JP2003512082A - Identification of cells for transplantation - Google Patents

Abstract

  (57) [Summary] Pluripotent cells suitable for transplantation therapy to repair nerve damage were identified by differential display, for example, from gene expression profiles for selected cells that can be compared to those obtained from control cells. You.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to the identification of cells suitable for transplantation into the vertebrate brain. More specifically, the present invention repairs nerve damage following transplantation into the brain.
It relates to the identification of multipotent neurons to obtain.

[0002] Damage to the background vertebrate brain invention, there is recognized to increase that may be repaired using cell transplantation technique. Typically, cells transplanted into a damaged brain are neural stem cells (eg pluripotent neuroepithelial stem cells) that can differentiate into cells with a neural cell phenotype. .

For example, Sinden et al., Neuroscience (1997) 81: 599-608, show that spatially learning after transplantation of conditionally immortalized hippocampal neuroepithelial stem cells into the ischaemia-lesioned hippocampus. It can be used to improve WO-A-9
See also 7/10329.

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

For example, MHP 36 cells (Sinden et al., Supra) promote repair, while apparently similar cell line MHP 15 does not. This difference is that both MHP cell lines are derived from the same tissue source (ie hippocampus) and both are pluripotent neural progenitor cell lines, ie both cell lines are neurons, astrocytes and rare cells. It appears despite the fact that it has the capacity to generate all of the brain cell types, including oligodendrocytes.

Therefore, it would be beneficial if cells could be identified at an early stage that are suitable for transplantation or have the ability to modify the cell line to achieve successful transplantation and repair. is there.

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 uses, for example, a technique called differential display (DD) to study the differences between repaired and non-repaired cell lines. Differential display is used to visualize differences in gene expression between two or more cell lines, and repairing cell lines have very similar gene expression profiles, but are much more It was found to have different profiles. This represents an important and unexpected finding, apart from their ability to repair, cell lines have morphology, growth characteristics.
This is because they are usually remarkably similar in racteristics and growth factor responsivity.

According to one aspect of the present invention, a method for selecting suitable cells for transplantation into a damaged vertebrate brain comprises: (i) is a cell having a neuronal phenotype? Or isolating cells capable of differentiating into it; (ii) obtaining a gene expression profile of the cell; (iii) using the expression profile of the cell from control cells known to be suitable for transplantation Comparing; 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 the MHP 36 cell line. Step (ii) can be performed by a differential display.

The present invention is not only useful for identifying cells suitable for transplantation, but may also be of relevance in determining whether nerve cells can or cannot be repaired. Can be used to identify

According to a further aspect of the invention, a method for identifying genes involved in determining whether nerve cells can promote repair in transplantation into a damaged brain is as follows: ( i) isolating a cell that has or is capable of differentiating into a cell having a neuronal phenotype; (ii) obtaining a gene expression profile of the cell; (iii) transferring the expression profile of the cell into a transplant. Comparing to that from a control cell line known to be suitable; and (iv) identical (or different) to that expressed by the control
Isolating the gene.

Using this method, it became possible to identify a large number of genes expressed by repaired cell lines as well as vice versa (and vice versa).

According to a third aspect, a method for selecting cells suitable for transplantation into a damaged brain is provided by any of the gene sequences identified herein as SEQ ID NOs: 1-5. Determining the presence of.

DESCRIPTION OF THE INVENTION The present invention provides a convenient method for identifying cells that cannot be used for transplantation and repair in a damaged vertebrate brain. Using differential display technology, cell lines can be screened against controls and suitable cell lines selected for further development.

Although other techniques for obtaining gene expression profiles are known, the differential display technique is preferred. In summary, the technique uses m from a population of cells.
By isolating RNA and using it to measure the expression level of a particular gene. This is most often done using reverse transcription to copy DN
A (cDNA), which is co-grown using specific and semi-quantitative primer sequences. The results are then compared to controls to assess differences in gene expression.

[0017]   Kits for performing differential displays are commercially available.

The cells used in the method should be capable of differentiating into cells having a neuronal phenotype, ie the differentiated cells should adopt either a neuron, astrocyte or oligodendrocyte phenotype. Is. Preferably, the undifferentiated cells are pluripotent (
multi-potent), ie, they have the capacity to develop into at least two of the above neuronal phenotypes. Pluripotent cells are known and procedures for obtaining such cells will be apparent to those of skill in the art.

Differential display technology can be performed on cells in a differentiated or undifferentiated state. However, it is preferred if the cells are maintained in an undifferentiated state during the technique. Conditions for maintaining the cells in an undifferentiated state are known in the art and include culturing methods using growth factors, and recombinant DNA technology, such as inserting oncogenes or condition-inducible oncogenes into cells.

The present invention provides a convenient method of identifying cells that can be used in transplantation and repair in the injured vertebrate brain. Using the methods of the invention, cell lines can be screened against controls and suitable cell lines selected for further development.

Genes that are differentially expressed in non-repair cell lines can be identified. Modification of the gene, for example by site-directed mutations, can be performed to inactivate the gene to provide additional cell lines that may be suitable for transplantation. For example, disruption of the Pax6 or 3R2C gene can be done to prepare cells for transplantation. Methods for modifying or inactivating the gene will be apparent to those of skill in the art.

The modified cell lines, or cell lines identified as suitable for transplantation by the methods of the present invention, can be used in conventional transplantation methods. For example, Sinden et al., Supra, shows the preparation of stem cells for transplantation into the mouse brain.

The invention comprises the use of cells according to the invention in the manufacture of a composition for the treatment of damage to the vertebrate brain. Suitable formulations for delivery of cells to the brain will be apparent to those of skill in the art having an interest in the properties of the cells for therapeutic use. Suitable amounts of cells for therapeutic use will be apparent to those skilled in the art based on conventional formulation methods, in addition to suitable excipients, diluents or carriers.

The following examples describe the cell lines MHP 36, MHP 15, MHP 3 and SVE.
11 shows a differential display experiment to identify 10 gene expression profiles.

Example Cell lines were grown at 33 ° C. in standard MHP medium containing bFGF in 175 cm ³ flasks. Cells were 90% confluent, then transferred to 39 ° C and cultured in the absence of interferon for an additional 7 days.

The cells were washed for 5 minutes with 20 ml HBSS (Gibco BRL). Cells were lysed by adding 20 ml Trizol (Life Technologies) and incubated at 37 ° C for 10 minutes. The lysate was then transferred to a 50 ml Falcon tube (Stardts) and 5 ml chloroform was added. The tubes were mixed vigorously for 1 minute and the phases were separated by centrifugation at 4,000 rpm for 15 minutes. The upper aqueous phase was transferred to a new 50 ml tube and 7 ml isopropanol was added. R
NA was precipitated by leaving the tube at -20 ° C for 1 hour. RN
A was pelleted by centrifugation at 4,000 rpm for 20 minutes. The pellet was washed in 70% ethanol (made with DEPC treated water), centrifuged as above and air dried at room temperature for 10 minutes. The pellet is then 439μ
Resuspended in 1 DEPC-water and transferred to RNase-free Eppendorf tubes.

In order to remove all contaminating DNA from the RNA sample, resuspend RNA
Added to the solution: MgCl ₂ , final concentration 5 mM; DDT, 100 mM; RNase inhibitor, 500 units, and DNase I, 700 units. The tubes were then incubated for 1 hour at 37 ° C. and RNA was added to an equal volume of acid phenol / chloroform / isoamyl alcohol (125: 24: 1) (with 1 minute vortexing) and 6 minutes for 14 minutes. 1,000
Purified by centrifugation at rpm (4 ° C). The upper water phase is a new Eppend
Transferred to an orf tube and 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 overnight at -20 ° C.

RNA was pelleted by centrifugation at 14,0000 rpm (4 ° C.) for 15 minutes; the pellet was washed with 1 ml 70% ethanol (DEPC) and air dried for 5 minutes. RNA was then resuspended in 100 μl DEPC water. RNA concentration and purity was measured by measuring the absorbance at 260 nm and 280 nm. The RNA was then diluted to give an aliquot at a concentration of 200 ng / μl, which was stored at -70 ° C.

Differential Display Analysis All 3'anchoring and 5'arbitrary primers were obtained from the Genomyx Hieroglyph mRNA Profile Kit. The differential display procedure can be divided into three steps: cd
NA synthesis, PCR amplification and gel electrophoresis.

First strand cDNA synthesis was performed using Qiagen Omnis
It was performed using the crip reverse transcriptase.

A pair of all 4 Hieroglyph arbitrary 5'primers were pairwise using one of the 12 Hieroglyph oligo (dT) anchored 3'primers and a 20 μl reaction using 400 ng of total RNA.
) In combination with the same oligo (dT) primer, duplicated
) Enough cDNA was generated for differential display PCR (DD-PCR).

2 μl of 200 ng / μl RNA solution was added to 2 μl of Hieroglyph T
7 (dT12) Uncard primer (AP) (2 μM) was added to a 0.2 ml thin-walled PCR tube. Then, this was heated at 70 ° C. for 5 minutes, and then allowed to stand on ice. To the denatured RNA / primer solution, the following was added: 10 × Omniscript reverse transcriptase (RT) buffer, 2 μl; 5 mM.
dNTP, 2 μl; 0.1 M DTT; 0.5 μl RNASEOUT RNase inhibitor (40 μ / μl) (Gibco BRL); 1 μl Omni
script (1 unit / μl) and DEPC-water up to 20 μl. The reaction mixture was then incubated at 42 ° C for 1 hour.

The differential display PCR for each sample was then
I went heavy. The DD-PCR reaction was performed using the Hieroglyc system (Genom).
yx), Clontech Taq polymerase mix, and [α- ³³ P].
Performed using dATP (3,000 Ci / mmol, Amersham).

For each cDNA sub-population, a suitable reverse transcriptase (RT)
Matching anchored prim
PCR core mix containing ers) (AP) was prepared in sufficient volume for the number of reactions required. The core mix contained all DD-PCR components except any primers used, which were aliquoted separately into suitable tubes.

[0035]   Each individual DD-PCR tube contained the components shown in Table 1.

[0036]

[Table 1]

Following DD-PCR, radiolabeled cDNA fragments were electrophoretically separated on polyacrylamide gels under denaturing conditions. This is Genomyx
Included using LR sequencer and LR-optimized HR-1000 polyacrylamide gel formulation. 4.5% and 6% HR-1000 gels were prepared according to the manufacturer's specifications; 4.5% gels were used to resolve fragments within the size range of 700 bp to 2 kb, while 6%. Using gel, 1
Fragments within the size range of 00bp to 600bp were isolated.

7 μl of each DD-PCR sample was added to 4 μl of sample loading dye (lo
ading dye) (Genomyx) and heated at 95 ° C. for 3 minutes, then cooled on ice. 3 μl of this heat-denatured sample was then added to the gel lane. Duplicate reactions were loaded in adjacent lanes and samples were generated with identical primer pairs in consecutive lanes. 6% gel for 6 hours at 2,500 V
, 100 W, 50 ° C .; and 4.5% gel for 16 hours at 1,500
V, 100W, used at 50 ° C.

Following electrophoresis, the gel is run on Genomyx according to the manufacturer's protocol.
-Dried on glass plate in LR sequencer. An autoradiograph of the gel was prepared by contacting a piece of BioMax MR (Kodak) ultra-high resolution film with the gel for 16 hours and letting it stand.

The autoradiograph showed bands corresponding to genes not expressed in repaired cell lines MHP 36 and MHP 3 but expressed in non-repaired cells SVE 10 and MHP 15. Bands corresponding to genes expressed in repaired cell lines but not in non-repaired cell lines were also present.

The autoradiograph was washed in 90% ethanol and dried before cutting the band from the gel. Autoradiograph, autorad (autorad
) Aligned on top of the gel with the help of a marker (Stratgene). The autoradiograph was fixed with tape along one long edge. Differentially expressed transcripts (DET) were identified and bands were cut according to the Genomyx protocol. 10 cut bands
Place in 0 μl elution buffer (EB, 10 mM Tris: HCl, pH 7.5) and elute the DNA for 6 hours at room temperature. The eluted DNA was stored at -20 ° C.

Single Strand Conformation Polymorphis
m) (SSCP) gel analysis was used to exclude possible false positives due to co-migration of fragments of the same size but different sequences. This procedure involves restriction reamplification of isolated fragments (SSCP-PCR) followed by SS
The separation of the products of this amplification on a CP gel was included. PCR conditions were similar to those used for DD-PCR except the number of cycles was reduced to 2 at low annealing temperature (50 ° C) and 10 cycles at high annealing temperature (60 ° C). 4 μl of SSCP-PCR reaction was mixed with 10 μl of SSCP-loading buffer (80% formamide, 0.01% bromophenol blue, 0.
01% xylene cyanol, 1 mM EDTA, 10 mM NaOH),
Then, it was denatured at 95 ° C. for 10 minutes and then loaded on an agarose gel. Samples in 0.6 × Trisborate EDTA buffer (TBE) for 16 hours at 8 W,
Electrophoresed on a Genomyx-LR sequencer. Following autoradiography, the region of the gel corresponding to the differentially expressed transcript candidates was cut and placed in 100 μl elution buffer (EB).

Sequencing and Identification of DET The recovered differentially expressed cDNA was reamplified by PCR to provide sufficient template material to allow direct sequencing. The PCR reaction is shown in Table 2.
It contained the components shown in.

[0044]

[Table 2]

The PCR product is then subjected to PCR purification kit (Q
iagen). The purified product was eluted in 60 μl elution buffer. Purified PCR products were sequenced using a Thermosequenase radiolabeled terminator cycle sequencing kit (Amersham). Reactions were performed using M13 reverse primer (-48) (Genomyx) according to the manufacturer's protocol. Sequencing products were loaded with 6% Lon
g Ranger (FMC), 8M urea, 1 × GTB (glycerol-tolerant buffer) loaded on gel and 2,500 V for 4 hours
, 100 W, 50 ° C. in 1 × GTB. Following autoradiography, the sequencing ladders were read manually and the sequencing data obtained was used to generate the GenBank database (public and ES
(Both T).

Table 3 shows the gene expression profile of the cell lines used in the experiment. M
HP 36 and MHP 3 are cells that are suitable for transplantation into the damaged brain. It is clear that the expression profiles in MHP 36 and MHP 3 are similar. Similarly, the profiles in MHP 15 and SVE 10 are similar. However, the profiles of MHP 36/3 and MHP 15 / SVE 10 are significantly different. This indicates a difference in the function of the two sets of cells as MHP 15 and SVE 10 cells have no ability to repair.

[0047]

[Table 3]

Analysis of expression products showed that non-repaired MHP 15 cells had Pax6 (Gotz et al., Neuron (199
8) 21; 1031-1044), which revealed that cells from MHP 36 do not express this gene.

Thus, neuronal cells expressing the Pax6 gene would not be expected to repair brain damage, while neuronal cell lines that do not express this gene may promote repair.

Pax6 is believed to be a positional specification gene that plays a role in determining the location of embryonic cells, the adaptive fate in the developing brain (Fernandez et al., Development (1998). 125: 2099-2111).

Further analysis revealed that non-repaired, non-pluripotent cells express the gene 3R2C (GenBank Accession No. D25216). MHP 36
Cells do not express this gene when cultured under permissive conditions.

[Sequence list]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Uvanogo Daffe             United Kingdom S5 58             London Denmark Windsor             Walk 1 Institute of               Sai Kai At Lee F-term (reference) 4B024 AA11 AA20 CA04 EA04 HA19                 4B063 QA13 QQ08 QR33 QR55 QS34

Claims (6)

[Claims] 1. A method for selecting cells suitable for transplantation into a damaged vertebrate brain, comprising: (i) cells having or capable of differentiating into cells having a neuronal phenotype. (Ii) obtaining a gene expression profile of the cell; (iii) comparing the expression profile of the cell with that from a control cell known to be suitable for transplantation; and (iv) A) selecting cells having a gene expression profile similar to that of the control. 2. A method for identifying a gene, the expression of which can determine whether a neuron can repair a damaged brain, comprising: (i) a cell having a neuronal phenotype. A cell that is or is capable of differentiating into it; (ii) obtaining a gene expression profile of the cell; (iii) comparing the expression profile of the cell with control cells known to be suitable for transplantation. And (iv) identical (or different) to that expressed by the control.
Isolating the gene. 3. The method according to claim 1 or 2, wherein the control cells do not express the gene Pax6 or 3R2C. 4. The method of any of the preceding claims, wherein the control cell is MHP36. 5. The method according to any of the preceding claims, wherein step (ii) is performed by a differential display. 6. A call suitable for transplantation into a damaged brain, comprising measuring the presence of any of the gene sequences identified herein as SEQ ID NOS: 1-5.
How to choose.