WO2001058255A1

WO2001058255A1 - Chimeric animal containing human blood

Info

Publication number: WO2001058255A1
Application number: PCT/JP2001/000961
Authority: WO
Inventors: Hiromitsu Nakauchi; Yutaka Fujiki; Kazuo Fukawa; Osamu Kudoh
Original assignee: Japan Science And Technology Corporation; National Federation Of Agricultural Cooperative Associations
Priority date: 2000-02-10
Filing date: 2001-02-09
Publication date: 2001-08-16
Also published as: JP2005229802A; AU3226401A

Abstract

A method of constructing a chimeric animal characterized in that hematopoietic liver stem cells are transplanted into an animal embryo; and a chimeric animal containing human blood.

Description

Description Chimera animal containing human blood

The present invention relates to a chimeric animal containing human blood and a method for producing the same. Background art

Hematopoietic stem cell activity in mice is measured by an in 'FO experimental system, a long term marrow repopulation method. It is considered that mouse hematopoietic stem cells have been almost purified by using this method, and this can be used as a material for further research >>. Until recently, there was no suitable animal model for human hematopoietic stem cells, and a method ^M for measuring colony forming ability by in F / 0 bone marrow culture was generally used. However, colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-IC) that are observed in colony forming cells 'TO bone marrow reconstruction

(repopurating capasity) is unclear. 1983 SCID; by (severe combined immune deficiency severe combined immunodeficiency) discovery of the mouse ^5), it is shown that can reconstruct the hematopoietic system of human Bok in in / TO, hereafter the study method was rapidly widespread . Ability to supply multilineage blood cells over long periods of time to detect hematopoietic stem cells

(long term multilineage engraftment) needs to be measured. Recently more efficiently capable of reconstructing the human hematopoietic system N0D / SCID mice have been developed ⁶⁾ is, ⁷ was to be frequently used in Atsusi - ^11)). This mouse-based system is an excellent system in that human blood cells can easily engraft it.However, the effect of transplantation by irradiation on peripheral tissues such as stromal cells will be affected, and the limitations of small animals. As a result, the life span is short for human blood to be assayed, the scale of the proliferation of human hematopoietic stem cells (OSC) is small, and differentiation into T cell lines cannot be generally observed. ^I — ¹³⁾ There are problems. Disclosure of the invention

An object of the present invention is to provide a chimeric animal containing human blood. The present inventors have conducted intensive studies to solve the above problems, and as a result, succeeded in producing an animal in which human blood circulates by transplanting human blood stem cells into the fetus of the animal. The invention has been completed.

That is, the present invention is a chimeric animal containing human blood. Examples of the animal include livestock animals such as bushu.

Furthermore, the present invention is a method for producing a chimera animal, which comprises transplanting hematopoietic stem cells into a fetus of the animal. Here, fetuses include those from 30 to 60 days of gestation.

Hereinafter, the present invention will be described in detail. This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2000-34276, which is a priority document of the present application. If large animals could be used to obtain human hematopoietic stem cells, it would be possible to more accurately assimilate the human hematopoietic system. Large animals are not known. Zanj an i et al. Have this problem solved by the intrauterine in utero) transplantation ^14). Xenografts may not be rejected during the fetal stage before the immune system matures, and xenografts may be performed in large animals. They are out of work a xenograft despite chimera by transplanting human hematopoietic cells in Hijji fetal, human hematopoietic stem cells for excellent doing the research ¹⁵ - ^18). Recently performs in Wero human hematopoietic stem cell transplantation in a similar manner to N0D / SCID mouse ¹³⁾ Ya I j fetal ^19), have been reported also made that it was to produce a chimera.

This technique is not limited to hematopoietic stem cell technology, but can be used in a variety of other applications. Improved rapid prenatal diagnostic techniques in recent obstetrics area, at present ^2fl is becoming possible to identify fetal abnormalities life pregnant娠早- immediately after birth for certain diseases with ² genetic background Bone marrow transplantation is effective for treatment, but it requires treatment to abolish bone marrow function (mye loabration), strong immunosuppression, and the problem of matching donors. Miscellaneous questions There is a title. On the other hand, if the method of bone marrow transplantation by intrauterine transplantation is completed, such a problem may be solved. Experimental intrauterine hematopoietic stem cell transplantation allogeneic the rodent ^24), catcher formate ²⁵ - ^28), a non-human primate ²⁹ - is discussed ^32), it was to be performed two of clinical in humans ³³ — ³⁴ ). Furthermore, by introducing a therapeutic gene into transplanted hematopoietic stem cells, the possibility of intrauterine fetal gene therapy can be expected35 ⁾ .

According to animal experiments and various clinical reports, achievement of blood chimeras (chimeras in which cells from different individuals are present in one individual at the same time) is known to result in tolerance of transplant immunity. and have ³⁶ - ^39). If this technique achieves micro-chimera in a large animal (a micro-chimera is so low that the state of the chimera is faintly detectable), the animal may have been tolerant to human organs. The present inventor has proposed that human blood chimeras can be used as an incubator for human organs in a xenotransplantation experimental system in which human hematopoietic stem cells are transplanted into fetuses. An experiment was conducted to determine whether or not it could be produced, and it was shown that hematopoietic stem cells can differentiate into multilineage blood cells and maintain the microchimera for a long time.

The present invention relates to chimeric animals capable of circulating human blood in the body, and particularly to livestock or non-human primates.

Hematopoietic stem cells to be transplanted in the present invention include those derived from human umbilical cord blood or bone marrow. Hematopoietic stem cells from umbilical cord blood and bone marrow can be collected by a commonly used method such as a specific gravity centrifugation method (for example, a method using Ficoil or Lymphoprep), a Banning method, or the like.

Among the human hematopoietic stem cells collected as described above, it is preferable to use CD34 positive and CD3 negative nucleated cells (MNCs) in the present invention.

CD34-positive nucleated cells can be collected by labeling the collected cells with an anti-human CD34 monoclonal antibody, and using a MACS or FACS device. On the other hand, CD3-negative nucleated cells are bound by reacting the collected cells with an anti-CD3 monoclonal antibody conjugated with biotin, and the resulting complex is labeled with streptavidin. Then, by removing the above complex with magnetic beads, CD3-negative nucleated cells can be obtained. Can be.

Hematopoietic stem cells for transplantation are used after dilution in an appropriate buffer (eg, PBS). The cell concentration 3. 9xl0 ⁵ ~l. 5xl0 ⁶ carbon atoms, preferably lxlO ⁶ pieces ~ 1. 5xl0 ⁶ per one animal.

The hematopoietic stem cells obtained as described above are transplanted into the fetus of an animal (an experimental animal other than a human). As animals, relatively large livestock animals such as horses, bushes, wild boars, donkeys, and goats, and non-human primates such as monkeys can be used. Busu is preferred in that respect. The varieties of puyu are not particularly limited, such as black varieties such as Meishan, Mongkai, Yucatan Microbuyu, brown varieties called Rokuwaku, brown varieties such as Duroc, and white varieties such as Great Yorkshire and Landrace. Varieties. Transplantation is performed as follows.

First, domestic animals are pregnant by artificial insemination or natural pregnancy by mating. The diagnosis of pregnancy can be made by ultrasound after artificial insemination. The date of transplantation is 30 to 81 days after pregnancy, preferably 30 to 60 days, and more preferably 35 to 52 days.

On the day of transplantation, after anesthesia is introduced into the pregnant animal, the abdomen is shaved, and the abdomen is thoroughly washed and disinfected with stone water.

The cells are transplanted into the fetus using an ultrasonic tomography device while observing the image projected on the monitor. The cells are implanted with a syringe needle or a puncture needle, and the injection is performed by injecting hematopoietic stem cells suspended in 0.2 ml of physiological saline per fetus through the puncture needle. The thickness of the puncture needle used for implantation is 22 to 23 gauge, preferably gauge. The cells are injected into the peritoneal cavity of the fetus. At the same time, it is preferable to insert a marker identified by X-ray for the purpose of identifying the transplanted fetus at birth. Then, if necessary, antibiotics are injected to complete the cell transplantation.

After surgery, it is preferable to administer antibiotics (eg, mesilin, chenum, etc.) for the purpose of preventing infection.

The fetus after transplantation is allowed to grow in the mother, and a chimeric animal can be obtained usually by delivery or cesarean section. Since the obtained animal circulates human blood, it is useful as an experimental animal or the like for developing a medicament for treating human diseases.

Further, the present invention is used as a proliferation system for human blood cells (or hematopoietic stem cells). This is useful for producing blood for transfusion and bone marrow for bone marrow transplantation. For example, autologous transplantation can be performed on leukemia patients by collecting their hematopoietic stem cells from the bone marrow of bush.

Furthermore, since the chimeric animal of the present invention induces immune tolerance in humans, the chimeric animal can be used as a place for maintaining and regenerating organs when transplanting human organs. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing an actual hematopoietic stem cell transplantation operation.

FIG. 2 is a diagram showing the abilities of the transplanted offspring.

FIG. 3 is a view showing the results of analysis of the liver of a fetal rat transplanted with CD3-depleted cells from which CD3 human T cells have been partially removed.

FIG. 4 is a diagram showing the results of analysis of the surface antigen of human T cells in the thymus of chimera. FIG. 5 is a diagram showing that human CD34-positive cells differentiate into various lineage blood cells in the human body.

FIG. 6 is a diagram showing the distribution of human blood cells in tissues.

FIG. 7 is a diagram showing the results of bone marrow analysis of offspring born after transplantation of human CD34-positive cells. FIG. 8 is a diagram showing the relationship between the number of days after gestation and the graft survival rate.

FIG. 9 is a graph showing the hematopoietic activity of human CD34-positive cells engrafted in bush bone marrow (colony forming ability, xenogeneic bone marrow reconstruction ability). BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these Examples.

[Example 1] Preparation of human blood chimera

(1) Preparation of hematopoietic stem cells

(1-1) Preparation of cord blood and bone marrow suspension

Human cord blood and bone marrow were used as sources of hematopoietic stem cells.

Human cord blood is more vaginal than normal pregnant women with informed consent Collected at the time of delivery. After delivery of the baby, the umbilical cord was cut, and the umbilical cord venous blood remaining in the umbilical cord on the placental side was aseptically collected by heparinization in a 50-ml syringe, and stored at room temperature or 4 ° C.

Human bone marrow was collected from human ribs. That is, the ribs are aseptically minced, the exposed bone marrow tissue is separated by pipetting in PBS (Phosphate-buffered saline), and a single cell suspension is prepared using a 25G needle, and then bone fragments, etc. The waste was removed through a nylon mesh to obtain a bone marrow cell suspension.

(1-2) Collection of nucleated blood cells

Human cord blood and bone marrow cell suspensions were each diluted 3-fold with PBS, gently layered on Lymphopre, and centrifuged.The mononuclear cell layer separated at a specific gravity of 1.077 g / ml was collected. Then, the plate was washed twice with PBS. As a result, nucleated cells (MNCs) of bone marrow (bonemarrow; BM) and cord blood (CB) were separated.

The ΔCs obtained as described above was diluted and stained with a Turk solution. After counting the number of cells, the cells were washed again with PBS, suspended in PBS, and used as ΔCs for transplantation.

(2) Separation and concentration of transplanted human hematopoietic stem cell fraction

(2-1) Purification of CD34 positive cells

Nucleated cells were labeled with an anti-human CD34 monoclonal antibody. Fluorescent dyes or magnetic beads were used for labeling. To purify human CD34-positive cells from nucleated cells, MACS (Direct CD34 isolat ion kit mlentyi Biotec Bergisch Gladbach, Germany) or FACS Vantage (Becton Dickinson, San Jose, CA) was used. Further, after counting the number of cells, the cells were washed with PBS and used for transplantation.

(2-2) Purification of CD3-negative nucleated cells

From the ΔCs obtained as described above, CD3 positive cells (CD3 + cells) were separated and removed by a magnetic bead method in order to mainly remove only T lymphocytes. A biotinylated anti-CD3 monoclonal antibody (PharMingen, San Diego, CA) was added to 丽 Cs, left on ice for 30 minutes, and then washed once with SM (staining medium). To this, BioMags treptavidin ultra-load part icles (PerSeptive Biosystems, Inc., Framinghani, Mass.) Washed with SM was added, and the mixture was similarly left on ice for 30 minutes to magnetize CD3 + cells. The gas beads were bound. Using a magnetic part concentrator (Dynal MPC-1, Oslo, Norway), CD3 + cells were separated by magnetic force, and nucleated blood cells (T-deplete) from which CD3 was partially removed were collected. After counting the number of cells, the cells were washed with PBS and used for transplantation.

(2-3) Purity check

Using a portion of the transplanted blood cells collected by the above procedure, the frequency of CD34 + cells and CD3 + cells contained in each was evaluated by FACS. Fluorescent immunostaining of the cells used for transplantation was performed using anti-human CD34-FITC and CD3-PE monoclonal antibody (mAbs), and the frequencies of CD34 + cells and CD3 ⁺ cells contained here were determined by flow cytometry. It was measured.

All nucleated cells, CD34-positive cells, and CD3-negative nucleated cells were used as fractions for hematopoietic stem cell transplantation.

(3) Transplantation of human hematopoietic stem cells into pig fetus

The experiment was conducted by the National Agricultural Cooperative Federation (JA All Agriculture) Feed and Livestock Central Research Institute (Tsukuba City, Ibaraki Prefecture), where bushus (Durok, Durok; D, or Large Yorkshire, Large White; W) was used. These stags were propagated and managed in an SPF environment through experiments. Pregnancy diagnosis was carried out on the apparent mating day using an ultrasonic diagnostic apparatus (Aloka SSD-500; Aloka Co., Ltd., Tokyo, Japan) between 25 and 30 days of gestation. The mother whose fetal heartbeat was confirmed was diagnosed as pregnant, and the pigs diagnosed with pregnancy were subjected to transplantation experiments. The experiments used were 31 to 78 days of gestation.

(3-1) Anesthesia

On the day of transplantation, azaperon 2.5 mg / kg (Stresnil; Sankyo Co., Ltd., Tokyo, Japan) was injected intramuscularly into pregnancy to induce anesthesia, followed by 3% isoflurane + laughter with a face mask (N ₂ 0) performs the inhalation anesthesia with +0 _2, was subjected to general anesthesia. Thereafter, the pig was fixed in the supine position on the operating table.

(3-2) Disinfection

After induction of anesthesia during pregnancy, the pig's abdomen is shaved and the skin is washed with a brush did. Then, it was sufficiently disinfected with Povidone-Iodine (Isodine solution; Meiji seika kaisha, Ltd., Tokyo, Japan).

(3-3) Ultrasound imaging of fetuses

Transplantation was performed percutaneously using the ultrasound tomography device Aloka ultrasound diagnostic equipment SSD-1000 (ALOKA CO., LTD Tokyo, Japan) for cell transplantation into the fetus. The ultrasonic probe used was a 7.5 MHz electronic convex sector probe (Aloka; UST-987-7-5) equipped with a sterile cover. The probe was equipped with a puncture adapter (Aloka MP-2458), and the fetus was drawn on the monitor.

(3-4) Puncture needle

The puncture needle used to introduce human hematopoietic stem cells into the fetus was a 22-gauge, 150 nmi percutaneous transhepatic bile duct drainage (P.T.C.D .; Percutaneous Transepatic Cholangio Drainage) needle (Top Tokyo, Japan). Cells were injected intraperitoneally into the fetus (described later), and at that time, a marker identified by radiography was inserted for the purpose of identifying the transplanted fetus at birth. The marker was prepared by cutting a P. T. C.D. needle needle into a 3 mm length and sterilizing it with an autoclave. At the time of fetal puncture, the P.T.C.D.D. $ 21 stylet was removed, this sterile marker was inserted into the outer cylinder, and the fetal abdominal cavity was punctured. Thereafter, the stylet was inserted and pressed out to insert the marker into the peritoneal cavity of the fetus.

(3-5) injection of cells and administration of antibiotics

The stylet was removed again, and hematopoietic stem cells suspended in 0.2 ml of physiological saline per fetus were injected into the fetus's peritoneal cavity through a PTCDD needle. Thereafter, 0.2 U of amniotic fluid was injected into the fetal abdominal cavity with 0.3 ml of antibiotic (5 mg / ml Metacilin; Takeda chemical industries, Ltd., Osaka, Japan), and the puncture needle was removed. On the day of pregnancy, IPM / CS lg was intramuscularly injected on the day of transplantation, and antibiotics were administered for 2 days thereafter. On pregnancy, antibiotics were administered intramuscularly over the next two days. This procedure allowed the transplantation of cells into up to four fetuses.

Most of the transplants were performed under ultrasound guidance, but in some cases the abdomen was opened under general anesthesia, the fetus was identified directly under the uterus, and cell transplantation was performed on the abdomen of the fetus.

(3-6) Delivery Delivery was by spontaneous vaginal delivery or cesarean delivery. The evening pregnancy period is usually 114 days. Natural vaginal delivery was at term. Some pigs exceeded the expected delivery date and were given a delivery inducer. At the time of delivery, the total number of offspring, the number of offspring, the number of stillbirths at the time of delivery, and the number of mummy offspring (in-utero fetal death during the first trimester) were recorded, and the newborns were labeled by ear stamping (numbering). X-rays of some live pups, stillborn pups and mummy pups were taken to identify markers present in the abdomen, and the recipients (transplanted pups) of the pups and dead pups were identified.

[Example 2] Analysis of offspring

1. Materials and Methods

(1) Collection of peripheral blood, bone marrow, and organs of newborns that have undergone human hematopoietic stem cell transplantation

The offspring were heparinized from their peripheral blood and bone marrow blood within the first few days of birth and at about one month after birth. Peripheral blood was collected from the jugular vein of Buena fixed in the supine position, and bone marrow blood was collected by suction from the iliac bone. In some pigs, hematopoietic tissues such as liver, spleen, and thymus were collected after euthanasia. Liver, spleen, thymus, etc. were collected from freshly collected pups that could be identified as recipients by markers. Analysis was not performed on bushus, which were stillborn as mummies, because it was difficult to collect organs.

Under anesthesia with Ketalal, peripheral blood was collected from the vein and bone marrow cells were collected from the pelvic sphenoid bone. After euthanasia under anesthesia, various organs were collected.

(2) Separation of nucleated blood cells

Peripheral blood and bone marrow blood collected from buses were lysed with 0.1% sodium bicarbonate added 0.83% ammonium chloride (pH 7.0) to remove red blood cells, and washed with PBS. Later, nucleated cells (Marshal Cs) were obtained. The collected organs were minced from the organ pieces, squeezed in PBS using a slide glass, and passed through a nylon mesh filter to remove contaminating foreign substances and obtain MNCs. Human blood cells were identified by analyzing 丽 Cs obtained from these porcine hematopoietic tissues by FACS and PCR.

(3) Identification of human blood cells in nucleated cells

Human blood cells were identified by flow cytometry and PCR. (3-1) Flow cytometry

The obtained nucleated cells were labeled with an anti-human CD45 antibody and an anti-pig pan-t issue antibody. Cells that are anti-human CD45 antibody positive and anti-negative pan-tissue antibody negative are determined to be human blood cells. Details are as follows.

An anti-human CD45-FITC (fluorescence isothiocyanate) monoclonal antibody ( _m Ab) was used as a marker for human blood cells. In order to confirm the differentiation antigen of human blood cells, mouse anti-human CD3, CD13, CD14, CD33, CD34, CD4K CD56, CD61 and glycophorin-A mAbs labeled with PE (Phycoerythrin) were used. An anti-porcine pan-tissue mAb (clone 1030h-l-19) was used as a marker for blood cells. The purified anti-butane antibody is labeled with biotin using the ECL protein biotinylation module (Amersham International pic, Buckinghamshire, Engl and) and detected by secondary staining with streptavidin-APC (a11 ophycocyanin). did.

Blood cells collected from the mouse were washed twice with PBS + 0.5% ゥ fetal serum + 0.05% sodium azide (st aining medium: SM), then anti-human CD45-FITC, anti-human 1 ineages -PE and anti-porcine pan-tissue-Piotin was mixed and left on ice for 30 minutes. Thereafter, the cells were washed once with SM, mixed with streptavidin-APC, and allowed to stand on ice for 30 minutes. Subsequently, after washing twice with SM, the cells were suspended in SM with propidium iodide (2 g / ml), and the dead cells were stained. Flow cytometric analysis was performed in four colors using FACS Vantage SE (Becton Dickinson, San Jose, CA), and samples were collected from at least 200,000 cells overnight.

② PCR

Genomic DNA was collected from the obtained nucleated cells using SepaGene (Sanko-jyunyaku Tokyo, Japan). The identification of the human genome was performed by designing a sequence-specific primer and amplifying it by PCR.

The primers are the following two types.

Primer 1: 5, -CTGGGCGCAAGAACGAGATTCTAT-3, (SEQ ID NO: 1)

Primer 2: 5'-CTCACTACTTTGTGACAGGTTCA-3 '(SEQ ID NO: 2)

The following reaction mixture was prepared so that the total amount was 50 \, and DNA was amplified using Gene Amp PCR System 9600 (Perkin Elmer). Reaction liquid composition:

DNA:

-1

Primer 2 1 xl

Polymerase 0.5 1

Buffer: 10 l

dNTP: 8 1

Distilled water: 24.5 1

50 / xl

In the PGR, after a reaction at 94 ° C for 60 seconds, 43 cycles of a reaction at 94 ° C for 20 seconds + 58 ° C for 20 seconds + 72 ° C for 30 seconds were performed, and finally the reaction was completed after a reaction at 72 ° C for 7 minutes. The obtained PCR product was observed by ultraviolet irradiation on bromide bromide on an agar mouth.

(4) Measurement of colony forming ability of human hematopoietic stem cells in pig fetus

To evaluate the function of human CD34 + cells established over 3 months after transplantation into the bone marrow, their proliferative potential was measured.

At least 3 months after transplantation, human blood cells could be identified in the bone marrow by FACS analysis. After removing erythrocytes by hemolysis, porcine bone marrow nucleated blood cells were washed twice with SM. Human CD34 + cells were collected using the MACS method. Further, the obtained cells are fluorescently stained with anti-human CD34-PE, anti-human CD45-FITC and anti-butane pan-tissue-biotin mAbs, and human hematopoietic progenitor cells showing the surface antigen of CD34 + CD45 + Pig "using FACS Vantage. Fractions were sorted and collected.

Thereafter, pig bone marrow fraction Cs was labeled with anti-human CD34 antibody and anti-human CD45 antibody, and the double positive cells were separated and recovered by FACS. The cells were cultured on a soft agar medium containing human blood growth factor (Methocult GF H4434; StemCell Technologies Inc. Vancouver, B.C.), and the colony forming ability after 2 weeks was observed.

(5) CFU-C Atsushi

The proliferation ability was observed by culturing 700 CD34 + CD45 + Pig-cells collected from pig bone marrow on a methylcellulose semi-solid medium (Methocult GF H4434; StemCell Technologies Inc., Vancouver, B.). Medium is 2-mercaptoethanol 1 (T ⁴ M, L- glutamine 2 mM, © Shi bovine serum (FBS) 30%, © shea serum albumin 1% containing Muhoka, recombinant human erythropoietin (rh-EP0), recombinant human stem It contains cellular factor (rh SCF), recombinant human granulocyte macula phage colony stimulating factor (rh GM-CSF) and recombinant human interleukin 3 (rh IL-3) cells are making it possible to form various colonies scratch. culture 5% C0 ₂ in an environment humidified by 37 ° C, was carried out 14 days. the resulting colonies the number and form by entity microscope In order to confirm that the formed colonies were derived from human cells, the collected colonies were collected and genomic DNA was extracted. Then, the same procedures as in the PCR method and agarose gel electrophoresis performed in (3) above were performed. Then, the human genome was confirmed.

(6) Immunodeficient mouse bone marrow reconstruction (SCID repopulation)

Using N0D / SCID mice, immunodeficient mouse bone marrow reconstruction was performed. As the N0D / SM scid-Jic mouse, an 8-week-old male mouse was used for the experiment. From the mouse tail vein pretreated with § ray whole body irradiation sublethal dose (300 cGy), were transplanted CD34 + CD45 + Pig- cells recovered from blanking evening by 6. 7 X 10 ⁴ cells / mouse. Five weeks after transplantation, mouse peripheral blood was collected, erythrocytes were lysed, and nucleated blood cells were subjected to fluorescent immunostaining with anti-human CD45-FITC mAb, and human cells were identified by FACS.

2. Result

(1) Transplant and analysis

As a preliminary experiment, the transplantation method reported in Higgie and Dog was adopted. First, the laparotomy was performed under anesthesia using 7 minibu mice on the 48th to 55th days of gestation, and human HSCs were implanted into the fetuses under direct observation of the uterus. The advantage of this method is that cell transplantation can be performed on all pregnant fetuses. However, this method resulted in miscarriage in 5 of 7 cases (71.4%) within 5 days after surgery, which appeared to be due to infection. Therefore, the present inventor has adopted a more non-invasive method, and has developed a method of performing transplantation from outside the mother's body to the fetus under ultrasound guidance without performing laparotomy. Transdermal human HSCs transplantation was performed.

It is possible to check the fetal heart rate of the fetus from around 25 days of pregnancy. After the day, the size of the fetus will be 20 X 10 X 10 thighs (20πιιη in length). It is from this time that transplantation is technically possible with the method of the present invention, and since the abdominal anterior-posterior diameter and left and right diameters are about 20 mm after 40 days of gestation, it is easy to perform puncture peritoneal puncture under ultrasound guidance. It can be enforced (Figure 1). However, even at this time, it was difficult to perform transplantation into the blood, such as in the heart cavity and umbilical cord blood vessels. When this technique was used, the number of fetuses that could be confirmed by ultrasound with intestinal gas from the mother and mother was several. Since the pig abdominal wall is considered to be extremely rich in bacteria, transplantation was carried out after sufficient disinfection of the abdominal wall, and miscarriage was prevented by administering antibiotics to the fetus, amniotic fluid, and the mother.

Busu is a multiple pregnancy animal, averaging 10-14 fetuses at a time. Only some pigs can be transplanted, and if stillbirth occurs, it is difficult to identify the recipient. Therefore, in order to identify and distinguish several recipients at birth, we attempted to distinguish the recipient from the other by soft X-ray imaging by implanting the marker with the cells in the recipient. For this reason, microwires were inserted into the fetal peritoneal cavity at the same time as cell injection, and radiographs were taken after birth. Human cells were identified by FACS and PCR. The detection sensitivity was 0.005% for FACS and 0.0005% for PCR, obtained by artificially mixing human and Buyotsu blood cells.

As a result, a marker was identified in neonates (Fig. 2), and it was confirmed that the presence of this marker did not affect fetal development.

(2) Engraftment of human cells

Human HSCs were transplanted to 96 fetuses in 37 pregnant women. Pregnancy was interrupted by cesarean section for the analysis of the two pregnancies, and the presence or absence of transplanted cells during pregnancy was confirmed. As a result, all of the three transplanted fetuses showed engraftment of human cells at 100 W. In addition, human HSCs were transplanted into five fetuses out of three pregnant females. Immediately after this, five newborns died, and the presence of human cells in the dead transplanted newborns was analyzed, and three (60.0%) found engraftment of human cells.

Thus, the reliability of ultrasound-guided cell transplantation was at least 60%. Twenty-two pregnancies delivered at term, and 18 of the 44 transplanted fetuses (40.9%) showed engraftment of human cells (Table 1). Summary of intrauterine transplantation results Shown in 1.

Table 1.Summary of human hematopoietic stem cell experiments on intrauterine fetal fetuses

Item Number of pregnancies Fetal recipient

Number of transplants 35 91

Impossible * 10 35

twenty four

Transplant survival 2 3

Premature birth 3 6

Transplant survival 3 3

Maturity delivery 20 52

Transplant survival 10 14

*: Died within 5 days after transplantation

In Table 1, “Number of pregnant women” means the total number of pregnant women who had a transplanted fetus in each item.

An anti-porcine pan-tissue antibody was used to efficiently identify low frequency human cells by flow cytometry. Since this antibody reacts with almost all blood cells of the septate blood cells, excluding the labeled cells by computer analysis results in good separation from human blood and a frequency of 0.01%. Even so, human cells have been accurately identified. PCR was also used as a method for identifying human cells in the case of lower chimerism. This detection sensitivity was possible when the frequency of human cells was 0.0001% or more.

Table 2 shows the relationship between the pregnancy time at the time of transplantation and the establishment of the chimera.

Table 2.Summary of cord blood transplant survival cases

Human cell identification symbol Transplant timing Transplant cell type Transplant cell number Engraftment period Maximum chimerism

(Day) (positive organ)

D3585 45 CD3 removal 3.0X10 ^Y 38 5.1% (FL)

D3433 45 CD3 removal 2.4X10 ⁸ 81 PCR (BM)

D3585 44 CD3 removal 1.0X10 ⁸ 110 PCR (BM) Volume 41 35 CD3 removal 1.ox 82 PCR (BM)

W502 42 CD3 removal 4.4X10 ⁷ 82 PCR (BM)

W393 45 CD34 1.0X10 ⁶ 111 PCR (BM)

D3476 46 CD34 1.5X10 ⁶ 123 PCR (BM)

D3584 43 CD34 3.9X10 ⁵ 123 PCR (BM)

D3571 42 CD34 6.0X10 ⁵ 57 PCR (BM) 1048 37 CD34 1.1X10 ⁶ 47 0.04% (BM)

W549 35 CD34 7.0X10 ⁵ 142 0.58% (BM)

D3735 52 CD34 1.0X10 ⁶ 103 PCR (PBL)

D3461 43 MNCs 3.5X10 ⁷ 62 PCR (spleen)

W502 45 丽 Cs 4.0X10 ⁷ 85 PCR (spleen)

CD3 removal: About 70% of CD3 positive cells were removed from umbilical cord blood band Cs.

丽 Cs: unpurified cord blood 丽 Cs

PCR: Human cells were detected by PCIU.

FL: fetal liver

BM: bone marrow

% Indicates the percentage of human cells in the blood by FACS. Table 2 shows the transplantation timing (days after pregnancy), transplanted cell type, number of transplanted cells (cells), period until transplantation analysis (engraftment period), and analysis results (chimerism) for successful transplantation cases . All those that survived for a long time after transplantation were transplanted before 52 days of gestation, and chimerism was particularly high when CD34-positive cells were transplanted between days 35 and 37 of gestation. % Indicates the percentage of human cells in the blood of the blood by FACS, and PCR indicates that human cells were identified by PCR with chimerism below the FACS sensitivity. (3) Involvement of transplantation time in the establishment of human hematopoietic stem cell transplantation

As a result of examining the appropriate pregnancy time for human HSCs in utero transplantation, engraftment of human blood cells was confirmed in transplants between 33 and 52 days of gestation. Transplants prior to 31st gestation had small fetuses and the transplant was uncertain. Transplantation on the 35th day of pregnancy resulted in high chimerism (> 0.1 ·) of human cells. The transplant survival rate (survival rate, number of blood chimera pups / transplanted fetuses) on pregnancy 32-52 days was 22/40 (55.0%) (FIG. 8).

From this, it is considered that the possibility of transplantation of human hematopoietic stem cells in utero to xenogeneic fetuses is preferable in the case of a fetus in the 33rd day of pregnancy.

Table 3 shows examples of engraftment.

Table 3 Relationship between engraftment and transplantation time

Transplant period Number of offspring Number of chimeric neonates

(Days after pregnancy)

31-39 10 3

40-49 30 8

50-78 12 3 *

*: Transplantation, went to all 52 day _t

(4) Examination of transplantation success cases

Human blood cells were identified in various organs of the fetus, and up to 315 days after transplantation, human cells were identified. Successful transplantation was observed in both 丽 Cs CD3 depleted 丽 Cs and CD34 + cells, but chimerism at a level that could be analyzed by long-term FACS was observed only in the case of CD34 + cell transplantation. On the other hand, the frequency of human cells in the blood could be detected by PCR (Table 2).

W1048 in Table 2 was obtained by suspending pregnancy by cesarean section on day 47 after transplantation of CD34 + cells and analyzing the fetuses. As a result, human blood cells were identified in fetal peripheral blood, bone marrow, liver, and spleen by FACS, and the presence of all blood cells (B cells, NK cells, myeloid cells, and megakaryocytes) except human T cells was confirmed. . Interestingly, human T cells were identified in the thymus that were not present in other organs, while no cells with other surface antigens were present (Figure 5, right column).

W549 pups contained 0.58% human blood cells in their bone marrow. Survived human blood Analysis of the differentiation antigens in the spheres (CM5 + cells) revealed that 6.5¾ was CD19 + (B cell lineage) and 96.5% was CD13, 14, 33+ (myeloid lineage). No cells were present. CD34, a marker for human hematopoietic progenitor cells, was expressed in 21.0% of human blood cells, confirming that human HSCs had engrafted and differentiated in human bone marrow (Fig. 7).

(5) Functional analysis of human CD34-positive cells present in bone marrow

On the 37th day of gestation, 1.1 x 10 ^s human CD34-positive cells were transplanted, and fetal thymus was analyzed by FACS 46 to 47 days after the transplantation. Engrafted human cells were found in all analyzed organs of peripheral blood, bone marrow, thymus, liver, and spleen (Fig. 6). Human CD3-positive T cells were identified in the thymus. The surface antigen was CD4 or CD8 single positive and had a Q! I3 TCR. This indicates that human CD34-positive cells can migrate to the thymus and differentiate into human T cells (Fig. 4). Human CD34-positive cells differentiate and engraft into the myeloid lineage (CD13, CD14, CD33), B lymphocyte lineage (CD19), NK cell lineage (CD56), platelet lineage (CD41, CD61) and nmlUlineage. did. Bone marrow progenitor cells (CD34) were also observed, indicating that CD34-positive cells were self-replicating or maintained in pig blood. CD3-positive cells were found in the thymus, indicating that CD34-positive cells could differentiate into human T cells in pigs (Figure 5).

FIG. 7 shows the results of bone marrow analysis of offspring born after transplantation of human CD34-positive cells. On transplantation of human CD34-positive cells on the 35th day of gestation, chimeras were born. Chimerism was 0.6% in bone marrow (88 days after transplantation). The pig then developed normally. Human cells differentiated into both myeloid and B cell lines as well as CD34-positive myeloid progenitors.

In summary, in the bone marrow and liver, all lineages of human blood cells except B cells, myeloid cells, NK cells, megakaryocytes, and T cells were identified. On the other hand, only CD3-positive human T cells were identified in the thymus, and no cells expressing other lineage human blood cell antigens were found. The T cells present in the thymus were CD3-positive and CM-positive, and were T cells with ai8 T-cell receptor (TCR).

Furthermore, the function of human CD34 + cells present in the bone marrow of pigs (W549 pups) was analyzed. CD45 + CD34 ⁺ Pig— cells were collected from the bone marrow by FACS sorting (Figure 9A). The colony forming ability and the SCID mouse bone marrow reconstruction ability were confirmed. As a result, these cells have the ability to form both myeloid and erythroid colonies (Fig. 9C, D), and have been transplanted into the bone marrow of sublethal irradiated N0D / SCID mice at least 5 weeks after transplantation. It survived (peripheral blood chimerism: 0.17 ± 0.23%, n = 3) (Fig. 9B).

3 Discussion

According to the present invention, a method has been developed in which hematopoietic stem cell transplantation into a fetus is performed non-invasively under ultrasound guidance without laparotomy of pregnant pigs, and a chimera is established even in xenotransplantation. Human CD34-positive cells engrafted in many blood-based tissues, and colony-forming cells were also maintained in the bone marrow. In the porcine thymus, human T cells differentiated from CD34-positive cells were accumulated.

It has been clarified that transplantation of // ero human hematopoietic stem cells into fetuses can produce human blood chimeras without immunosuppression or bone marrow abolition treatment. The chimera state lasted up to 142 days after transplantation, confirming long-term engraftment.

In the case of long-term engraftment of human cells, the transplantation period was before 52 days of gestation. This indicates that it is important to transplant before the immune system develops. According to a report examining the immune system ⁴¹ ), lymphocytes were identified from day 28 onward, and T cells were found around day 50 in spleen and peripheral blood. slgM-positive cells are first identified in the liver on day 44 ( ⁴² ). This report is consistent with the timing of transplantation in this experiment. The transplanted human CD34-positive cells were shown to differentiate into multilineage blood cells including T cells in the fetus. In the system using SCID mice, differentiation into T cells is not generally observed. This indicates that the large animal system, Busu, is useful for assessing the differentiation of human T cells by Ww. Human blood cells that migrated to the thymus may have been educated in pigs thymus ⁴³ - ^45).

On the other hand, some fetuses were suspected of having graft-versus-host disease (GvHD) by intrauterine transplantation of human blood cells. Markers were confirmed in fetuses that had already passed long after birth at birth, and in one case, a large amount of ascites was present at birth and died. Analysis of pregnancy revealed that T cells predominantly proliferated in した Cs-transplanted cells (Table 2; D3585) Therefore, it may be desirable to remove T cells from transplanted cells. Alternatively, by using this in reverse, it may be possible to use it as a GvHD model.

In the Higgies system, the presence of T cells is said to have both an effect of promoting the engraftment of human cells and an effect of causing GvHD46 ⁾ . However, the method of the present invention is characterized in that it is non-invasive by not performing laparotomy, unlike the system using a sheep or dog. In addition, Wero transplantation can safely produce neonatal chimera without bone marrow abolition treatment. This point is also considered important in considering clinical applications. The creation of chimeras is simpler than that of neonatal bone marrow transplantation, which requires strict postnatal care. In transplantation experiments, microchimerism at the PCR level is also effective for immunological tolerance. Therefore, animals that have become human blood chimeras may be tolerant to human transplant organs, and future applications are possible. Since Buyu is a multi-fetal animal, there is an advantage that various assays can be performed simultaneously by transplanting various cells with different markers. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety. References

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The present invention provides a chimeric livestock animal containing human blood and a method for producing the same. The animal of the present invention is useful as an experimental animal or the like for developing a drug or the like for a human disease. Sequence Listing Free Text SEQ ID NO: 1: Synthetic DNA SEQ ID NO: 2: Synthetic DNA

Claims

The scope of the claims

1. Chimeric animals containing human blood.

2. The chimeric animal according to claim 1, wherein the animal is a pig.

3. A method for producing a chimeric animal, which comprises transplanting hematopoietic stem cells into an embryo of the animal.

4. The method according to claim 3, wherein the fetus is from 30 to 60 days of gestation.