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Identification and Isolation of Multipotent Cells From Non-Osteochondral Mesenchymal Tissue

a mesenchymal tissue and multipotent cell technology, applied in the field of isolated multipotent cells, can solve the problems of high ethical burden, low yield, pain in the process of obtaining bone marrow,

Inactive Publication Date: 2007-10-25
AUTONOMOUS UNIVERSITY OF MADRID +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about a type of adult cell that can be isolated from non-osteochondral mesenchymal tissue. These cells have the ability to differentiate into different cell lineages, such as bone, muscle, and nerve cells. The isolated cells express specific markers and lack other markers, making them easy to identify and isolate. The invention also includes a method for obtaining and identifying these cells. The technical effect of this invention is the ability to isolate and characterize a population of multipotent adult cells that can be used for regenerative medicine and tissue engineering.

Problems solved by technology

By their nature, adult stem cells can be used in an autologous setting, and as such, they are immunologically compatibles and their use does not raise any ethical concerns.
The process of obtaining bone marrow is painful and the yield is very low, a substantial increase in the number of cells being necessary by ex vivo expansion, to obtain clinically relevant amount.
This step increases cost and makes the procedure time consuming, as well as increases the risk of contamination and loss of material.
However, except for MAPC, none of these populations has been, until present, sufficiently characterized at the phenotype level.
Therefore, although the presence of multipotent adult cells has been described in different connective tissues, in the current state of the art, it is not possible to identify and unequivocally distinguish between different multipotent cell types obtained from soft tissue, or to obtain a substantially pure population.
The uncertainty about which markers are associated with the stem cells to allow them to be distinguished from those cells that show a greater degree of differentiation, along with the low percentage of stem cells present in adult cells, has made it difficult to identify and purify populations of multipotent adult mesenchymal cells.
A significant disadvantage in using multipotent adult cells resides in the fact that most of the current sources for obtaining multipotent adult cells are contaminated with other cell types, complicating the process of identification, isolation and characterization of the populations of multipotent adult cells with the objective of using them for therapeutic or other ends.

Method used

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Examples

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example 1

Isolation of Multipotent Adult Cells from Soft Tissue and Characterization of Surface Markers

[0125] The isolation of multipotent adult cells from soft tissue was performed by selecting those cells with a capacity for proliferation and differentiation, characterized in that they show adhesion to the plastic container of the cell culture. Then, the cells were characterized by monitoring by flow cytometry the expression of a series of surface markers on the recently isolated cells and during the course of the culture development in vitro.

[0126] The isolation of the multipotent adult cells was carried out from sub-dermal adipose tissue, obtained by liposuction from 3 healthy human donors (donors 1, 2 and 3). First, the sample from the sub-dermal adipose tissue was washed with phosphate buffered saline solution (PBS). To achieve destruction of the extracellular matrix and the isolation of the cells, an enzymatic digestion was performed with type 11 collagenase in saline solution (5 mg / ...

example 2

In Vitro Differentiation of Multipotent Adult Cells from Human Non-Osteochondral Mesenchymal Tissue into Bone Phenotype Cells

[0144] In the differentiation assay, characterized human multipotent adult cells of the present invention were used. The cells were isolated from the 3 samples of analyzed lipoaspirates, each corresponding to a healthy donor (Example 1). The multipotent adult cells were isolated and characterized as mentioned in Example 1. A sample of mesenchymal stem cells (MSC) of human bone marrow was used as positive control.

[0145] The isolated cells were seeded at a density of 10,000 cells / cm2 onto 6-well plates (one plate per sample), and were incubated in standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic). After two days of culturing, the culture medium of one of the wells (control) is replaced with fresh medium, and the remaining wells with osteogenesis inducing medium, which contains the standard culture medium with the following added:

[0146] ...

example 3

In Vitro Differentiation of Multipotent Adult Cells from Human Non-Osteochondral Mesenchymal Tissue into Muscle Phenotype Cells

[0151] In the differentiation assay, characterized human multipotent adult cells of the present invention were used. The cells were isolated from the 3 samples of analyzed lipoaspirates, each corresponding to a healthy donor (Example 1). The multipotent adult cells were isolated and characterized as mentioned in Example 1. A sample of MSC of human bone marrow was used as positive control.

[0152] The isolated cells were seeded at a density of 10,000 cells / cm2 into standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic). After two days of culturing, the culture medium of one of the wells (control) was replaced with fresh medium, and the remaining wells with myogenesis inducing medium (Wakitani et al., 1995), which contains the standard culture medium with the following added:

[0153] Ascorbate-2-phosphate 0.1 mM

[0154] Dexamethasone 0.01 μM

[0...

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Abstract

Identification and isolation of multipotent cells from non-osteochondral mesenchymal tissue. This invention relates to the identification and isolation of multipotent cells from non-osteochondral mesenchymal tissue. Specifically, it relates to an adult multipotent cell or a cell population or composition comprising said cell, isolated from non-osteochondral mesenchymal tissue, characterized in that it is positive for the following markers: CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 and because it lacks expression of the following markers: CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and CD133.

Description

FIELD OF THE INVENTION [0001] The present invention relates to isolated multipotent adult cells which are isolated from non-osteochondral mesenchymal tissue and are characterized by the presence and absence of a set of cell surface markers. The invention also relates to a method for identifying and isolating a population of said cells, as well as to the applications thereof, for example, in the manufacture of a pharmaceutical composition for the repair and regeneration of tissues. BACKGROUND OF THE INVENTION [0002] Stem cells show differential characteristics as they are able to sustain themselves and differentiate into one or more cell type. Although research into stem cells and their applications is still in its early stages, adult stem cells in bone marrow have been used in transplants for more than 30 years. Nevertheless, in recent years, stem cell technology has made large advances such that stem cells are currently considered as a promising source of tissue and organs, with an...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/06A61K39/00G01N33/53A61K35/12A61K35/32C12N5/074C12N5/0775
CPCC12N5/0667C12N5/0607A61P19/00A61P21/00A61P25/00A61P43/00C12N5/0662C12N5/0652A61K35/28C12N5/0619C12N5/0654C12N5/0658C12N2506/1384C12N2510/00G01N33/5008
Inventor GARCIA CASTRO, ROSA ANAFERNADEZ MIGUEL, MARIA GEMAGARCIA ARRANZ, MARIANOGONZALEZ DE LA PENA, MANUEL ANGEL
Owner AUTONOMOUS UNIVERSITY OF MADRID
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