KR20020065492A - Identification of cells for transplantation - Google Patents

Abstract

Multifunctional cells suitable for transplantation treatment and repairing nerve damage have been identified from gene expression profiles of selected cells that can be compared with those obtained from control cells, for example, by differential presentation.

Description

Identification of cells for transplantation

There is increasing recognition that cell transplantation techniques can be used to repair damage to vertebrate brains. Typically, the cells transplanted into the damaged brain will be multifunctional neuroepithelial stem cells that can differentiate into neural stem cells, eg, cells with a neuronal phenotype.

For example, Neuroscience (1997) 81: 599-608 by Sinden et al. Describes that conditionally-immortalized hippocampal neuroepithelial stem cells can be used to improve spatial learning after transplantation into ischemia-disorder hippocampus. See also International Publication No. 97/10329.

However, it has been found that not all neural stem cells can be transplanted for successful repair of nerve damage.

For example, MHP36 cells (Sinden et. Al., Supra) aid in repair, but MHP15, a seemingly similar cell line, cannot repair. Both MHP cell lines originate from the same tissue source (ie the hippocampus), and both cell lines produce multifunctional neural precursor cell lines, ie, the complete complement of brain cell types, including neurons, astrocytes and oligodendrocytes. Despite the fact that it has the ability, the difference occurs.

Thus, it would be beneficial if it was possible to identify cells suitable for transplantation at an early stage, or to have the ability to modify the cell line to achieve successful transplantation and repair.

The present invention relates to the identification of cells suitable for implantation into the vertebrate brain. More specifically, the invention relates to the identification of multifunctional neurons capable of repairing nerve damage following transplantation to the brain.

Summary of the Invention

The present invention is based on the recognition that cells suitable for transplantation and repair can be identified based on their gene expression profile.

The present invention employs a technique called differential display (DD), for example, which studies the differences between repaired and non-recovered cell lines. Differential expression has been used to visualize differences in gene expression between two or more cell lines, and it has been found that restoring cell lines have very similar profiles with cell expressions of very similar profiles but not repairing. Apart from their ability to repair, the above findings are important and unexpected because cell systems are generally remarkably similar in morphology, growth characteristics and growth factor responsiveness.

According to one aspect of the invention, a method of selecting cells suitable for transplantation into the brain of an injured vertebrate is:

(i) isolating cells that are, or can differentiate into, cells having a neuronal phenotype;

(ii) obtaining a gene expression profile from said cell;

(iii) comparing the expression profile of said cell with the profile of a control cell known to be suitable for transplantation; And

(iv) selecting cells having an expression profile similar to that of the control.

In one embodiment of the invention, the control cells are from MHP36 cell line. Step (ii) may be carried out by differential presentation.

The present invention is not only useful for identifying cells suitable for transplantation, but can also be used to identify genes or gene products involved in determining whether neurons are repaired or not.

According to another aspect of the present invention, a method for identifying genes involved in determining whether neurons can aid repair in transplantation into a damaged brain is:

(i) isolating cells that are, or can differentiate into, cells having a neuronal phenotype;

(ii) obtaining a gene expression profile from said cell;

(iii) comparing the expression profile of said cell with that of a control cell known to be suitable for transplantation; And

(iv) separating the same (or different) gene as the gene expressed by the control.

The method can be used to identify multiple genes expressed by the repairing cell line but not by the non-recovering cell line (and vice versa).

According to a third aspect of the invention, the method of selecting a cell suitable for transplantation into an injured brain consists in determining the presence of any of the gene sequences identified herein as SEQ ID Nos. 1-5.

Description of the invention

The present invention provides a convenient method for identifying cells that may not be used for transplantation and repair in an injured vertebrate brain. Using differential expression techniques, cell lines are selected from the control and suitable cell lines can be selected for further development.

Although other techniques for obtaining gene expression profiles are known, differential expression techniques are preferred. In brief, the technique relies on separating mRNA from the cell population and using it to determine the expression level of a particular gene. The above is often accomplished using reverse transcription to obtain copy DNA (cDNA) that is co-amplified using specific semi-quantitive primer sequences. The results are then compared to the control to verify differences in gene expression.

Kits for performing differential presentations are commonly available.

The cells used in the method must be able to differentiate into cells with neuronal phenotypes, ie the differentiated cells must receive one of the neurons, astrocytic or bloated cell phenotypes. Preferably, the cells in the undifferentiated state are multifunctional, ie have the ability to develop into at least two of the neuronal phenotypes described above. Multifunctional cells are well known and the procedure for obtaining such cells will be apparent to those skilled in the art.

Differential expression techniques can be performed in cells in differentiated or undifferentiated conditions. However, it is desirable if the cells remain undifferentiated during the differential presentation technique. Conditions for maintaining longitudinals in an undifferentiated state are well known in the art and include culture methods using growth factors and recombinant DNA techniques, such as inserting tumor genes or conditionally-induced tumor genes into cells, for example. .

The present invention provides a convenient way to identify cells that can be used for transplantation and repair in an injured vertebrate brain. Using the methods of the present invention, cell lines can be selected for the control and suitable cell lines can be selected for further development.

Genes differentially expressed in non-recoverable cell lines can be identified. For example, modification of a gene by specific local mutagenesis can be carried out to inactivate a gene that provides another cell line suitable for transplantation. For example, disruption of Pax6 or 3R2C genes can be performed to produce cells for transplantation. Methods for modifying or inactivating genes will be apparent to those skilled in the art.

Modified cell lines, or cell lines that are identified as suitable for implantation by the methods of the present invention, can be used in conventional transplantation methods. For example, the Sinden et al indicate the preparation of stem cells for transplantation into the mouse brain.

The invention includes the use of the cells according to the invention in the preparation of a composition for treating damage to the brain of a vertebrate. Formulations suitable for cell delivery to the brain will be apparent to those skilled in the art who are interested in the properties of the cells for therapeutic use. In addition to suitable excipients, diluents or carriers, the appropriate amount of cells for therapeutic use will be apparent to those skilled in the art based on conventional formulation methods.

The following examples show differential expression experiments for identifying gene expression profiles of cell lines MHP36, MHP15, MHP3 and SVE10.

Cells were grown at 33 ° C. in standard MHP medium containing bFGF in 175 cm 3 flasks. The cells were allowed to 90% fusion before delivery to 39 ° C. and incubated for an additional 7 days in the absence of interferon.

The cells were washed with 20 ml HBSS (Gibco BRL) for 5 minutes. 20 ml of Trizol (Life Technologies) was added to lyse the cells and incubated at 37 ° C. for 10 minutes. The lysate was then transferred to 50 ml Falcon tubes (Stardts) and 5 ml chloroform was added. The tubes were mixed vigorously for 1 minute and the phases separated by centrifugation at 4000 rpm for 15 minutes. The upper aqueous phase was transferred to a fresh 50 ml tube and 7 ml of isopropanol was added. The tube was left at -20 ° C for 1 hour to precipitate RNA. The RNA was pelleted by centrifugation at 4000 rpm for 20 minutes. The pellet was washed with 70% ethanol (made with DEPC treated water), centrifuged as described above, and air dried at room temperature for 10 minutes. The pellet was then resuspended in 439 μL of DEPC-water and transferred to an Eppendorf tube without RNase.

MgCl ₂ to a final concentration of 5 mM to remove all contaminating DNA from RNA samples; DDT to a final concentration of 100 mM; 500 units of RNase inhibitor and 700 units of DNase I were added to the resuspended RNA solution. The tube was then incubated at 37 ° C. for 1 hour and vortexed for 1 minute by addition of the same volume of acid phenol / chloroform / isoamyl alcohol (125: 24: 1) and 14000 rpm (4 minutes). RNA was purified by centrifugation). The upper aqueous phase was transferred to a new Eppendorf tube and the phenol / chloroform extraction was repeated until the interface was clear. 8M LiCl was added to the final aqueous layer to a final concentration of 2.5M and the tube was left at -20 ° C overnight.

The RNA was pelleted by centrifugation at 14000 rpm for 15 minutes, and the pellet was washed with 1 ml of 70% ethanol (DEPC) and allowed to air dry for 5 minutes. The RNA was then resuspended in 100 μl of DEPC-water. The concentration and purity of RNA were determined by measuring absorbance at 260 nm and 280 nm. The RNA was then diluted to provide an aliquot at a concentration of 200 ng / μl stored at -70 ° C.

Differential expression analysis

All 3'anchoring and 5'arbitrary primers were obtained from the Genomyx Hieroglyph mRNA profile kit. The differential presentation process can be divided into three steps: cDNA synthesis, PCR amplification, and gel electrophoresis.

Strand cDNA synthesis was first performed using Qiagen Omniscript reverse transcriptase.

Replicated differential expression with the same oilgo (dT) primer paired with all four Hieroglyph random 5 'primers in 20 μl reaction with one of 3' primers immobilized with 12 Hieroglyph oligo (dT) and 400ng total RNA It was used to form enough cDNA for PCR (DD-PCR).

2 μl of 200 ng / μl RNA solution is added to a 0.2 ml thin-wall PCR tube along with 2 μl Hieroglyph T7 (dT12) immobilized primer (AP) (2 μM). Then it was heated to 70 ° C. for 5 minutes before placing it on ice. 2 μl of 10 × Omniscript reverse transcriptase (RT) buffer in denatured RNA / primer solution, 2 μl of 5 mM dNTP's, 0.1 M DTT, 0.5 μl RNASEOUT RNase inhibitor (40 μ / μl) (Gibco BRL), 1 μl Omniscript (1 unit / μl ) And DEPC-water were added to 20 μl. The reaction mixture was then incubated at 42 ° C. for 1 hour.

Then, differential expression PCR was performed on each sample in duplicate. The DD-PCR reaction was carried out using the Hieroglyph system (Genomyx), Clontech Taq polymerase mixture and [α- ³³ P] dATP (3000 Ci / mmole, Amersham).

For each cDNA sub-subgroup, a PCR core mixture containing an appropriate reverse transcriptase (RT) mixture and matching the immobilized primer (AP) was prepared in an amount sufficient for the required number of reactions. The core mixture contained all DD-PCR components separately aliquoted into appropriate tubes except for any primers to be used.

Each individual DD-PCR tube contained the components shown in Table 1.

DD-PCR Components [Save] 1x reaction volume (20 μl) [final] water - 9.35 μl - PCR buffer 10x 2 μl 1x dNTP (1: 1: 1: 1) 250 μM 2 μl 20 μM 5'ARP Primer 2 μM 2 μl 0.2 μM 3'AP primer 2 μM 2 μl 0.2 μM cDNA - 2 μl - Taq 50x 0.4 μl 1x [α- ³³ P] dATP 10 μCi / μl 0.25 μl 0.125 μCi / μl

After DD-PCR, radioisotope labeled cDNA fragments were electrophoretically separated on polyacrylamide gels under denaturing conditions. This involves the use of Genomyx LR synthesizers and LR-optimized HR-1000 polyacrylamide gel formulations. 4.5% and 6% HR-1000 gels were prepared according to the manufacturer's instructions; A 6.0% gel was used to separate fragments in the size range of 100 bp to 600 bp, while a 4.5% gel was used to digest the fragments in the size range of 700 bp to 2 kb.

7 μl of each DD-PCR sample was mixed with 4 μl of sample containing dye (Genomyx) and heated at 95 ° C. for 3 minutes before cooling on ice. Then 3 μl of this heat-modified sample was added to the gel lanes. Duplicate reactions were performed in adjacent lanes and samples were generated with the same primer pairs in successive lanes. 6% gels were used at 2,500V, 100W and 50 ° C for 6 hours and 4% gels were used at 1,500V, 100W and 50 ° C for 16 hours.

After electrophoresis, the gel was dried on glass plates in the Genomyx-LR synthesizer according to the manufacturer's instructions. A piece of BioMax MR (Kodak) ultra high resolution film was placed in contact with the gel for 16 hours to generate a radiograph of the gel.

The radiographs showed bands corresponding to genes expressed in non-recovery cells SVE 10 and MHP15 but not expressed in repair cell lines MHP36 and MHP3. There was also a band corresponding to a gene expressed in the repairing cell line but not in the non-recovering cell line.

Before incision of the band in the gel, the radiographs were washed in 90% ethanol and dried. Radiographs were aligned on top of the gel using an autorad marker (Stratgene). Radiographs were fixed along the long edge of the tape. Differentially expressed transcripts (DET) were identified and bands were excised according to Genomyx guidelines. The cleaved bands were placed in 100 μl of elution buffer (EB, 10 mM Tris: HCL, pH7.5) and allowed to elute DNA at room temperature for 6 hours. Eluted DNA was stored at -20 ° C.

Single Strand Conformation Polymorphism (SSCP) gel analysis was used to eliminate false confidence that could be caused by co-migration of fragments of the same size but different sequences. This process involves separating the product of amplification on an SSCP gel, followed by limited reamplification of the isolated fragment (SSCP-PCR). PCR conditions are similar to those used for DD-PCR except that the number of cycles is reduced to 2 at low annealing temperatures (50 ° C.) and the number of cycles is reduced to 10 at high annealing temperatures (60 ° C.). . 4 μL of SSCP-PCR reaction is added to 10 μL of SSCP-containing buffer (80% formamide, 0.01% bromophenol blue, 0.01% xylene cyanol, 1 mM EDTA, 10 mM NaOH) and on agarose gel It was denatured at 95 ° C. for 10 minutes before feeding in. Samples were electrophoresed at 8 W for 16 hours in 0.6x Tris Borate EDTA Buffer (TBE) on a Genomyx-LR synthesizer. After the radiograph, the areas of the gel corresponding to the candidate differentially expressed transcripts were excised and placed into 100 μl of elution buffer (EB).

Sequencing and Identification of DETs

The recovered differentially expressed cDNA was reamplified by PCR to provide an important template material that allows direct sequencing. The PCR reaction contained the components shown in Table 2.

DD-PCR Components [Save] 1x reaction volume (100 μl) [final] water - 49 μl - PCR buffer 10x 10 μl 1x dNTP (1: 1: 1: 1) 250 μM 10 μl 20 μM 5'ARP Primer 2 μM 10 μl 0.2 μM 3'AP primer 2 μM 10 μl 0.2 μM SSCP-cDNA - 10 μl - Taq 50x 1 μl 1x

The PCR product was then purified using the PCR Purification Kit (Qiagen) according to the manufacturer's instructions. The purified product was eluted in 60 μl of elution buffer. Purified PCR products were sequenced using a Thermosequenase radioisotope labeled terminator cycle sequencing kit (Amersham). The reaction was performed according to manufacturer's instructions using M13 reverse primer (-48) (Genomyx). Sequencing products were transferred to 6% Long Ranger (FMC), 8M urea, 1 × GTB (glycerol-resistant buffer) gel and electrophoresed at 2500 V, 100 W and 50 ° C. in 1 × GTB for 4 hours. After the radiographs, the sequencing ladder is read manually and the sequencing data generated is used to examine the GenBank database (both published and EST).

Table 3 shows the gene expression profiles of the cell lines used in the experiment. MHP36 and MHP3 are suitable cells for transplantation into the damaged brain. It is clear that the expression profiles in MHP36 and MHP3 are similar. Similarly, the profiles are similar in MHP15 and SVE10. However, the profiles of MHP36 / 3 and MHP15 / SVE10 are significantly different. This illustrates that the functional difference of two sets of cells, such as MHP15 and SVE10 cells, is incapable of repairing.

Cell line Homologue MHP3 MHP15 MHP36 SVE10Clone 23 gene % DD-band SEQ ID No. 1A +++++ - +++++ - Human EST More than 68bp 88% SEQ ID No. 2B +++++ - +++++ - Glogin-245 100% SEQ ID No. 3C +++++ - +++++ - Mouse EST 175bp or more 95% SEQ ID No. 4D +++++ - +++++ - Mouse EST 438bp or more99% SEQ ID No. 5E +++++ - +++++ - Heavy Chainimmunoglob ' 108bp or more 94% SEQ ID No. 6F - +++++ - +++++ Mouse EST 84 bp or more 91% SEQ ID No. 7G - +++++ - +++++ Neuroprotective Protein, Adnp 100%

Analysis of the expression product indicates that cells derived from MHP36 do not express the Pax6 gene, whereas non-recovery MHP15 cells express the gene Pax6 (Gotz et al, Neuron (1938), 21; 1031-1044).

Thus, neurons expressing Pax6 genes are not expected to repair brain damage, whereas neuronal systems that do not express the genes may help repair.

Pax6 is a position-specific gene, which is thought to play a role in determining where embryonic cells are destined for adoption in brain development (Fernandez et al, Development (1998) 125: 2099-2111).

Another analysis revealed that non-repaired, non-multifunctional cells express gene 3R2C (GenBank Accession Number D25216). MHP36 cells did not express these genes when cultured under accepted conditions.

Claims (6)

By selecting the cells that are suitable for transplantation into the damaged vertebrate brain, (i) selecting cells that are or may be capable of differentiating into cells having a neuronal cell phenotype; (ii) obtaining a gene expression profile of said cell; (iii) comparing the expression profile of said cell with an expression profile derived from a control cell known to be suitable for transplantation; And (iv) selecting cells having a gene expression profile similar to the gene expression profile of the control. Its expression is a way of identifying genes that can determine whether neurons can repair a damaged brain, (i) selecting cells that are or may be capable of differentiating into cells having a neuronal cell phenotype; (ii) obtaining a gene expression profile of said cell; (iii) comparing the expression profile of said cell with an expression profile derived from a control cell known to be suitable for transplantation; And (iv) separating the same (or different) gene as the profile expressed by the control. The method of claim 1 or 2, wherein the control cell does not express the gene Pax6 or 3R2C. The method of any one of the preceding claims, wherein the control cell is MHP36. The method according to any one of the preceding claims, wherein step (ii) is performed by differential expression. A method of selecting a cell suitable for transplantation into an injured brain, the method comprising determining the presence of any of the gene sequences identified by SEQ ID Nos. 1-5.