JPH05502385A - Growth, production and composition of megakaryocytes and platelets - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] bihano, biha Cross 1 relation to the gall This application is part of Application No. 593.688 filed on October 5, 1990. This is a continuation application.

theory 1 Tori Public The field of the invention relates to the proliferation and production of hematopoietic cells, more particularly megakaryocytes and platelets. do.

l-view Thrombocytogenesis is the differentiation and maturation of megakaryocytes and platelets.

Research has been done to understand the process of thrombopoiesis and the factors involved in thrombopoiesis. Despite the tremendous effort that has been put into The confirmation still remains. Factors that appear to have effects in vitro are: It appears to have little or no effect in vivo. thrombocytogenesis and The activity is poorly understood and has been isolated in pure form and characterized. should be attached. megakaryocyte precursors that provide for their maturation and formation of platelets Efforts to provide long-term culture are also uncertain.

Thrombocytogenesis is a very important process for an individual's health. Platelets are seeds in many protective processes, supplying various factors, participating in clot formation, and the like. Play a vital role. thus providing a method that will aid in the understanding of thrombopoiesis. and provide long-term production of macroglobules and platelets that can be applied as a therapeutic method There is a real interest in that.

■Arbitration welcome Blood Ce1l, Volume 15, No. 1, 1989 refers to megakaryopoiesis . Hill and Levin, Blood Cells (1989) 15 :141-166 describe the regulation of thrombopoiesis. heparin by endothelial cells Is the discussion about the role of Thorntor 3? , Sc 1ence (1983) 222: can be found at 6'23-5. WO90US1725 describes megakaryocyte proliferation. We report a growth-promoting activator protein. Thrombin glycosaminoglycan Regulation of megakaryocyte differentiation has been reported.

Kenjiho 2ohm - required 2ho By using a medium that promotes megakaryocyte precursors and megakaryocyte proliferation, megakaryocyte growth can be achieved over a long period of time. Methods are provided for growing karyocytes and platelets in culture. premegakaryocyte Various sources of progenitor cells are used; megakaryocyte growth factor, megakaryocyte precursors and megakaryocytes Provided to enhance proliferation. The assay system uses megakaryocyte precursors and megakaryocytes. To assay the effects of factors on sphere proliferation and maturation and platelet formation provided to. Megakaryocytes and platelets are treated with thrombopoietic treatments, compounds or compositions. It is used to identify karyocyte proliferation activity and understand the mechanism of megakaryocyte proliferation.

Waiting f'I Methods and methods for culturing and in vivo proliferation of megakaryocyte precursors, megakaryocytes and platelets A composition is provided. Megakaryocytes are treated with heparin and megakaryocyte growth factor or the presence of human plasma. can be grown in the presence of In culture, megakaryocytes are progenitors from exogenous sources. Can be maintained for long periods without cell replenishment.

A bioassay for monitoring megakaryocyte proliferation and factors that affect it. provided. Megakaryocyte precursors, megakaryocytes and platelets are suitably an indicator of megakaryocyte proliferation. Fibroblast proliferation while inactivating inhibitors, especially those produced by platelets. Human progenitor cells are grown in a suitable nutrient medium under conditions that do not support, preferably inhibit, proliferation. can be produced by growing it in culture. The source of progenitor cells can be any convenient sources such as bone marrow, fetal liver, non-adherent cells from bone marrow, sorted cells, white Blood cells, e.g. buffy coat without red blood cells, CD34+ fraction from bone marrow, low density cells , class I [HLA positive cells or the like. However, the generation of megakaryocytes There should be an initial precursor that can maintain the

For long-term culture, the cell composition used may desirably contain hematopoietic stem cells. Probably. The cell composition may be from a fetus, neonate or adult. major The methodology used with respect to any primate hematopoietic cell composition comprising stem cells. However, the methodology is also used with human cell compositions if desired.

For many purposes, proliferation of megakaryocytes, megakaryocyte precursors and platelets is desired. subordinate Therefore, having a system that can proliferate antikaryocyte cells is important for megakaryocytes. It is very important in providing a source of cells and platelets. What's next , describes a method for propagation in culture of antikaryotic and platelet cells.

In many cases, the containers used for culture growth are designed to enhance fibroblast formation. coated for protection. According to the invention, in order to avoid promoting fibroblast formation Uncoated or treated containers are used. Containers without coatings are commercially available or from Falcon, Corning or similar sources. Obtained by requesting such a container. The container has a plurality of wells l/- 1-, including roller bottles, fermenters, trays, or the like.

The medium used should contain the necessary salts, minerals and nutritional supplements, such as amino acids, Any convenient medium that provides glycose and vitamins. Also, a small amount of raw A physically acceptable reducing agent is used, such as 2-mercaptoethanol.

Mercaptoethanol is about 1.0-' to 10-6M, preferably about 10-'M will generally exist.

Non-seed additives do not promote or inhibit fibroblast formation and proliferation, or are preferred. or may be used to promote megakaryocyte proliferation.

Additives that may be used not to promote fibroblast formation in the presence of fetal bovine serum is heparin, citrate or other additives that interfere with platelet aggregation and degranulation. includes. The amount of heparin is about 50 μg/d, preferably at least about 100 μg/d. μg/-, and usually does not exceed 4 ■/-, more usually does not exceed 2 ■/d. , preferably in the range of about 0.5 to 1.5 mg/-. The amount of citrate present If so, it will generally be in the range of about 0.1 to 1%. Other additives in the system They will be used according to their activity against and any adverse effects. The medium is Generally, low levels of plasma or serum, human plasma or serum or fetal bovine serum are also used. , preferably about 10% or less, more preferably about 5%, and -C at least about 1% in the absence of a specific cytokin in a defined medium.

The amount of serum and other additives can be optimized where antikaryotic cells are stimulated to proliferate. and fibroblast proliferation is inhibited.

MCDB 107° IMDM, RPMI, usually enhanced by low levels of serum Any complex eukaryotic growth medium can be used, including , EX-CEL, etc.

Desirably, factors produced by platelets and that inhibit megakaryocyte proliferation are present at low I/vectors. will be maintained in the file. The specific factors are platelet factor 4 (PF-4) and including the decomposition product tromposbondin. Heparin binds to PF-4 and can reduce its inhibitory activity on megakaryocyte proliferation. discovered. Therefore, if heparin is maintained in the medium, the heparin -4 and its decomposition products ■Prevent them from having harmful effects. Absence of heparin Below, other methods can be used to reduce levels of PF-4. This is a culture Continuously changing the medium and removing PF-4 and its degradation products from the medium Accordingly, addition of an antibody against PF-4, dialysis to remove PF-4, and The same can be achieved.

To increase preservation of platelets formed in culture, calcium levels and um/phosphate ratio can be adjusted. Generally, calcium levels are present in the range of about 0-10mM5 and the phospho-1-concentration ranges from about 0-10mM. It exists within the surroundings. ADP levels also provide a system for converting ADP to ATP. It can be adjusted by supplying Useful system, other systems are also used creatinine phosphate and creatinine phosphate kinase. Cree Atinine kinase and 1. /atinine kinase phosphate concentration is determined according to the desired AD P/ATP levels will be maintained within the range that provides. These concentrations are critical There is no system, and it is primarily intended for the convenience of gaining production capacity in the system. blood Platelet preservation can also be achieved with dextrose acid sylate.

The megakaryocyte cell culture also comprises at least one growth factor as the megakaryocyte growth factor of interest. When used together, they can be grown in the substantial absence of serum (51%).

The growth factor is present at a relatively high concentration, usually about 25 ng/d or more, preferably about 50 ng/d. /- or more and especially 1100 n/d or more, interleukin TL-1, -3, -6 and Eri) Roboetin, including IL-6 and Erythroboietin in poetry. Contains. High levels of cofactors, such as vitamins and other components, such as β-carbohydrates Chin 1, insulin, etc., such as x-vyv○ and EX-CEL (J.

A defined enriched medium with R, Sc 1ent f ic) should be used It is.

The cell composition obtained in the culture medium is highly concentrated for megakaryocyte precursors, megakaryocytes and platelets. More than 10%, usually more than 50%, of the liquid culture medium is megakaryocytes/blood. This is a platelet-bearing strain. At least 90% by weight of megakaryocyte blastoblasts and megakaryocytes (wt. cells in liquid culture, excluding platelets in counting), especially megakaryocytes A composition is obtained. Therefore, cells that are committed to the megakaryocyte/platelet lineage, provide factors. to stabilize adherent populations of cells and appropriate growth factors, platelets that can be fed containing substances to inhibit fibroblast proliferation and, where appropriate, substances to inhibit fibroblast proliferation. A culture comprising a conditioned medium that supports megakaryocyte proliferation is obtained.

Cells of interest can be isolated from culture media or bone marrow. Cells of interest also have surface membranes Isolated from blood by using separation techniques related to proteinaceous markers The pia mater can be manually removed. The cells of interest are CD34+, -41+, -41- and Normally, it would be classified as -33+. These cells are derived from polyploid adult megakaryocytes. Since they can be distinguished, they are diploid. Therefore, magnetic beads, positive and negative selection, Pre-megakaryocytes by separation using FAC3, affinity columns and similar Anyone can obtain cell compositions highly enriched in precursors. enriched meganucleus More rapid maturation and production of platelets is achieved by using a population of bulb precursors can be done.

Factors present in the conditioned medium associated with megakaryocytes are typically at ambient temperature of 41°C or below. It has been found that it can be stabilized for more than two weeks at a temperature higher than that.

When performing megakaryocyte and platelet generation, it is usually about 1 x 10s to 1 x 1 per ld. 07 human bone marrow cells, preferably 1X10' cells per relative, or about 2X A buffy coat unit from an adult containing 10B white blood cells (but not including red blood cells) , will be inoculated. under conventional physiological conditions, i.e. with approximately 5% carbon dioxide. Approximately 37°C in a humidified chamber containing used to grow cells, where oxygen is at least about 5%, usually less It would be about 20%. Higher oxygen levels were reported to inhibit fibroblast proliferation. high oxygen labeling, usually no more than 90%, is necessary for cell proliferation. may be used depending on the other conditions used. Conveniently, a 6-10cm cork wells or 96-well plates are used, and it Allow cells to proliferate. Within about 7 days, megakaryocytic cells are observed and the culture Nutrition may continue for six months or more. If dialysis is not used, the medium is Change regularly, generally at least twice without regression, preferably at least every two days. It will be done. Cell islands with deposited adherent cells were observed, and this Differentiate into mature megakaryocytes observed as lobular cells. Megakaryocytes and adherent (interstitial) cells An adherent layer, which is a combination of cells, is observed. Cell islands are megakaryocyte precursors (megakaryocyte buds) It has the characteristic CD41 marker for megakaryocytes, supporting cells that are (and is mononucleated) appears on the adhesion layer. The progenitor cells also contain the CD34 marker. Carr will also support it. Platelets are present in the medium and in the medium associated with the adhesion layer. There will be. Megakaryocytic cells contain the surface protein marker CD41 (I[b/ IIIa) (integrin) and CD42 (Ib). and when the cells are stained by Wright-Giemsa, the megakaryocytes Displays multilobular or polyploid characteristics. Progenitor cells, by comparison, have CD41 and CD41 34 markers and will be mononucleated.

Megakaryocyte cells are Megacolor (Cytocolor).

They can be further differentiated by staining with M.D., Inc., Hinckley, OH). Huge In karyocytes, the cytoplasm stains deep purple. Additionally, electron microscopy reveals As such, the majority of cells in culture are actively proliferating platelets.

On the other hand, the level of src protein expression and its kinase activity is significantly increased in megakaryocyte precursors, It can be used as a measure of megakaryocyte cell proliferation, measuring megakaryocytes and platelets. Ah relatively homogeneous or heterogeneous phenotype selected by one or more markers or by cell lysates of populations, which can be carried out, for example, by specific antibodies. The rc kinase can be isolated and then one or more washes performed. labe labeled nucleotide triphosphates, e.g. labeled double isotopes. By using phosphorylation as an indication of the presence of cells associated with megakaryocyte proliferation, level can be measured. On the other hand, Western plot or SRCELI SA can be used to quantify src protein.

As the culture matures, the concentration of platelets continues to rise, so that after about a week , the concentration of platelets is about 10'1 platelets/relative, and about 106-109 of platelets/d. The level of platelet production is determined by continued growth and growth of the culture. can be maintained by maintaining the medium with appropriate composition of ingredients and removing fluids. can be enhanced by enrichment of platelets using a variety of techniques.

Cells are treated with chelating agents such as EDTA, sonication, enzymes such as proteases, etc. using enzymes such as trypsin, collagenase, plasmin, etc., or similar methods. can be harvested by mechanically separating the adherent layer. specific used The embodiment is not critical to the invention. Next, megakaryocyte buds and megakaryocytes are extracted using various techniques, For example, cell rotation (cytospin), FAC3, affinity separation, density It can be isolated using separation, magnetic bead separation and similar methods. As a result, the real The essentially pure cell composition confers megakaryocyte proliferation, i.e. megakaryocyte precursors, megakaryocytes. Obtained from cells that give rise to spheres and platelets.

The composition may contain terminal materials that do not involve the isolation of such cell compositions from bone marrow or blood. It will not contain components of peripheral blood. Additionally, individual cell types are virtually pure. It can be isolated in the purest form, i.e. megakaryocyte blastema, megakaryocyte and platelet, which is commonly typically at least 75% of the same cell types, usually at least about 90% of the same cell types. It comprises the same cell type and up to several 100% of the same cell type.

Conditioned medium or human plasma is used for isolation of factors involved in megakaryocyte proliferation. obtain. Fractional ammonium sulfate precipitation enriches the protein fraction for growth factors. used for In addition, enrichment can be performed using a gel to separate the two components of various sizes. by using a permeation method and then assaying for both enriched fractions. achieved. The enriched fraction is then treated with n-propanol or acetonitrile. with an aqueous acidic medium, such as 0.1% trifluoroacetic acid, or IM acetic acid. Further enrichment is performed using γP-HPLC, which is eluted with γP-HPLC.

Purification of cartilage activity was further achieved by HPLC using a cationic column. where the column buffer is present at a pt+ of about 7.5-9 and the sample is eluted with a high ionic strength eluent, e.g. 2M inorganic salt, e.g. NaCl. It is eluted using a linear gradient in ram buffer. Its activity is linear gradient is observed at both ends of the highest peak and is generally about 1.25 to It is observed in the fractions eluting in both 1.6M salt fractions.

Fractions are bioassayed and enriched fractions are subjected to gel electrophoresis or other separation. can be further purified by techniques.

Megakaryocyte growth factor is added to the medium from the culture or plasma through a heparin column. Obtained by sorting. The factor is at least about 1 /st, preferably Conventional complex eukaryotic enrichment containing heparin at a concentration of about 3 μ/d and at 4°C In the soil medium, heparin can be transferred from heparin-sepharose to heparin. Characterized by bonds. Heparin-Sepharose beads:medium ratio is common at least about 1 g + 2 ml, preferably about 5 ml, and about 1 g + 10 d No more expensive than. factor, the absence of which substantially inhibits the proliferation and maturation of cells of the megakaryocytic lineage. cell proliferation of megakaryocytic lineages in culture systems such as those described above. further characterized by supporting.

When measured in solution, the molecular weight of the factor is greater than approximately 40 Kt). Seem. It is also stable at 56°C in intact plasma. it is It also appears to be species-specific and is comparable to being effective on human cells. is not effective on mouse cells under these conditions. Finally, it is Affi-Gel Do not combine blue commercials.

Factors of interest provide 1,000-fold or more enhancement in concentration and specific activity Further purification may be performed by conventional techniques such as those described above.

To assess the response of megakaryocyte precursors to various factors, factors Body culture medium assay compositions, such as megakaryocyte proliferation and methyl comprising factors that support Cellulose assay compositions can be used to evaluate cellulose assay compositions. Methyl cellulose used is generally at least about 1.5% in water and about 3% (v/v ) will not exceed. The cell source is the same as that used for culture. obtain. Also, at least about 15%, preferably about 20% fetal bovine serum, or plasma will be included in the culture. A convenient volume for performing the assay is 2 Approximately 200 μl is the volume used for a 4-well plate.

To support megakaryocyte proliferation, high concentrations of cytokines IL-1, -3 and -6 are required. , as well as Eri loboetin, are generally at least 7 to about 065 times or more It is encompassed by the saturation of .

Other factors, such as 31 or metal factors, may be included in significant concentrations. Saturation means dark An additional increase in temperature has a significant effect on the number of cell colonies produced. It means not having. Conveniently, individual factors of about 1100 n/d are used. obtain.

The cells that are inoculated include CD41+, CD34+ cells, and acts as a megakaryocyte precursor. Cells are intact bone marrow or CD41+, CD34+ There may be an enriched fraction comprising cells. Growth pattern in forming colonies The cells largely do not contain the CD34 marker and usually have the CD33 marker. and has the morphology of mature megakaryocytes.

The target factor or composition contains all of the above factors, or (1) or more than one of the above factors. studied by adding such factors or compositions to media lacking obtain. Therefore, megakaryocyte proliferation or other factors or cytokines to provide for it. The inherent ability of the composition along with fetal bovine serum to stimulate its ability to interact with power can be determined. The assay lasts for at least about 5 days, usually less than 5 days. Both are carried out for 7 days to v'J21 days, preferably for about 7 days to 14 days. Asse The conditions for this are that the liquid medium or methylcellulose is Conditions are maintained at 37°C and 5% CO2.

By using varying doses of such compositions or agents, activity can be determined. In this embodiment, rapid screening whether the product or factor is useful for megakaryocyte proliferation or platelet production; can be carried out to determine.

Megakaryocyte precursors and/or megakaryocytes alone or with infusion of platelets can cause thrombocytopenia. It can be used for the treatment of diseases. In many cases, e.g. surgery, chemotherapy, etc. In this case, autologous megakaryocytic cells are expanded in culture and returned to the host to develop megakaryocytes. Other effects of treatment that nuclear cells and/or platelets can provide or minimize bleeding be done. The cells are impregnated with a physiologically acceptable medium, e.g. saline, and and generally at least about 105 cells/-1 usually about 106 to 10a cells/-1 It can be present in concentrations ranging from 100 cells/d. The dose is generally about 10 will vary between ''' and 108a cells/kg host. Typically, the composition will be at least 25% by weight, more usually at least 50% and sometimes less It will also contain 90% by weight megakaryocyte precursors and megakaryocytes. One or more as necessary. Several injections are required. Platelets are harvested according to conventional means and can be given.

Megakaryocyte cells can be used for gene therapy, where the cells are one normal megakaryocyte cell. maintains function under adverse conditions or utilizes mechanisms normally unavailable to megakaryocyte cells. can act to provide functionality. For example, multiple drug resistance (mdr) ) a phenotype is introduced into the cell, where it makes it resistant to chemotherapeutic agents and Allows for continuous normal megakaryocyte response during therapy treatment. If the deficiency occurs in a particular episode or episodes in which it is desired to increase the proliferation of megakaryocytes or other megakaryocytic cells. Growth factors, which when present between them, provide an enhanced source of growth factors in the bone marrow A gene encoding can be introduced. Various methods provide for the transformation of cells. known for stable extrachromosomal elements or homologs or random integration. provide Viral vectors that cannot be reproduced have been previously described in the literature; This can be used for dyeing, 1-transfection and incorporation. For culture By being able to proliferate megakaryocytic cells in cells, anyone can transform such cells. have the opportunity to do so, and be able to expand and clone the desired transformants. Wear.

The following examples are for illustrative purposes and are not intended to limit the invention.

Fruit - Dragon ■Earthworks Hevalin-containing bovine fetal bone marrow culture.

A, Cells: 265 fetal bone marrow, 2X.

Medium: IMDM + 5% Gibco FC3 + 10-'M 2-mercaptoeta Nord. Fe2 and 2-mercaptoethanol are added to fresh individual media. It was an exchange body.

Heparin: Sigma 1781J/ng, stock solution 200mg/d.

Plate: 10011Tll Falcon plate.

Plate 1: No heparin was added.

Plate 2: 1000μg/iI! Heparin Sigma concentration of 1.

Both plates were subjected to 10-day removal of medium and hydration of medium and heparin almost daily, and occasionally every other day. Controlled by the addition of phosphorus. By the 10th day of soil, there was a marked increase in cell quality. The difference was clear. A light micrograph was taken to clearly demonstrate its morphology. Plate 1 is , fibroblasts that form large ball-shaped cells that are often expelled from the plate. It had sheets of similar cells. Plate 2 is a confluent layer of round cells with various morphologies. It had No sheets of fibroblasts were observed.

B. The above study was repeated. However, 5% normal human plasma was used instead of Fe12. used. Egg-shaped morphology indicative of antikaryocytic type cells was observed on both plates. , in which normal human plasma was used in place of Fe2, and heparin The effect was shown to be related to Fe2.

Inflammation I: High heparin concentration effect on fetal bone marrow cultures.

t: K 266 and 267 samples were canted in half along the long axis, and the Incubate with lagenase or trypsin at 37"C and 0.5 h. Proteins were prepared by mixing in the dark for 3 hours at intervals and then washing three times with medium. It was peeled off from. Cell concentration was 2 x 105/-.

Plate: I100n Falcon plate.

Medium: IMDM + 5% FC3 + 10-'M 2-ME.

Heparin: if used, 1000 μg/Sigma (Sigma) diluted in sterile water did.

Four plates were prepared, two plates each containing 266 and 26 7 and the other pair of plates contains plates with heparin and controls. .

As observed in previous experiments, plates without heparin were In two weeks, it became hyperproliferated with expanded and elongated shaped cells. Contains added heparin The plate shows the ``egg-shaped'' cell proliferation seen above. These cultures Nutrition was controlled as described above by changing the HaboQOD medium.

General: Effects of different plates on megakaryocyte and fibroblast proliferation.

The methods described in Examples 1 and 2 were used with 275 in the cell preparations. 4 pieces plates were prepared, two plates were from Corning; The other two plates are from Falcon, where the individual vs. The samples included control and heparin-containing media.

Cultures were managed in the same manner as described above.

Corning's control plates provide rapid growth of adherent cells with fibroblastic morphology. It showed proliferation. Within 7-10 days, the culture is overgrown on the surface and the cells are The cells were detached from the plate. These cells, often ball-shaped, float in the medium. It was found to be playable. The medium turns yellow rapidly despite occasional changes. It became. In Corning's heparin-containing media, a mixed pattern of Proliferation was observed for both fibroblast-like cells and cells with an “ovoid” morphology. It was noticed. The proliferation of fibroblast-like cells was very significantly reduced and the culture The overall cellularity of was reduced compared to control plates. In about 2 to 3 weeks, fibroblasts Anticular cells predominated in the culture and the number of oval shaped cells was reduced. whole Generally speaking, the number of non-adherent cells was minimal.

In Falcon plates and control plates, rapid growth of fibroblast-like cells Overgrowth was present. These cells were found in Corning's control plates. confluency is not reached as quickly and in any of the Corning plates. There were more "egg-shaped" cells. These cells are fibroblast-like cells Form clusters on surfaces that appear to exclude. Finally, heparin-containing medium Falc including ground.

In the n-plate, the growth of oval cells to confluence was replaced by clumps of multinucleated cells. observed with fruit formation. The adhesion layer of antikaryocytes and stromal cells is formed by antikaryotic fibroblasts. A supporting colony with cells was formed.

±±: Fluorescence-activated cell sorter scanning.

Cells from K266 and Ni267 cultures were removed by gently sucking them up with a pipette. were collected and contained approximately 105 cells/-. The antibodies used were 0. I Anti-CD7 and anti-CD42 in PBS and 2% FC3. cells Incubate with antibody 20 μ2 on ice for 20 min and then with Fe2 Washed. 1: FITC Rahelized goat anti-mouse Ig diluted 100 ( 20 μl/sample) was incubated with the above mixture on ice for 20 minutes, Cells were subsequently washed with Fe2. Next, cells were separated on FAC3c an analyzed. The scan is ungated standard limit 52 (arbitrary units) and amplified was 1. 266 showed a sharp peak in the culture, where the CD7 peak was 42 was substantially displaced from Beak. Similar results were obtained for Crab 267. .

By comparison, the control is the fluorescence intensity obtained on unadded antibody (self-fluorescence). showed no significant increase.

Godan: Methylcellulose assay.

In the next study, the methylcellulose assay was performed using 20% fetal bovine serum and Using IL-1, -3° and -6 and erythropoietin at 1100 n/d, respectively. The test was carried out under conventional conditions. Complete bone marrow is used at various inoculum levels; And the selected cells were based on CD34 and CD41. CD34+, CD4 1+ was 0.5-0.9% of the complete bone marrow population.

The following table shows the results: cell cell colony cluster WBM CD34 C1) 41 Concentration No,”/10’±-lXl0” d 0-1-3 - + -1xlO'd 1-2-0 - ± + 1xlo' d 10-7-19 4-5~7- - - lX1 0'd 0-0-1- - + lX10'm 0-1-0+ -2xlO' d 0-1-5 2-3-2 ~ + -2xlO' d 1-2-1 1-1- 1-10 + 2X10' d 34-40~40 9-8-12- - - 2X10'ag Q-0-0 - + 2xlO' d 0-0-3± -l Xl05d 15-16-15 3-9-7 *These homotypic colonies are more than 50 5, and the cluster contains 20 or more cells.

Colonies consisted of morphologically identifiable antikaryocytes. Therefore, the colony Low concentrations were observed in CD34+ and CD41+ cells maturing into karyocytes. moreover, If complete bone marrow is used at a seeding level of 105 cells/-, it will provide antikaryocytes. A significant number of colonies were observed. Furthermore, the CD34+ and CD41+ populations were was found to be sensitive to the factors identified, and as a result, removing one factor , and by substituting such factors into the test composition, anyone can The effect of such test factors on the formation of spheres can be determined.

Godan: Proliferation of antikaryocytes from the pia mater.

One unit of white blood cells (buffy coat) from an adult contains approximately 2×10+1 white blood cells. That single The red blood cells in the position were placed in a buffered ammonium chloride solution in Tris-MCI. The cells were disrupted by incubation at 37°C for 10 minutes. next, Leukocytes were cultured under standard heparin culture conditions as described in Example I. Cells/10 cm Bras, 5 x 10' cells/61 plates or 105 cells cells/microtiter plate at a volume of 10 d, 5 d or 100 μ2. Approximately 20 10CI plates can be prepared.

Obtain a confluent monolayer of antinuclear spheres and precursors after 1% bipolar interval according to the embodiment described in Example 1. Ru. Most other cell types no longer exist. The original implant was 0.1% antikaryon. Assuming that the precursors are included, this preparation will allow a confluent 10 cm dish to contain 107 strips. Since it contains cells, it has been expanded 103 times.

On the other hand, an aliquot of 1-2X107 fresh white blood cells was added to 10% DMSO and 2 Can be frozen in liquid nitrogen in 0% FC3. Those vials are It can then be thawed for use in heparin culture.

[7: Anti-nuclei by measuring src protein by E L X S A Sphere assay.

Antikaryocyte cultures were established with 8 x 10' cells in 96 well plates I. stand up Each growth factor source is tested in quadruplicate (4 wells). of proliferation After 10 days, the medium is removed from the wells and the culture is allowed to air dry overnight.

src ELISA was performed by rehydrating cells in PBS for 5 minutes. It is done. src antibody (2-17) (mouse monoclonal) in PBS+ Dilute 800 in 1% BSA. , 40 μC to individual wells, and and incubate for 30 minutes. Wash wells three times with PBS. 2nd anti goat alpha mouse Ig (alkaline phosphatase conjugated) (Calt, ag) 1:250 in PBS+1% BSA. Add 40 μl to each and incubate for 30 minutes. Transfer the well to PBS 3 Wash frequently. One tablet of P-nitrophenyl phosphate was added to dietaboramine buffer. (Sigma) 51d and add 100u4 to individual wells.

The plate is left in the dark for 30 minutes and read at 405 nm.

Five main points: Isolation of megakaryocyte growth factor.

Heparin-Sepharose CL-6B (P h a rrna c i a) 5 g was swollen in PBS to a final volume of 25-g, and washed lengthwise with PBSIJ2. Purified. Pooled fresh human plasma (25d) from three donors was collected at 50- to the swollen beads in a tube and mixed overnight at 4°C. plasma removed and filtered (referred to as "consumed").

The beads were then incubated at 4"C with IMDM25d containing 3"/d of heparin. Mixed for an hour. The medium was decanted and filtered (referred to as “eluate”). ).

Heparin cultures were then grown using either bone marrow or buffy coats as described in Example and 6. plasma in various forms; (1) used without treatment; (10%); (2) depleted plasma; or (3) depleted plasma + lysate. The exudate (30% final volume) was used in place of fetal bovine serum as described in Example 1. It was. A src kinase assay was performed on cultures grown for two weeks.

The total protein in the dish was measured: CPM in the SrC band was Bands were excised from and determined after scintillation counting. The result is It is as follows: 1) I (L-60600, 801% 2) FIEL 837 1.1) O13% 3)) lc, 10% 4,860 0 ．． 73 100%P 4) HC, 10% 316 0.25 19% HeSε 5) HC, 10% 3,090 0.50 93% HeSe+F *FIL-60 control line: myeloid leukemia HEL human erythroleukemia type HC heparin culture HP human plasma He S E Heparin-Sepharose column depleted plasma F Heparin -Anti-karyocyte growth factor eluted from the Sepharose column (eluate) +expressed as % of culture #3.

■ Engineering: Purification of megakaryocyte growth factor.

Human plasma protein (0.65 g) was added to buffer A, (Tris-HC in IOIIM). l, pH 8, 0, 0, 1 iM EDTA) Place on DEAE-cellophane column of nlI and add buffer A and buffer A. It was eluted with a linear gradient of 2M NaCl. The flow rate was 2M1/min. and collected both parts of 5-. Biological activity was determined during salt gradient elution. Its activity is found to be present at higher salt concentrations or below the main peak. The major portion of was present in the fraction from about 1.25-1.6M NaCl.

Both portions were assayed in 96 titer well plates with 4 replicates. can be done. Both aliquots are dialyzed against PBS. X-Vivo (Wh 5-10% and 1/- heparin to be assayed with were seeded with 5xio' cells/well and fed every 3 days. supply. Two weeks later, cells were examined for antikaryotic morphology and Megac. Stain with olor.

An effective and economical method for producing human megakaryocytes in culture has been provided. , it is clear from the above results. Therefore, a continuous source of platelets is required for treatment and platelet production. Supplied for the study of functions as well as their formation. In addition, antikaryocyte proliferation has been studied. can be done. In addition, factors associated with antikaryocyte proliferation are supplied, and other factors are It can be isolated in several ways.

All publications and patent applications mentioned herein will be fully acknowledged by those skilled in the art to which this invention pertains. This is an indication of the level of All publications and patent applications are separate publications. or as if the patent application were specifically and individually incorporated by reference. Incorporated herein by way of example.

Although the invention has been fully described, 2. Many changes and modifications are within the scope of this invention. It can be done.

Summary book Antikaryocytes and platelets promote antikaryocyte proliferation while interfering with fibroblast formation produced in culture by using conditions. In particular, human blood along with fetal bovine serum Plasma or heparin supports antikaryosphere formation in vessels lacking a fibroblast-supporting coat. It was found that Factors related to antikaryocyte proliferation are supplied.

Submission of translation of written amendment (Article 184-7, Paragraph 1 of the Patent Act) June 3, 1998 Antikaryocytes and platelets proliferation, production, and Amori 3 Patent applicant Address: California, USA 94303. Baro Alto West Bayshore Road 3,400 people Name: Systeminx, Inc. Id 5 Date of submission of written amendment March 13, 1992 (reception date) 6 List of attached documents (1) One translation of the written amendment 7 / March 2nd / 3rd edition 1.ヱD　75￥＄　? □(Took 1 Yun J-un for 14 , a factor capable of supporting antikaryocyte proliferation, present in plasma; Stable at 50°C in plasma for at least 2 weeks; binds and can be eluted by heparin-containing IMDM medium; supports antikaryotic proliferation; and A factor characterized in that antikaryocyte proliferation is reduced in the absence of said growth factor.

15. The factor according to claim 14, wherein the factor is a Hi-factor.

16, grown in liquid culture and harboring CD34 and 41, and CD14. A hematopoietic cell composition enriched for cells lacking.

17. Claim 16, wherein said cells are enriched for cells containing CD33. Cell compositions described in Section.

18. At least 50 cells grown in liquid culture and conferring antikaryotic proliferation % (wherein at least 10% of the cells are antikaryocyte precursor cells) Cellular composition of cells.

international search report 11. , l　4-6-11*　PCTE/US911071.5(+

Claims (15)

[Claims] 1. A method for producing megakaryocyte proliferation, the method comprising: human hematopoietic progenitor cells or their megakaryocyte precursors; The enriched fraction was incubated in a nutrient medium comprising human plasma under conditions supporting megakaryocyte proliferation. , and/or culturing under fibroblast proliferation inhibiting conditions. 2. Claims wherein said culture is in a container devoid of capsule-supported fibroblast proliferation. The method described in box 1. 3. 2. The method according to claim 1, wherein the medium contains heparin and fetal bovine serum. Method. 4. A claim wherein said nutrient medium comprises up to about 20% human plasma or fetal bovine serum. The method described in item 1. 5. The method of claim 1, wherein the medium comprises additional megakaryocyte growth factor. . 6. CD34+, -41+, -14- fraction enriched with the human hematopoietic precursor cells The method according to claim 1, wherein the method is a hematopoietic cell fraction. 7. Enriched structures for cells that have CD34 and 41 and lack CD14 Blood cell composition. 8. Claim 7, wherein said cells are enriched for cells having CD33. Cellular composition of. 9. At least 50% of the cells contributing to megakaryocyte proliferation (at least 5% of the cells) A cell composition of human cells comprising 0% megakaryocyte precursor cells. 10. Claim 9, wherein said cells are at least 90% megakaryocyte precursor cells. Cell compositions as described. 11. At least 50% by weight of cells other than platelets are precursors or megakaryocytes that give rise to megakaryocytes. A composition of cells in liquid medium that are karyocytes. 12. 1. A method for assaying factors that regulate megakaryocyte proliferation, the method comprising: Serum, factors of interest and IL-1, -3, -6 or erythropoietin. Both bone marrow cells or buffy coats are placed in a liquid or semi-solid nutrient medium comprising one cytokin. inoculated with white blood cells; and The number of cells growing under the same conditions in the absence of the factor of interest is approximately 5 days or longer. A method comprising determining the number of superproliferating megakaryocyte cells. 13. 1. A method for determining the level of megakaryocyte proliferation in a cell population, the method comprising: lysing the cells to produce a supernatant; other proteins in the supernatant; semi-releasing the src kinase from; and Detecting the amount of src kinase activity as a measure of the megakaryocyte population in the cell population. A method comprising: 14. A factor that can support megakaryocyte proliferation and is present in plasma; Stable at 50°C in plasma for at least 2 weeks; binds to heparin-Sepharose. combined and eluted with heparin-containing IMDM medium; supports megakaryocyte proliferation; and A factor characterized in that megakaryocyte proliferation is reduced in the absence of said growth factor. 15. 15. The factor of claim 14, wherein said factor is a human factor.