WO2020218579A1 - Method for producing pluripotent stem cells conditioned for induction of differentiation - Google Patents

Abstract

The present invention provides a method for producing pluripotent stem cells that are conditioned for the induction of differentiation, the method comprising: a step (A1) of forming spherical cell masses in the individual recesses of a culture container, by subjecting pluripotent stem cells to suspension culture for 6 to 48 hours, the culture container having a nonadhesive or low-adhesive culture surface as an interior bottom surface, the culture surface having a plurality of recesses that are identical to each other and densely disposed so as to abut each other, each of the recesses having an inner wall surface that forms a funnel-shaped inclined surface and having a bottom surface that forms a recess-shaped curved surface that smoothly connects to the inner wall surface; and a step (A2) of subjecting the spherical cell masses obtained in step (A1) to flat adhesion culture. The present invention also provides a method for producing differentiated cells, the method comprising a step (B1) of subjecting the conditioned pluripotent stem cells provided by the aforementioned method to adhesion culture in a medium that induces differentiation to a predetermined differentiated cell (for example, hemogenic endothelial cells); and a step (B2) of selecting, from a cell population containing the predetermined differentiated cells and yielded by the step (B1), the differentiated cells using as an indicator a differentiation marker specific for the differentiated cells. The present invention further provides a method for producing differentiated cells (for example, hematopoietic progenitor cells) by the further differentiation of the selected differentiated cells (for example, hemogenic endothelial cells).

Description

Method for producing pluripotent stem cells acclimatized for differentiation induction

The present invention is a differentiated cell that combines a method for conditioned pluripotent stem cells for inducing differentiation, induction of differentiation by adhesion culture from pluripotent stem cells acclimated by the method, and sorting using a differentiation marker. Regarding the purification method of.

To differentiate pluripotent stem cells (PSCs) into a variety of cell types, approaches such as mimicking embryogenesis through gradual exposure to morphogens, forcibly expressing master transcription factors, or combining them. Has been taken. For example, as a method for inducing hematopoietic progenitor cells (hereinafter, also referred to as “HPC”) from human PSC, HPC is induced by introducing a transcription factor that can promote hematopoiesis in combination during the induction of blood cell differentiation by morphogen. A method of doing so has been reported (Non-Patent Document 1).

HPC induction is roughly divided into two stages: induction of hematopoietic endothelial cells (HE) from PSC and subsequent induction of HPC, and the former is further conditioned to use the stocked PSC for differentiation induction (conditioning). It is divided into a process of inducing differentiation of acclimated PSC into HE. When considering cell therapy for future patients, in order to stably obtain a sufficient number of cells, establish a culture system that can stably supply HE, which is the basis for inducing HPC differentiation, in high purity and in large quantities. Is essential.

Currently, the standard for inducing HPC differentiation from PSC is a modified method of embryoid body formation by Gordon Keller et al. (Non-Patent Documents 1 and 2), but it is difficult to develop into cell therapy due to the following points. In the situation.
(1) Stable production is difficult due to variations in the size and density of the embryoid body (EB).
(2) It is difficult to sufficiently dissociate cells during the induction of differentiation by EB formation, and it is difficult to stably obtain high-purity HE by magnetically activated cell separation (MACS).
(3) The culture conditions for inducing differentiation from HE to HPC are not optimized, and the induction efficiency is low.

Therefore, an object of the present invention is to provide a culture system for producing HPC from PSC, which can solve the above three problems and produce a large amount of HPC stably and at low cost to the extent that clinical application is possible. is there.

As a result of diligent studies to solve the above problem (1), the present inventors considered that PSC conditioning is important to improve the variation in EB size and density. Conventionally, normal colonies are picked up from the stocked PSC colonies, subcultured in an undifferentiated maintenance medium, adherently cultured, and normal colonies are selected from the grown colonies and used for EB formation / differentiation induction. It took about a week for conditioning. However, this method has a problem that the size differs between colonies and the differentiation induction varies. Therefore, the present inventors have reported that embryoid bodies of uniform size can be efficiently formed from human iPS cells, which is a microfabrication culture vessel EZSPHERE (registered trademark; AGC Technograss) (Sci. Rep. , 6: 31063 (2016), WO 2017/047735, WO 2017/164257, Nature, 545: 432-438 (2017)) were used for the earlier PSC conditioning. That is, normal colonies are once adherently cultured in an undifferentiated maintenance medium from the stocked PSC colonies, then dissociated into single cells, seeded in EZSPHERE®, and cultured for a short period (about 1 day). did. As a result, we succeeded in producing a spherical iPS cell mass with uniform size and number of cells, which maintained an undifferentiated state as a whole, at high density.

Next, in response to the above-mentioned problem (2), the present inventors subjected the obtained spherical iPS cell mass to planar adhesive culture instead of suspension culture, and the cell mass spontaneously flattened and was substantially flattened. It has a two-dimensional structure. After exchanging the medium with the HE differentiation-inducing medium and culturing, the cell mass was detached from the dish, dissected, and subjected to MACS with CD34 positive as an index. As a result, the cell mass could be dissociated efficiently, and high-purity HE was successfully obtained in a stable manner. Furthermore, it was also found that HE can be efficiently converted to HPC by the subsequent differentiation-inducing culture without purifying HE from the cell mass after planar adhesive culture.

Furthermore, the present inventors have succeeded in efficiently and stably converting HE into HPC by using a combination of a specific medium composition and extracellular matrix for the problem (3) above.

With the above ingenuity, the conventional method could efficiently induce HPC only about once every five times, whereas according to the method of the present inventors, the probability is 3 to 4 times out of 5 times. We succeeded in constructing a stable and highly reproducible differentiation induction system that achieves differentiation induction to HPC.

Among the above protocols, the adoption of short-term suspension culture by EZSPHERE (registered trademark) in the PSC conditioning process has made it possible to provide uniform and high-quality conditioned PSCs, which makes HPC stable. It was considered that it greatly contributed to the construction of a culture system capable of inducing a large amount. In the past, it was common general knowledge that PSCs were easily differentiated when they were made into a sphere (spheroid). Since the conditioning of PSC is to refresh the PSC to a state suitable for inducing differentiation while maintaining the undifferentiated state, it was a completely unexpected idea to use EZSPHERE (registered trademark) in the process. .. By setting the period of this suspension culture to a short period of 6 to 48 hours, for example, about 1 day, the present inventors have a spherical PSC mass having the same size and number of cells while maintaining the undifferentiated state of PSC. Succeeded in producing a large amount and stably.
Therefore, PSC conditioning using EZSPHERE (registered trademark) can be widely applied not only to mesoderm-series cells such as HPC, but also to induce differentiation into endoderm / ectoderm-series cells. In addition, a spherical PSC mass obtained by suspension culture using EZSPHERE (registered trademark) is subjected to planar adhesive culture, the cell mass is changed to a substantially two-dimensional structure, and then differentiation induction is performed to obtain a desired differentiated cell. Since it can be efficiently purified by a sorting technique known per se, it is possible to obtain a more stable and large amount of arbitrary differentiated cells by combining the techniques.
As a result of further research based on these findings, the present inventors have completed the present invention.

That is, the present invention provides the following.
[1] A method for producing pluripotent stem cells acclimatized for inducing differentiation.
(1) A culture vessel having a non-adhesive or low-adhesive culture surface as an inner bottom surface, in which a plurality of recesses that are the same as each other are densely arranged so as to be adjacent to each other. Pluripotent stem cells are suspended for 6 to 48 hours using the culture vessel having an inner wall surface having a funnel-shaped slope and a bottom surface having a concave curved surface smoothly connected to the inner wall surface. A method comprising culturing and forming a spherical cell mass in each recess; and (2) a step of planar adhesive culturing the spherical cell mass obtained in step (1).
[2] The method according to [1], wherein the pluripotent stem cells to be subjected to the step (1) are adherently cultured using a culture vessel coated with an extracellular matrix having a known composition.
[3] The method according to [2], wherein the extracellular matrix is laminin or a fragment thereof.
[4] The method according to any one of [1] to [3], wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
[5] The method according to any one of [1] to [4], wherein the pluripotent stem cells are of human origin.
[6] A method for producing a predetermined differentiated cell from pluripotent stem cells.
(1) A step of adhering and culturing the conditioned pluripotent stem cells obtained by the method according to any one of [1] to [5] in a medium that induces differentiation into the differentiated cells; 2) A method comprising a step of selecting the differentiated cells from a cell population containing the predetermined differentiated cells obtained in the step (1) using a differentiation marker peculiar to the differentiated cells as an index.
[7] The method according to [6], wherein the step (2) is performed by magnetically activated cell separation.
[8] The method according to [6] or [7], wherein the predetermined differentiated cell is a mesoderm cell.
[9] The method according to [8], wherein the predetermined differentiated cell is a blood cell or a progenitor cell thereof.
[10] The method according to [9], wherein the progenitor cell is a hematopoietic endothelial cell or a hematopoietic progenitor cell.
[11] A method for producing hematopoietic progenitor cells.
A method comprising (1) providing the selected hematopoietic endothelial cells obtained by the method according to [10]; and (2) inducing differentiation of the hematopoietic endothelial cells into hematopoietic progenitor cells.
[12] In the step (2), hematopoietic endothelial cells are cultured in a differentiation-inducing medium using Stemline (registered trademark) Stemline II medium as a basal medium using a culture vessel coated with fibronectin, laminin or a fragment thereof. The method according to [11], which is carried out by the above.
[13] The method according to [8], wherein the predetermined differentiated cells are skeletal muscle cells, chondrocytes, renal cells, cardiomyocytes or adipocytes, or progenitor cells thereof.
[14] The method according to [6] or [7], wherein the predetermined differentiated cell is an ectoderm or endoderm cell.
[15] The method according to [14], wherein the ectoderm cells are nervous system cells, sensory system cells or epidermal cells, or progenitor cells thereof.
[16] The method according to [14], wherein the endoblastic cells are pancreatic β cells, hepatocytes, intestinal cells, lung cells or thyroid cells, or progenitor cells thereof.

According to the PSC conditioning method of the present invention, a spherical PSC mass having a uniform size and density can be obtained in a shorter period of time (about 4 days) than before. Further, since the dissociation of the cells subjected to the differentiation induction treatment after that is facilitated, a desired differentiated cell can be produced in a large amount and stably by combining cell selection such as MACS in the middle. In particular, in the induction of HPC differentiation via HE, the efficiency of HPC induction can be dramatically improved by combining the selection of CD34-positive HE and the subsequent optimized culture conditions. Can be done.

(A) A cross-sectional view of a microfabricated culture vessel used in the PSC conditioning method of the present invention, and (B) a diagram schematically showing a state of preparation of a spherical PSC mass using the culture vessel from the production of the culture vessel. (Reprinted from Supplementary Fig. 2 of Sci. Rep., 6: 31063 (2016) with some modifications). It is a schematic diagram of the PSC conditioning method of this invention. PSC maintained in mTeSR1 medium on iMatrix-511 was exfoliated from the dish on day -4 (the start date of differentiation induction to HE was set to day 0), dissociated into TrypLE Express single cells, and then Y. It was seeded in mTeSR1 medium supplemented with -27632 and cultured overnight to form a spherical cell mass. -On the 3rd day, the spherical cell mass was seeded on iMatrix-511 in mTeSR1 medium and cultured for 3 days. On day 0, the spherical cell mass spontaneously flattened into a substantially two-dimensional structure. (A) It is a schematic diagram of the differentiation induction method of HE and HPC used in Example 1. On day 0, the medium was replaced with Essential 8 containing CHIR99021, BMP4 and VEGF 165 and cultured in a 5% O _2.5 % CO ₂ incubator. On the second day from the start of differentiation, the medium was replaced with Essential 6 containing SB431542, VEGF 165 and SCF. On day 4, CD34-positive cells were screened by MACS to enrich HE and Stemline® added on fibronectin, SCF, TPO, Flt-3L and IL-6 / IL-6Rα, ITS-X and Glutamax. Incubated in Stemline II medium for 6 days. (B) Flow cytometry plot of HE-containing cell population selected and enriched by MACS. (A) It is a typical phase difference image of hematopoietic progenitor cells 7 days after stimulation with a hematopoietic cocktail. (B) Flow cytometric plots of representative CD45 and CD34 of the entire culture 7 days after stimulation with a hematopoietic cocktail. (C) It is a typical phase difference image of granulocytes / macrophage progenitor cells generated by CFU assay from hematopoietic progenitor cells 11 days after the start of differentiation induction. (D) The colony forming units of granulocytes / macrophage progenitor cells (CFU-M, CFU-G, CFU-GM) generated by the CFU assay are shown. It is a schematic diagram of the conditioning of PSC used in Example 2 and the method of inducing differentiation of HE, HPC and various blood cells. After conditioning PSC and inducing hematopoiesis (mesoderm differentiation) in the same manner as in Example 1, differentiation induction was continued without performing cell recovery or selection of CD34-positive cells at the 4th day of differentiation. From the 4th day after the start of differentiation, culture was continued using a combination of different cytokines as shown below according to the type of target cells. -Induction of hematopoietic progenitor cells (Fig. 6A): On the 4th day after the start of differentiation, the medium was changed to Stemline® Stemline II medium supplemented with VEGF and SCF and cultured for 2 days. After 6 days from the start of differentiation, the cells were replaced with Stemline® Stemline II supplemented with SCF, TPO, Flt-3L, ITS-X and Glutamax and cultured for 4 days, and the floating cell fraction was fractionated on the 10th day after the start of differentiation. CD34 ⁺ CD45 ⁺ hematopoietic progenitor cells were obtained in the cells. -Induction of monocytes and macrophages (Fig. 6B): On the 4th day after the start of differentiation, the medium was changed to StemPro®-34 medium supplemented with VEGF and SCF and cultured for 2 days. After 6 days from the start of differentiation, the cells were replaced with StemPro®-34 medium supplemented with SCF, TPO, Flt-3L, IL-3, and M-CSF and cultured for 7-10 days. After that, the cytokines were further changed to 3 types of Flt-3L, GM-CSF, and M-CSF, and the culture was continued for 7 days to obtain CD14 ⁺ CX3CR1 ⁺ monocyte cells in the floating cell fraction. -Induction of erythroblastic / myeloid cells (Fig. 6C): Stemline (registration) in which VEGF, SCF, TPO, Flt-3L, IL-3, IL-6, and EPO were added to the medium on the 4th day after the start of differentiation. The medium was changed to Stemline II medium and cultured for 2 days. After 6 days from the start of differentiation, the cells were replaced with Stemline® Stemline II supplemented with SCF, TPO, Flt-3L, IL-3, IL-6, and EPO and cultured for 3 days. The floating cells were collected in the differentiation after 9 days, after which the culture was continued for 7 days in a medium of the same composition was transferred as it is to the new culture dish without the cell sorting, CD235 ⁺ CD33 ^- erythroid cells and CD235 ^- CD33 ⁺ Myeloid cells were obtained. -Induction of NK cells (Fig. 6D): On the 4th day after the start of differentiation, the medium was changed to Stemline® Stemline II medium supplemented with SCF and Flt-3L and cultured for 8 days. After 12 days from the start of differentiation, the cells were replaced with Stemline® Stemline II medium supplemented with SCF, Flt-3L, IL-7, and IL-15 and cultured for 36 days to obtain CD56 ⁺ CD314 ⁺ NK cells. In Example 2, (A) a flow cytometry plot showing differentiation into hematopoietic progenitor cells (CD34 positive and CD45 positive) on the 10th day from the start of differentiation, and (B) monocytes on the 21st day from the start of differentiation. Macrophages (CX3CR1 positive and CD14 positive), (C) Red blast lineage (CD235a positive and CD33 negative) 16 days after the start of differentiation, Myeloid lineage (CD235a negative and CD33 positive) and (D) 48 days after the start of differentiation It is a flow cytometry plot showing the differentiation into NK cells (CD314 positive and CD56 positive) of the eye.

(I) Method for Producing Pluripotent Stem Cells Acclimated for Inducing Differentiation The present invention relates to a method for producing pluripotent stem cells acclimatized for inducing differentiation (hereinafter referred to as "PSC conditioning method of the present invention"). I will provide a.
Here, "conditioning of PSC" means that when PSC is subjected to differentiation induction, the starting PSC is refreshed while maintaining undifferentiation. After thawing, the cryopreserved PSC stock is adherently cultured on a flat medium to wake it up, and normal colonies are picked up and used for differentiation induction. Since the efficiency is low, it is common to perform conditioning to bring the cells into a state suitable for differentiation induction, and then to perform differentiation induction. Conventionally, a dormant PSC is subcultured on a flat medium and adherently cultured in an undifferentiated maintenance medium, and when colonies that maintain undifferentiation are formed to some extent, normal colonies are selected and used for differentiation induction. Was there.

In the PSC conditioning method of the present invention, the sleeping PSC is not subcultured on a flat medium and has the same plurality of fine recesses (also referred to as “microwells”), and the surface of the microwells is non-adhesive or low. By seeding in a special finely processed culture vessel that is adhesive and suspending and culturing for a short period of time to form a spherical cell mass, the spherical cell mass is adherently cultured on a flat medium to spontaneously culture the spherical cell mass. Flatten it so that it has a substantially two-dimensional structure.
That is, the PSC conditioning method of the present invention is
(1) A culture vessel having a non-adhesive or low-adhesive culture surface as an inner bottom surface, in which a plurality of recesses that are the same as each other are densely arranged so as to be adjacent to each other. Pluripotent stem cells are suspended for 6 to 48 hours using the culture vessel having an inner wall surface having a funnel-shaped slope and a bottom surface having a concave curved surface smoothly connected to the inner wall surface. It includes a step of culturing and forming a spherical cell mass in each recess; and (2) a step of plane-adhesive culture of the spherical cell mass obtained in step (1).

(A) Preparation of PSC The PSC used in the present invention may be an undifferentiated cell having "self-renewal ability" capable of proliferating while maintaining an undifferentiated state and "differentiation pluripotency" capable of differentiating into all three embryonic stem lineages. The present invention is not particularly limited, and for example, establishment of embryonic stem (ES) cells, artificial pluripotent stem (iPS) cells, embryonic reproductive (EG) cells derived from primordial reproductive cells, and GS cells from testis tissue. Examples thereof include multipotent germline stem (mGS) cells isolated in the culture process, multipotent adult progenitor cells (MAPC) isolated from bone marrow, and MUSE cells. ES cells may be ES cells generated by nuclear reprogramming from somatic cells. ES cells or iPS cells are preferable. The pluripotent stem cell is not particularly limited as long as it is derived from a mammal, but is preferably a human-derived pluripotent stem cell.

ES cells can be established by removing the inner cell mass from the blastocyst of a fertilized mammalian egg and culturing the inner cell mass on a fibroblast feeder. In addition, cells are maintained by subculture using a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). It can be carried out. For methods of establishing and maintaining ES cells in humans and monkeys, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92: 7844-7848; Thomson JA, et. al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc . Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99: 1580-1585; Klimanskaya I, et al. (2006), Nature. 444: 481-485, etc.
For human ES cell lines, for example, WA01 (H1) and WA09 (H9) are from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan). It is available from.

iPS cells can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or proteins, with properties similar to ES cells, such as pluripotency and self-renewal proliferative potential. It is an artificial stem cell derived from somatic cells (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International release WO 2007/069666). Here, the "somatic cell" means any animal cell (preferably a mammalian cell including human) except a germline cell such as an egg, an egg mother cell, an ES cell, or a differentiation pluripotent cell, and a fetal (pup). ) Somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells, passaged cells, and established cells. Will be done. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, (3) lymphocytes, and epithelium. Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, renal cells And differentiated cells such as fat cells are exemplified.

When the obtained iPS cells are used for human regenerative medicine, somatic cells should be collected from the patient or another person with the same or substantially the same HLA type from the viewpoint of not causing rejection. Is particularly preferable. Here, the HLA type "substantially the same" means that when cells obtained by inducing differentiation from iPS cells derived from the somatic cells are transplanted into a patient by using an immunosuppressant or the like, the transplanted cells are transplanted. It means that the HLA types match to the extent that they can survive. For example, the main HLA (for example, 3 loci of HLA-A, HLA-B and HLA-DR, or 4 loci including HLA-C) may be the same.

Reprogramming factors are genes that are specifically expressed in ES cells, their gene products or non-coding RNAs, or genes that play an important role in maintaining undifferentiated ES cells, their gene products or non-coding RNAs, or It may be composed of low molecular weight compounds. Genes contained in reprogramming factors include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15. -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc. are exemplified, and these reprogramming factors may be used alone or in combination.

PSC can be acclimatized for differentiation induction by the PSC conditioning method of the present invention immediately after preparation, but can be maintained and cultured by a method known per se before being subjected to the PSC conditioning method of the present invention. As the basic medium for maintenance culture, for example, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, minimum essential medium (MEM), Eagle MEM medium, αMEM medium, Dalbeco modification Examples include Eagle's medium (DMEM), Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM / F12 medium, ham medium, RPMI 1640 medium, Fischer's medium, and a mixture of these. Not limited to. Alternatively, a commercially available medium for pluripotent stem cells (eg, primate ES cell medium, mTeSR1, StemFit AK02N, StemFit AK03N, Essential 8 and the like) may be used.

The medium can be a serum-containing medium or a serum-free medium. Preferably, a serum-free medium can be used. Serum-free medium (SFM) means a medium that does not contain any untreated or unpurified serum, and thus includes media containing purified blood-derived components or animal tissue-derived components (such as growth factors). Can be. Concentrations of serum (eg, fetal bovine serum (FBS), human serum, etc.) are 0-20%, preferably 0-5%, more preferably 0-2%, most preferably 0% (ie, serum-free). Can be. SFM may or may not contain any serum substitute. Serum substitutes include, for example, albumin (eg, albumin substitutes such as lipid-rich albumin, recombinant albumin, plant starch, dextran and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin. , Collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol or equivalents thereof may be mentioned as appropriate. Such serum substitutes can be prepared, for example, by the method described in WO 98/30679. Further, for the sake of simplicity, a commercially available product can be used. Examples of such commercially available substances include Knockout ™ Serum Replacement (KSR), Chemically-defined Lipid concentrated, and Glutamax (Invitorogen).

The medium may contain other additives known per se. For example, growth factors (eg, insulin, etc.), polyamino acids (eg, putresin, etc.), minerals (eg, sodium selenate, etc.), sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), Amino acids (eg, non-essential amino acids (NEAA), L-glutamine, etc.), reducing agents (eg, 2-mercaptoethanol, etc.), vitamins (eg, ascorbic acid, d-biotin, etc.), steroids (eg, [beta] -Estradiol, progesterone, etc.), antibiotics (eg, streptomycin, penicillin, gentamycin, etc.), buffers (eg, HEPES, etc.), nutritional additives (eg, B27 supplement, N2 supplement, StemPro®, etc.)-Nutrient Supplement, etc. ) Can be mentioned. It is preferable that each additive is contained in a concentration range known per se.

PSCs may be cultured in the presence or absence of feeder cells, but considering clinical application to humans, it is desirable that PSCs be cultured in the absence of feeder cells. Therefore, in a preferred embodiment of the present invention, PSCs are cultured under feeder-free conditions.

The culture vessel used for maintaining and culturing PSC is not particularly limited, but is limited to a flask, a tissue culture flask, a dish, a petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, and a multi. Well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles can be mentioned. The culture vessel can be cell adherent. The cell adhesion culture vessel can be coated with any cell adhesion substrate such as extracellular matrix (ECM) for the purpose of improving the adhesion of the surface of the culture vessel to cells. The cell adhesion substrate can be any substance intended for adhesion of PSCs or feeder cells (if used). Substrates for cell adhesion include laminin, collagen, gelatin, poly-L-lysine, poly-D-lysine, poly-L-ornectin, and fibronectin and their mixtures, such as matrigel, and lysed cell membrane preparations. Preparations) (Klimanskaya I et al 2005. Lancet 365: p1636-1641). These cell adhesion substrates are coated on the culture vessel at the concentrations normally used for culturing PSC, depending on their type.

In this culture, the PSC plated on the culture vessel, for example, a cell density of about 10 ⁴ to 10 ⁵ cells / cm ^2, under an atmosphere of _{1 ~ 10% CO 2/99} ~ 90% air, about in an incubator It can be cultured at 30-40 ° C, preferably about 37 ° C.

In the process of culturing PSC, the medium can be exchanged in the middle of the culturing period. The medium used for the medium exchange may be a medium having the same components as the medium before the medium exchange, or a medium having different components. Preferably, a medium having the same components is used. The time of medium replacement is not particularly limited, but is carried out, for example, every 1 day, 2 days, 3 days, 4 days, or 5 days after the start of culturing in a fresh medium.

The PSC produced (and maintained) as described above can be cryopreserved by a method known per se. For example, PSC is collected in a centrifuge tube or the like, cells are centrifuged and pelletized, a cryopreservation solution containing a cryoprotectant is added to suspend the cells, and the suspension is placed in a cryopreservation tube at -80 ° C. Examples include, but are not limited to, a method of freezing in a freezer and then storing in a gas phase or liquid phase nitrogen tank. As the cryoprotectant, for example, DMSO, glycerin, antifreeze protein, antifreeze glycoprotein and the like can be appropriately used. A commercially available cryopreservation solution (eg, STEM-CELL BANKER) can also be used.

As a method for thawing the cryopreserved PSC, a method well known in the art can be used (for example, Freshney RI, Culture of Animal cells: A Manual of Basic Technique, 4th Edition, 2000, Wiley-Liss, Inc., See Chapter 19). Preferably, it is thawed rapidly in a hot water bath at about 37 ° C. When using a highly cytotoxic cryoprotectant such as DMSO, it is desirable to dilute the DMSO concentration with an appropriate diluent immediately after thawing to the extent that it does not adversely affect the cells, and centrifuge the supernatant. It is desirable to remove toxic cryoprotectants and the like by removing them. As the diluent, for example, serum-containing or serum-free medium, physiological saline or PBS can be used.

The thawed PSC is put to sleep by adhesive culture on a flat medium. After removing the supernatant, the cell pellet is tapped and disrupted, and undifferentiated maintenance medium (eg, primate ES cell medium, mTeSR1, StemFit AK02N, StemFit AK03N, Essential 8 etc.) (ROCK inhibitor (eg, Y) if necessary. -27632) may be added) to add laminin, collagen, gelatin, poly-L-lysine, poly-D-lysine, poly-L-ornectin, and fibronectin and mixtures thereof, such as matrigel, and Seed on a culture vessel coated with a substrate for cell adhesion such as lysed cell membrane preparations, and cultured in a 5% CO2 incubator at 37 ° C. As a substrate for cell adhesion, an extracellular matrix having a known composition, for example, laminin or fibronectin, is used rather than a matrix gel or a lysed cell membrane preparation in which various factors such as TGFβ are mixed in order to maintain the undifferentiated state of PSC. And fragments thereof, more preferably laminin or a fragment thereof (for example, laminin 511 E8 fragment (eg, iMatrix-511)) and the like are desirable. Medium exchange can be performed the day after the passage and every other day thereafter. Incubate until 70-80% confluent and pick up grown colonies.

(B) Micro-processed culture container The micro-processed culture container used in the step (1) of the PSC conditioning method of the present invention is a culture container having a non-adhesive or low-adhesive culture surface as an inner bottom surface, and the culture is described above. A plurality of recesses (microwells) that are the same as each other are densely arranged on the surface so as to be adjacent to each other, and each recess has a funnel-shaped inclined inner wall surface and a concave shape that is smoothly connected to the inner wall surface. It is characterized by having a bottom surface which is a curved surface of.
Here, "identical to each other" means that when cells are seeded in each recess at the same density, the plurality of spherical cell clusters formed in each recess have a uniform size and number of cells. It means that the size (opening diameter, depth, etc.) and shape of the recesses are similar to each other, and do not have to be exactly the same. In the present invention, when the plurality of recesses are "same size to each other" and "same shape to each other", it means that they are "same" in the same meaning as described above.
"Densely arranged" means that the total opening area of the recesses occupies a larger proportion of the culture surface (that is, such as a square matrix arrangement or a finely arranged (honeycomb) arrangement). It means a state in which the recesses are arranged so as to be as close to each other as possible and adjacent to each other so that the ratio of the partition wall portion between the recesses becomes smaller).
A "funnel-shaped slope" is a slope whose opening diameter decreases from the opening toward the bottom surface, such as the inner slope of a mortar.
The "concave curved surface" is a concave curved surface such as a hemispherical concave surface or a parabola-shaped concave surface that can promote cell aggregation.

FIG. 1 (A) shows an enlarged view of a cross section of the microfabricated culture vessel used in the present invention, and FIG. 1 (B) shows the steps of the PSC conditioning method of the present invention from the production of the microfabricated culture vessel. The situation up to 1) is schematically shown. In the microprocessed culture vessel used in the present invention, a plurality of fine recesses (microwells) having the same shape are densely formed on the bottom surface of the culture vessel usually used for culturing cells by laser irradiation (eg, CO ₂ gas laser). Manufactured by arranging and providing in. Examples of the shape of the culture vessel include, but are not limited to, dishes (eg, 10 mm, 35 mm, 100 mm, etc.) and multi-well plates (eg, 6-well, 96-well, etc.). The material of the culture vessel is not particularly limited as long as a desired microwell can be provided by laser irradiation, and examples thereof include polymer materials such as plastic and polystyrene. The dimensions of the microwell that can be laser-machined are an opening diameter of about 200 to 2000 μm and a depth (length from the opening to the bottom surface) of about 100 to 900 μm. However, in the PSC conditioning method of the present invention, Since it is necessary to form a spherical cell mass while maintaining the undifferentiated state of the entire PSC mass, the diameter of the spherical PSC mass after suspension culture for 6 to 48 hours is 10 to 800 μm, preferably 20 to 500 μm. It is desirable to have an opening diameter and depth such that preferably 40 to 100 μm, for example, the average diameter of the openings is 200 to 2000 μm, preferably 200 to 1000 μm, more preferably 200 to 500 μm, still more preferably 400 to 400. 500 μm, the average depth is 100 to 900 μm, preferably 100 to 400 μm, and more preferably 100 to 200 μm.

The culture surface of the microfabricated culture container is non-adhesive or low-adhesive so that PSC does not adhere to the culture surface. Such a culture surface can be formed by subjecting a surface treatment using a cell adhesion inhibitor (protein low adhesive). Examples of the cell adhesion inhibitor include phospholipid polymers (such as 2-methacryloyloxyethyl phosphorylcholine), polyhydroxyethyl methacrylate, fluorine-containing compounds, and polyethylene glycol. In addition to exerting suppression of cell adhesion in the coat layer, the culture vessel may be molded with a resin having an effect of suppressing cell adhesion, such as a silicone resin.

It has a non-adhesive or low-adhesive culture surface, an inner wall surface in which microwells are funnel-shaped slopes, and a concave curved surface surface that is smoothly connected to the inner wall surface. As a result, the PSC seeded as a single cell drops evenly into each microwell, and adjacent cells bind to each other to form a spherical cell mass having a uniform size in a short time without adhering to the bottom surface. Can be done.
It is preferable that the inner wall surfaces of the recesses adjacent to each other are smoothly connected to each other so that there is no flat surface between the recesses adjacent to each other. This allows most of the seeded cells to fall into the wells and form spherical cell clusters without loss of cells.

The microfabricated culture vessel can be produced, for example, by the method described in WO 2017/047735. In addition, as such a microfabrication culture container, commercially available ones (eg, EZSPHERE (registered trademark) manufactured by AGC Technograss, AggreWell ^TM manufactured by StemCell Technologies, Elplasia (registered trademark) manufactured by Kuraray, etc.) are used. You can also do it.

(C) Step (1)
In the step (1) of the PSC conditioning method of the present invention, the PSC prepared as described in (a) above is seeded in the microfabricated culture vessel of (b) above, and the PSC is suspended-cultured for a short period of time. A spherical cell mass is formed in the recess (microwell).
For example, normal PSC colonies obtained by thawing and sleeping the cryopreserved PSC (the colony morphology is normal, the boundaries between the cells forming the colony are clear, etc. are observed and selected. ) Was picked up and dissociated to the single cell level using an appropriate enzyme dissociation solution (eg, Accutase, TrypLE Select, TrypLE Express, etc.), and then a ROCK inhibitor (eg, Y-27632) was not added. Suspend in differentiation maintenance medium (eg, primate ES cell medium, mTeSR1, StemFit AK02N, StemFit AK03N, Essential 8, etc.) and inoculate in a microprocessed culture vessel. The seeding density of PSC may be such that the number of cells per microwell is 100 to 1000 cells, preferably 100 to 400 cells, and more preferably 150 to 300 cells. For example, when using a 96-well plate type EZSPHERE®, since about 95 microwells are provided per well, for example, about 10,000 to about 100,000 cells per well, preferably about 10,000 to About 40,000 cells, more preferably 15,000 to 30,000 cells, can be seeded.

The culture period of PSC in the step (1) is not particularly limited as long as it is necessary and sufficient for the PSC to form a spherical cell mass in a state where the whole cell mass remains undifferentiated, but the culture period is 3 days or more. If this is the case, the size of the spherical PSC mass may increase and undifferentiated state may not be maintained, so it is desirable that the time is less than 3 days. It is preferably within 48 hours, more preferably within 36 hours. When PSC is seeded in a microprocessed culture vessel, spherical cell clusters are formed rapidly (about 3-6 hours), so the lower limit of the culture period is 3 hours, but the adhesion culture in step (2) and it In the subsequent induction of differentiation, in order to obtain differentiated cells stably and efficiently, it is preferably 6 hours or more, and more preferably 12 hours or more. Considering the shortening of the conditioning period and the subsequent efficiency of differentiation induction, a preferable range of the culture period can be, for example, 6 to 48 hours, more preferably 12 to 36 hours, and particularly preferably about 24 hours.

When the culture period is 48 hours or less, the size of the obtained spherical PSC mass varies depending on the seeding density of PSC and the size of the recess (microwell) of the microfabrication culture vessel. In order for the spherical PSC mass to maintain its overall undifferentiated state, it is desirable to adjust the diameter of the cell mass to be 10 to 800 μm, preferably 20 to 500 μm, and more preferably 40 to 100 μm. For example, as a microfabrication culture vessel, 96-well plate type EZSPHERE® with an opening diameter of 400 to 500 μM and a depth of 100 to 200 μM (in this product, about 95 recesses (microwells) for each of 96 wells). The number of PSC seeded cells per microwell is 100 to 400, preferably 150 to 300, so that a spherical PSC mass of a desired size can be obtained by culturing for 1 to 2 days. Can be prepared. In this case, when converted to the number of seeded cells per well (each of 96 wells in which microwells are arranged), about 10,000 to about 40,000 cells, preferably about 15,000 to about 30,000 cells are seeded. It will be.

(D) Step (2)
In the step (2) of the PSC conditioning method of the present invention, the spherical cell mass obtained in the step (1) is subjected to planar adhesive culture to spontaneously flatten the cell mass, and the cell has a substantially two-dimensional structure. Turn into a lump.
Spherical PSC masses are collected from microprocessed culture vessels by pipetting, etc., suspended in undifferentiated maintenance medium (eg, primate ES cell medium, mTeSR1, StemFit AK02N, StemFit AK03N, Essential 8 etc.), and laminin, collagen , Gelatin, poly-L-lysine, poly-D-lysine, poly-L-ornectin, and fibronectin and mixtures thereof, such as Matrigel, and coated with cell adhesion substrates such as lysed cell membrane preparations. Seed on the culture medium and incubate in a 5% CO2 incubator at 37 ° C. As a substrate for cell adhesion, an extracellular matrix having a known composition, such as laminin and fibronectin, is used rather than a matrix gel or a lysed cell membrane preparation containing various factors such as TGFβ in order to maintain the undifferentiated state of PSC. And fragments thereof (eg, laminin 511 E8 fragment (eg, iMatrix-511)) and the like are desirable. The seeding density is not particularly limited, but may be, for example, 1 to 10 cell clusters / cm ² , preferably 2 to 5 cell clusters / cm ² .

The culture period of the PSC mass in the step (2) is not particularly limited as long as it is sufficient for the PSC mass to spontaneously flatten and form a substantially two-dimensional structure, but for example, 2 to 4 days. It can be preferably about 3 days.

Since the PSC mass obtained in step (2) has a substantially two-dimensional structure, the cell-cell adhesion is not strong unlike EB having a spheroid structure, and it can be easily dissociated to the single cell level by enzyme treatment. .. Therefore, during or after the induction of differentiation, the cell population is subjected to enzymatic treatment, and the differentiated cells or the progenitor cells are selected using the differentiation marker specific to the target differentiated cell or its progenitor cell as an index. It is possible to obtain a highly pure differentiated cell population with less contamination other than the target cells.

(II) Method for Producing Predetermined Differentiated Cells from Pluripotent Stem Cells The present invention also provides highly purified target differentiated cells or progenitor cells thereof from the conditioned PSCs obtained by the PSC conditioning method of the present invention. (Hereinafter, also referred to as “the method for inducing differentiation of the present invention”) is provided.
That is, the method for inducing differentiation of the present invention
(1) A step of adhering and culturing the conditioned PSC obtained by the PSC conditioning method of the present invention in a medium that induces differentiation into a desired differentiated cell (or a progenitor cell thereof); and (2) From the cell population containing the differentiated cells (or their progenitor cells) obtained in (1), the differentiated cells (or their progenitor cells) are selected using the differentiation marker specific to the differentiated cells (or their progenitor cells) as an index. Includes the process of
Here, the "target differentiated cell" means the differentiated cell to be finally produced, and the "progenitor cell" means a multi-step process during differentiation of the PSC into the target differentiated cell. When a differentiation induction step is required, it means a cell that passes through during the differentiation induction. In the sense that the progenitor cells are inevitably induced in the process of obtaining the differentiated cells to be finally produced, the progenitor cells can also be said to be "target cells". Including progenitor cells, they are collectively referred to as "target differentiated cells" and "predetermined differentiated cells".

The PSC mass obtained by the PSC conditioning method of the present invention is, for example, a medium for inducing differentiation into target differentiated cells by removing the medium from the culture medium used in step (2) of the method (differentiation induction medium). ) Is added and the adhesion culture is continued as it is, so that the step (1) of the differentiation induction method of the present invention can be performed.

Examples of the basal medium for differentiation induction medium include Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, minimum essential medium (MEM), Eagle MEM medium, αMEM medium, and Dalveco modification. Examples include Eagle's medium (DMEM), Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM / F12 medium, ham medium, RPMI 1640 medium, Fischer's medium, and a mixed medium thereof. Commercially available media such as Essential 8, Essential 6, and Stemline® Stemline II can also be used.

The medium can be a serum-containing medium or a serum-free medium. Preferably, a serum-free medium can be used. Serum-free medium (SFM) may contain any serum substitute.
The medium may contain other additives known per se, such as growth factors (eg, insulin), polyamino acids (eg, putrecin), minerals (eg, sodium selenate), sugars (eg, glucose), organic. Acids (eg, pyruvate, lactic acid, etc.), amino acids (eg, non-essential amino acids (NEAA), L-glutamine, etc.), reducing agents (eg, 2-mercaptoethanol, etc.), vitamins (eg, ascorbic acid, d- Biotin, etc.), steroids (eg, [beta] -estradiol, progesterone, etc.), antibiotics (eg, streptomycin, penicillin, gentamicin, etc.), buffers (eg, HEPES, etc.), dietary supplements (eg, B27 supplement, N2) Supplement, StemPro®-Nutrient Supplement, etc.) can be contained. It is preferable that each additive is contained in a concentration range known per se.

The differentiation-inducing medium contains a differentiation-inducing factor that can induce differentiation into the desired differentiated cells. As such a differentiation-inducing factor, those known per se can be appropriately selected and used according to the type of the desired differentiated cell.

For example, mesoderm cells can be mentioned as the target differentiated cells. The cells of the mesenchymal lineage may be any cells belonging to the mesodermal lineage as long as the differentiation induction system from PSC is established, and for example, blood cells, vascular endothelial cells, skeletal muscle cells, chondrocytes. , Renal cells, myocytes, fat cells, or precursor cells thereof. Here, the "blood cell" means an arbitrary cell contained in the blood that differentiates from a hematopoietic stem cell, and is an erythrocyte, a platelet, a neutrophil, an eosinocyte, a lymphocyte, a macrophage, an NK cell, or a dendritic cell. , T cells, B cells, and their progenitor cells (eg, erythrocytes, myeloblasts, monospheres, lymphoid progenitor cells, etc.) and the like.

Alternatively, endoderm cells can be mentioned as the target differentiated cells. The endometrial lineage cell may be any cell belonging to the endometrial lineage as long as the differentiation induction system from PSC is established, and for example, pancreatic β cell, hepatocyte, intestinal cell, lung cell, thyroid. Examples thereof include cells such as cells and their precursor cells.

The mesoderm system and endoderm system use mesoderm cells as a common precursor. Therefore, differentiation into mesoderm cells is induced in the early stage of the step of inducing differentiation into mesoderm or endoderm cells. Differentiation into middle endoderm cells can be confirmed by examining the expression of middle endoderm markers such as T, Foxa2, Gsc, Mixl1 and the like.

Alternatively, ectoderm cells can be mentioned as the desired differentiated cells. The ectodermal cells may be any cells belonging to the ectodermal system as long as the differentiation induction system from PSC is established, and for example, neural cells (eg, nerve cells, glial cells, etc.), Examples include sensory cells or epidermal cells (eg, skin cells) or precursor cells thereof.

Methods for inducing differentiation of PSCs into any of the above mesodermal cells, endoderm cells or ectoderm cells are well known in the art, for example, Nakatsuji and Suemori, "Experimental Medicine Separate Volume ES. "iPS Cell Experiment Standard" (published by Yodosha, 2014) describes methods for inducing differentiation into various trigerm layer cells, and those skilled in the art can refer to these books to obtain PSC. It can induce differentiation into various trigermoid cells.

In a preferred embodiment of the present invention, the differentiated cell of interest is a hematopoietic endothelial cell (HE). To induce differentiation into HE, for example, (i) the PSC mass obtained by the PSC conditioning method of the present invention is adherently cultured in a medium containing BMP, GSK3β inhibitor and VEGF, and then (ii) VEGF, SCF and It can be carried out by adhesion culture in a medium containing a TGFβ inhibitor.

The BMP used in the above step (i) is at least one BMP selected from the group consisting of BMP2, BMP4 and BMP7, and is preferably BMP4. BMP is preferably of human origin. For example, when BMP4 is used, the concentration of BMP4 is not particularly limited, and examples thereof include 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml, and 20 ng / ml to 80 ng / ml.

Examples of GSK3β inhibitors include BIO (also known as GSK-3 {3 inhibitor Ix; 6-bromoinzylpine 3'-oxym), which is an indylpine derivative, and SB216763 (3- (2,4-), which is a maleimide derivative. Dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (4-dibromoacetophenone), which is a phenylαbromomethylketone compound , L803-mts, a cell membrane penetrating phosphoric peptide, and CHIR99021 (6- [2- [4- (2,4-Dichlorophenyl) -5- (4-methyl-1H-imid azol-2), which has high selectivity. -yl) pyrimidin-2-ylamino] ethylamino] pyridine-3-carbonitrile). These compounds are commercially available from, for example, Calbiochem, Biomol, etc., and can be easily used. Preferably, it can be CHIR99021. For example, when CHIR99021 is used, the concentration of CHIR99021 is, for example, 1nM, 1OnM, 50nM, 1OOnM, 500nM, 750nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 15μM, 20μM, 25μM. , 30 μM, 40 μM, 50 μM or these concentrations, but are not limited to these.

There are several splicing variants of VEGF (mainly 121, 165, 189, 201 amino acid lengths in humans), any of which may be used, or a combination of several, but other subs. VEGF165 can be preferably used because it transmits a stronger signal than the type. The concentration of VEGF is not particularly limited, and examples thereof include 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml, and 20 ng / ml to 80 ng / ml. VEGF is preferably of human origin.

Culturing in step (i) can be carried out in a 5% CO ₂ incubator at 37 ° C. (preferably under hypoxic conditions of 5% O ₂ ) for 2-3 days.

Examples of the concentration of VEGF used in the above step (ii) include the same concentration as in the step (i). Examples of the SCF concentration include 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml, or 20 ng / ml to 80 ng / ml. The SCF is preferably of human origin.

Examples of TGFβ inhibitors include SB431542, SB202190 (RKLindemann et al., Mal. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276. (Lilly Research Laboratories), A-83-01 (WO 2009/146408), etc. These compounds are commercially available from, for example, STEMGENT, and can be easily used. Preferably, it can be SB431542. For example, when SB431542 is used, the concentrations of SB431542 are, for example, 1nM, 1OnM, 5OnM, 1OOnM, 5OOnM, 750nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 15μM, 20μM, 25 μM, 30 μM, 40 μM, 50 μM or these concentrations, but not limited to these.

BFGF can be further added to the medium used in the above step (ii). The concentration of bFGF is not particularly limited, and examples thereof include 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml, and 20 ng / ml to 80 ng / ml. The bFGF is preferably derived from humans.

Culturing in step (ii) can be carried out at 37 ° C. in a 5% CO ₂ incubator (preferably under hypoxic conditions of 5% O ₂ ) for 2-3 days.

Induction of differentiation from PSC to HE is described in JP-A-2017-23019, WO 2017/164257, Nature, 545: 432-438 (2017), Nat. Cell Biol., 17 (5): 580-591 (2015). It can also be carried out according to the method described in.

In the step (2) of the differentiation induction method of the present invention, the differentiated cells are selected from the cell population containing the differentiated cells obtained in the step (1) using the differentiation marker peculiar to the differentiated cells as an index. As the differentiation marker, those known per se can be used depending on the target differentiated cells. For example, in the case of HE, CD34 positive can be used as an index for selection.

For example, FACS or MACS can be used as a means for selecting the desired differentiated cells from the cell population, but it is more preferable to use MACS from the viewpoint of less damage to the cells.

When the high-purity differentiated cells selected by the differentiation-inducing method of the present invention are progenitor cells of the cells of the final target, the selected differentiated cells are further subjected to a differentiation-inducing method known per se. The final target differentiated cells can be obtained in large quantities and stably. For example, when HE is induced by the differentiation induction method of the present invention as described above, a large amount of HPC can be stably produced by further subjecting the selected high-purity HE to the HPC induction method. it can.
That is, the present invention also
Provided is a method for producing HPC, which comprises (1) a step of providing a selected HE obtained by the differentiation induction method of the present invention; and (2) a step of inducing differentiation of the HE into HPC.

As a method for inducing differentiation of HPC from HE, for example, the method described in WO2017 / 164257, Nature, 545: 432-438 (2017), Nat. Cell Biol., 17 (5): 580-591 (2015). Etc., but the present invention is not limited to these, and any method known per se can be applied.

For example, the selected HEs are, for example, laminin, collagen, gelatin, poly-L-lysine, poly-D-lysine, poly-L-ornithin, and fibronectin and mixtures thereof, such as Matrigel, and lysed cell membrane preparations ( Seed on a culture container coated with a substrate for cell adhesion such as lysed cell membrane preparations), for example, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, minimum essential medium ( MEM), Eagle MEM medium, αMEM medium, Dalbeco's modified Eagle medium (DMEM), Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM / F12 medium, ham medium, RPMI 1640 medium, Fischer's medium, Alternatively, commercially available media such as Essential 8, Essential 6, Stemline (registered trademark) Stemline II, StemPro (registered trademark) -34, and a mixed medium thereof, for example, SCF, TPO, Flt-3 ligand, IL-6 / HE can be induced to differentiate into HPC by adding a hematopoietic cytokine cocktail such as IL-6Rα and culturing it. Alternatively, after culturing in the presence of VEGF and SCF for 1 to 3 days, the hematopoietic cytokine cocktail can be added and cultured.

In the method for producing HPC of the present invention, the culture in step (2) can be carried out in a 5% CO ₂ incubator at 37 ° C. (preferably under hypoxic conditions of 5% O ₂ ) for 5 to 10 days. The HPC obtained in this step can be confirmed and selected by FACS, MACS, etc. using CD34-positive and CD45-positive as indicators, and a functional HPC has been obtained by a colony forming assay known per se. You can confirm that.

The HPC obtained as described above can be further differentiated (matured) into various blood cells by a method known per se. For example, in the basal medium exemplified in the induction of differentiation from HE to HPC, for example, SCF, TPO, Flt-3 ligand, IL-6 / IL-6Rα, IL-3, IL-11, IGF-1, EPO, Factors such as VEGF, bFGF, BMP4, SHH, and angiotensin II can be added in appropriate combinations according to the target blood cells, and HPC can be cultured to differentiate into the target blood cells. The culture can be carried out in a 5% CO ₂ incubator at 37 ° C. (preferably under hypoxic conditions of 5% O ₂ ) for about 5 to 20 days, but is not limited thereto. For example, how to induce differentiation into megakaryocyte progenitor cells and platelets is described in detail in WO 2018/038242.

The various blood cells obtained as described above have the presence or absence of cell surface molecules specific to each blood cell (for example, CX3CR1 positive and CD14 positive in the case of monocytes / macrophages; CD33 in the case of erythroid cells). Negative and CD235a positive; in the case of myeloid cells, CD33 positive and CD235a negative; in the case of NK cells, CD314 positive, CD56 positive, etc.) can be used as an index for confirmation and selection by FACS or MACS.

As shown in Example 2 described later, in the differentiation induction method of the present invention, when various blood cells are induced to differentiate via HE and HPC, a cell population containing HE is transferred to HPC without selecting HE. The obtained HPC may be subjected to differentiation induction, and after confirmation and selection by FACS or MACS, for example, using CD34-positive and CD45-positive as indicators, differentiation may be induced into various blood cells, or obtained. The cell population containing HPC may be used for inducing differentiation into various blood cells without selecting the HPC. By using the PSC conditioned by the PSC conditioning method of the present invention, it is possible to relatively efficiently induce differentiation into HPC and various blood cells without selecting HE derived from the PSC.
Therefore, in another preferred embodiment of the present invention, in the method for inducing differentiation of the present invention, the differentiated cell of the present invention is a blood cell, and the differentiation into the blood cell is induced via HE and HPC. This method includes a step of selecting the cells using a differentiation marker peculiar to HPC or a target blood cell as an index.

Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited to the following Examples.

Example 1
1. Material
1-1. Cell line
409B2, 201B7 (317-9) (both iPS cell lines derived from healthy people)

1-2. Reagents Each reagent was prepared as follows.
PSC Plate Medium: Before use, mTeSR1 and 10 μM Y-27632 were mixed and stored at 4 ° C. Spheroid plate medium: mTeSR1 and 625-1250 ng / mL iMatrix-511 (depending on the cell line) were mixed. Day 0 Differentiation Medium: Essential 8 was mixed with 2 μM CHIR99021, 80 ng / mL BMP4 and 80 ng / mL VEGF 165. Day 2 Differentiation Medium: Essential 6 was mixed with 1 μM SB431542, 80 ng / mL VEGF 165, and 100 ng / mL SCF. 1 mM EDTA: 1 mL of 0.5 M EDTA was added to 500 mL of PBS (calcium / magnesium free). EHT Medium: Stemline® Stemline II and 50 ng / mL SCF, 20 ng / mL TPO, 50 ng / mL Flt-3L, 20 ng / mL IL-6 / IL-6Rα, ITS-X , Glutamax and antibiotics-antifungal agents were mixed. FACS buffer: PBS (calcium / magnesium free) mixed with 2% FCS and 1 mM EDTA.

2. PSC colony forming In a 37 ° C incubator with 5% CO ₂ , hPSCs were grown on 0.5 μg / cm ² iMatrix-511 in mTeSR1 from 70% to 80% density.

Dissociation of PSC (4 days ago)
The medium was aspirated and the culture surface was washed twice with PBS (Ca / Mg free). The cells were then treated with TrypLE Express and incubated at 37 ° C. for 15 minutes (during which the cells were not dried). The cells were suspended in mTeSR1, the suspension was transferred to a tube of appropriate size, the cells were centrifuged at 200 xg for 3 minutes, the supernatant was aspirated and the cells were suspended in PSC plating medium. The PSC suspension was plated on EZSPHERE at 48,000-70,000 cells / cm ² (depending on the cell line) and incubated overnight in a 5% CO ₂ 37 ° C. incubator. At this time, a 100 μL / well cell suspension was seeded on a 96-well EZSPHERE plate. In general, seeding 96 wells with about 20,000 cells / well yields about 100 spheroids.

PSC Spheroid Plating (3 days ago)
PSC spheroids were collected in 15 mL conical tubes by gentle pipetting with a P1000 Pipetman and allowed to settle by allowing the spheroids to stand at room temperature for 2 minutes. The supernatant was aspirated, suspended in spheroid plate medium, the suspension was dispensed to a density of 4-spheroid / cm ² , and cultured in a 37% incubator with 5% CO ₂ for 3 days.

3. Hematopoietic induction (mesoderm differentiation) (day 0)
The medium was aspirated, Essential 8 containing 2 μM CHIR99021, 80 ng / mL BMP4 and 80 ng / mL VEGF 165 was added and cultured in a 37 ° C. incubator (hypoxic incubator) with 5% O ₂ and 5% CO ₂ . After culturing in Essential 8 for 2 days, the medium was aspirated, then Essential 6 containing 1 μM SB431542, 100 ng / mL SCF and 80 ng / mL VEGF 165 was added and cultured in a hypoxic incubator.

4. Isolation of hematopoietic endothelium (day 4)
After 2 days, the medium was aspirated and the surface of the medium was washed twice with PBS (calcium / magnesium free). Cells were treated with TrypLE Express and incubated for 30 minutes in a 5% CO ₂ 37 ° C. incubator. The cells were gently suspended in 1 mM EDTA, dissociated to the single cell level, the suspension was transferred to a 50 mL conical tube and the cells were centrifuged at 200 xg for 3 minutes. The supernatant was aspirated and the cell pellet was suspended in 300 μL MACS buffer. 100 μL of CD34 microbeads were added, gently pipetted and then incubated at room temperature for 30 minutes. The suspension was filtered through a 40 μm cell strainer. CD34 ⁺ cells were isolated using an autoMACS Pro separator.

5. Induction of hematopoietic cells from hematopoietic endothelial cells (Endothelial-to-Hematopoietic Transition (EHT))
5-1. Fibronectin coating By diluting 1 mg / mL fibronectin in PBS (calcium / magnesium-free), a required amount of 5 μg / mL fibronectin coating solution was prepared. 0.5 mL of the coating solution was dispensed into each well of the 24-well culture plate. The plates were incubated at room temperature for 30 minutes.

The sorted CD34 ⁺ cells were centrifuged at 200 xg for 3 minutes. The cell pellet was resuspended in 1 mL of EHT medium. Next, the viable cell density was determined by the number of cells. The cell density was adjusted to 200,000 cells / mL by adding EHT medium. The coating solution was aspirated and discarded from the fibronectin coated wells. 0.5 mL of CD34 ⁺ cell suspension was dispensed into each fibronectin-coated well. Incubated in a hypoxic incubator.

6. Flow cytometry assay for hematopoietic cells
6-1. Recovery of floating cells The medium was transferred to a 15 mL conical tube and centrifuged at 200 × g for 3 minutes. The supernatant was aspirated.

6-2. Recovery of adherent cells The culture surface was washed twice with 0.5 mL of PBS (Ca / Mg-free). 200 μL of TrypLE Express was added, the entire surface was covered with TrypLE Express, and excess liquid was sucked. The plate was incubated in a 37 ° C. incubator for 5 minutes. The cells were resuspended in 1 mL FACS buffer.

Floating cells and adherent cells were mixed, the cells were centrifuged at 200 × g for 3 minutes, and the supernatant was aspirated. The cells were then resuspended in 50 μL FACS buffer and reacted with the anti-CD34 and anti-CD45 antibodies for 1 hour in the dark at room temperature. The cells were washed twice with PBS (Ca / Mg free) and centrifuged at 200 xg for 3 minutes. The supernatant was aspirated and resuspended in 0.5 mL FACS buffer containing 0.5 μg / mL DAPI. The expression of CD34 and CD45 was measured by LSR Fortessa.

7. Hematopoietic cell colony forming unit (CFU) assay
7-1. Recovery of hematopoietic cells The medium was transferred to a 15 mL conical tube, and the wells were gently washed twice with PBS (Ca / Mg-free).

The cells were centrifuged at 200 xg for 3 minutes and the supernatant was aspirated. The cells were resuspended in 1 mL of EHT medium and the number of cells was used to determine the number of cells. To 3 mL of MethoCult H4435 supplemented with antibiotics, a suspension corresponding to 10,000 cells was added and mixed well 5 times with a 16 G or 18 G syringe. The entire Methocult suspension was then dispensed into each well of a 6-well plate and cultured in an incubator at 5% CO _2, 37 ° C for 2 weeks while maintaining humidity. The colony is sensitive to movement, so I tried not to shake the dish. Two weeks later, colonies were counted under a microscope.

Results The schematic process of forming PSC colonies is shown in FIG. PSC spheroids were formed on EZSPHERE for 1 day. In the presence of iMatrix-511, these spheroids spontaneously flattened into almost two-dimensional colonies.

The schematic process of hematopoietic cell induction is shown in FIG. 3 (A). After growing the PSC colonies to a diameter of 750 μm, the medium was sequentially changed to induce mesoderm organoids. By sequentially changing the medium until the 4th day, the PSC colonies gradually became a sunny side-up structure during differentiation. On day 4, hematopoietic endothelial cells were purified from mesoderm organoids by magnetic selection (Fig. 3 (B)), suspended in EHT medium, and then seeded on fibronectin. By stimulating these cells with hematopoietic cytokines (SCF, TPO, Flt-3L and IL-6 / IL-6Rα) for 7 days, morphological changes from endothelial cells to hematopoietic cells occur, and hematopoietic cell colonies are observed. (Fig. 4 (A)). Flow cytometry was performed on the hematopoietic cells induced in this manner, and the expression of CD34 and CD45 was confirmed (Fig. 4 (B)). In addition, CFU assay confirmed colony formation of granulocytes / macrophage progenitor cells derived from CD34 ⁺ CD45 ⁺ hematopoietic progenitor cells (CFU-M = 13, CFU-G = 12, CFU-GM = 13.5, all of which are CD34. ⁺ CD45 ⁺ number of colonies formed from 10,000 cells) (FIGS. 4 (C) and 4 (D)).

Example 2
After performing PSC colony formation and hematopoietic induction (mesoderm differentiation) in the same manner as in Examples 2 and 3, differentiation was performed without cell recovery or selection of CD34-positive cells at the 4th day of differentiation. The induction was continued. A schematic diagram of the culture protocol of this example is shown in FIG. From the 4th day after the start of differentiation, culture was continued using a combination of different cytokines as shown below according to the type of target cells.
-Induction of hematopoietic progenitor cells (Fig. 6A): On the 4th day after the start of differentiation, the medium was changed to Stemline® Stemline II medium supplemented with VEGF and SCF and cultured for 2 days. After 6 days from the start of differentiation, the cells were replaced with Stemline® Stemline II supplemented with SCF, TPO, Flt-3L, ITS-X and Glutamax and cultured for 4 days, and the floating cell fraction was fractionated on the 10th day after the start of differentiation. CD34 ⁺ CD45 ⁺ hematopoietic progenitor cells were obtained in the cells.
-Induction of monocytes and macrophages (Fig. 6B): On the 4th day after the start of differentiation, the medium was changed to StemPro®-34 medium supplemented with VEGF and SCF and cultured for 2 days. After 6 days from the start of differentiation, the cells were replaced with StemPro®-34 medium supplemented with SCF, TPO, Flt-3L, IL-3, and M-CSF and cultured for 7-10 days. After that, the cytokines were further changed to 3 types of Flt-3L, GM-CSF, and M-CSF, and the culture was continued for 7 days to obtain CD14 ⁺ CX3CR1 ⁺ monocyte cells in the floating cell fraction.
-Induction of erythroblastic / myeloid cells (Fig. 6C): Stemline (registration) in which VEGF, SCF, TPO, Flt-3L, IL-3, IL-6, and EPO were added to the medium on the 4th day after the start of differentiation. The medium was changed to Stemline II medium and cultured for 2 days. After 6 days from the start of differentiation, the cells were replaced with Stemline® Stemline II supplemented with SCF, TPO, Flt-3L, IL-3, IL-6, and EPO and cultured for 3 days. The floating cells were collected in the differentiation after 9 days, after which the culture was continued for 7 days in a medium of the same composition was transferred as it is to the new culture dish without the cell sorting, CD235 ⁺ CD33 ^- erythroid cells and CD235 ^- CD33 ⁺ Myeloid cells were obtained.
-Induction of NK cells (Fig. 6D): On the 4th day after the start of differentiation, the medium was changed to Stemline® Stemline II medium supplemented with SCF and Flt-3L and cultured for 8 days. After 12 days from the start of differentiation, the cells were replaced with Stemline® Stemline II medium supplemented with SCF, Flt-3L, IL-7, and IL-15 and cultured for 36 days to obtain CD56 ⁺ CD314 ⁺ NK cells.

Results After hematopoietic induction (mesoderm differentiation), the cell population of mesoderm organoids containing HE is directly induced to differentiate into HPC without purifying CD34-positive cells (HE), and further differentiated into various blood cells. (Mature). As a result, it was found that the differentiation into CD34-positive and CD45-positive HPC was efficiently induced by the induction of hematopoiesis for 6 days (10 days from the start of differentiation) (Fig. 6A). Furthermore, by stimulating with the various mature cocktails mentioned above, CX3CR1-positive and CD14-positive monocytes / macrophages, CD33-negative and CD235a-positive erythroblasts, CD33-positive and CD235a-negative myeloid lines, CD314-positive and CD56-positive Differentiation into NK cells was confirmed (FIGS. 6B to D).

According to the present invention, it is possible to stably produce a large amount of arbitrary differentiated cells such as HE and HPC from PSC, and to produce a disease model and a robust drug efficacy / toxicity evaluation system for drug discovery. Extremely useful for construction and even clinical application.

This application is based on Japanese Patent Application No. 2019-086767 (Filing date: April 26, 2019) filed in Japan, the contents of which are all included in the present specification.

Claims (12)

1. A method for producing pluripotent stem cells acclimatized for inducing differentiation.
   (1) A culture vessel having a non-adhesive or low-adhesive culture surface as an inner bottom surface, and a plurality of recesses that are the same as each other are densely arranged on the culture surface so as to be adjacent to each other. Pluripotent stem cells are suspended for 6 to 48 hours using the culture vessel having an inner wall surface having a funnel-shaped slope and a bottom surface having a concave curved surface smoothly connected to the inner wall surface. A method comprising culturing and forming a spherical cell mass in each recess; and (2) a step of planar adhesive culturing the spherical cell mass obtained in step (1).
2. The method according to claim 1, wherein the pluripotent stem cells to be subjected to the step (1) are adherently cultured using a culture vessel coated with an extracellular matrix having a known composition.
3. The method according to claim 2, wherein the extracellular matrix is laminin or a fragment thereof.
4. The method according to any one of claims 1 to 3, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
5. The method according to any one of claims 1 to 4, wherein the pluripotent stem cells are derived from humans.
6. A method for producing a predetermined differentiated cell from pluripotent stem cells.
   (1) A step of adhering and culturing the conditioned pluripotent stem cells obtained by the method according to any one of claims 1 to 5 in a medium that induces differentiation into the differentiated cells; 2) A method comprising a step of selecting the differentiated cells from a cell population containing the predetermined differentiated cells obtained in the step (1) using a differentiation marker peculiar to the differentiated cells as an index.
7. The method according to claim 6, wherein the step (2) is performed by magnetically activated cell separation.
8. The method according to claim 6 or 7, wherein the predetermined differentiated cell is a mesoderm cell.
9. The method according to claim 8, wherein the predetermined differentiated cell is a blood cell or a progenitor cell thereof.
10. The method according to claim 9, wherein the progenitor cells are hematopoietic endothelial cells or hematopoietic progenitor cells.
11. A method for producing hematopoietic progenitor cells
    A method comprising (1) providing the selected hematopoietic endothelial cells obtained by the method according to claim 10; and (2) inducing differentiation of the hematopoietic endothelial cells into hematopoietic progenitor cells.
12. The step (2) is performed by culturing hematopoietic endothelial cells in a differentiation-inducing medium using Stemline (registered trademark) Stemline II medium as a basal medium using a culture vessel coated with fibronectin, laminin or a fragment thereof. The method according to claim 11.

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