelly and umbilical cord intima tissue. Treat tissue with collagenase and seed cells into cell...

Embodiment 2

[0116] Both HUC WJ and CL produced a large number of MSCs. Table II shows the number of passage 0 MSCs and SSEA3+. The average concentration of MSCs and SSEA3+ cells per gram of tissue was 3.7 ± 0.55 × 10 4 WJ-MSC, 1.89±1.67×10 3 WJ-SSEA3+, 3.00±0.80×10 4 CL-MSC and 2.24±2.00×10 3 CL-SSEA3+ cells. Heavier umbilical cords have more WJ MSCs (R 2 =0.64, p=0.01 Figure 4 ). The 99WJ group had an abnormally high 42.37% SSEA+ cells at passage 0. However, cord weight was not associated with CL-MSC / WJ-SSEA3+ / CL-SSEA3+. The number of WJ-MSCs did not correlate with CL-MSCs or WJ-SSEA3+. There was also no correlation between CL-MSCs and CL-SSEA3+.

[0117] WJ and CL cells were cultured alone, and the percentage of SSEA3+ in multiple passages was compared (see Figure 5). In the P0 group, more than 98% of the total cells from both WJ and CL were positive for CD105, and it was even higher in P1 and P2. At P0, the percentages of SSEA3+ cells in WJ and CL were 4.97%±4.30% and 5.26%±...

Embodiment 3

[0123] Two adult Sprague-Dawley rats with 12.5-mm T11 spinal cord weight drop contusion were transplanted with HUC SSEA3+ and CD105+ cells into the spinal cord 2 weeks after spinal cord injury (SCI). The cells are injected into the dorsal root entry zone of the spinal cord above and below the injury site. Cells survived 4 weeks after transplantation. Rats were not immunosuppressed. Transplanted cells were stained with antibodies against human nuclei (Stem 101+), but were negative for Nestin, GFAP, NeuN, NF155, and Iba1. When transplanted into the brain and spinal cord, human MUSE cells can survive for a long time and will not be immune rejected (Uchida H et al. Stem Cells. 2016; 34(1): 160-173; Uchida H et al. Stroke. 2017; 48(2):428-435).

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Abstract

This disclosure describes efficient methods for separating desired populations of cells, including Multilineage-Differentiating Stress-Enduring (MUSE) cells. Also described are the methods for isolating and enriching MUSE cells through a sorting, expanding, and re-sorting procedure. The enriched cells or cell populations can be used for treating cancer, repairing various tissues, and treating various degenerative or inherited diseases.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 831,491, filed April 9, 2019. The aforementioned applications are incorporated herein by reference. [0003] field of invention [0004] The present invention generally relates to methods of enriching desired cell populations, and more particularly to methods of enriching desired cell populations comprising multilineage differentiated sustained stress (MUSE) cells and uses thereof. [0005] Background of the invention [0006] Multilineage-Differentiating Stress-Enduring (MUSE) cells are a subtype of mesenchymal stem cells (MSCs), which express state-specific embryonic antigen 3 (SSEA3). MUSE cells can spontaneously differentiate into endoderm, ectoderm, and mesoderm lineages in vitro, or can be induced to generate cell types from all three lineages. They can renew themselves, but do not form teratomas in the body. M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073A61K35/28A61P25/28A61K35/51
CPCA61K35/51A61K35/28C12N5/0605A61P25/28C12N2501/115C12N2502/1114C12N2502/1164C12N2502/45
Inventor W·杨孙东明I·塔德莫里冷子宽出泽真理
Owner RUTGERS THE STATE UNIV
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