Detailed Description
The present disclosure provides a culture medium composition for differentiation from pluripotent stem cells to hematopoietic stem progenitor cells, methods of use, and applications thereof, as may be accomplished by appropriate modification of process parameters by one of skill in the art, given the benefit of this disclosure. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.
In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components. The term "a," "an," and "the") includes plural referents. The term "plurality" refers to two (species) or more. The terms "such as," "for example," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.
In this disclosure, the term "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.
In this disclosure, when a range of values is provided, it is understood that unless the context clearly indicates otherwise, each intervening value, between the upper and lower limit of that range and any other stated or intervening value, and any lower range between that stated range, is encompassed within that range.
In this disclosure, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.
In this disclosure, "one implementation," "one embodiment," "some implementations," "a particular embodiment," "related embodiments," "an embodiment," "some embodiments," "additional embodiments," or "further embodiments," "further implementations," or "another embodiment," "other embodiments," means that at least one feature or characteristic description is included in the correlation with the embodiments. Thus, the above-described phrases are not necessarily all referring to the same embodiment throughout the present disclosure. Furthermore, the particular features may be combined in any suitable manner in one or more embodiments.
In this disclosure, unless otherwise indicated, the terms "comprises" and "comprising" and variations thereof are to be understood to include the stated component, feature, element, or step, or combination of components, features, elements, or steps.
In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. The definition of common terms of molecular biology can be found in Lewis's GENES, twelfth Edition, jocelyn E, krebs, elliott S. Goldstein, stephen T. Kilpatrick, verlag Jones & Bartlett Learning. The definition of biochemical terms can be found in LEHNINGER PRINCIPLES of Biochemistry, weight Edition, david l. Nelson, michael m. Cox, publishers: w.h. Freeman. The definition of common terms of cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Press GARLAND SCIENCE. The definition of common genetic terms can be found in Genetics: analysis of Genes and Genomes, weight Edition, daniel L, hartl, MARYELLEN RUVOLO, press: jones & Bartlett Learning.
Unless otherwise specified, the experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular Biology, microbiology, cell Biology, genomics and recombinant DNA, which can be found in, for example, standard books of molecular cloning experimental guidelines (Molecular Cloning: A Laboratory Manual), cell Biology laboratory manuals (Cell Biology: A Laboratory Handbook), and the like.
Definition:
The term "pluripotent stem cell" or "PSCs" refers to a cell that has the ability to differentiate into more than one differentiated cell type under different conditions, and preferably into a cell type that has all three germ cell layer characteristics. The main feature of pluripotent stem cells is their ability to differentiate into multiple cell types, preferably into all three germ layers, using, for example, a nude mouse teratoma formation assay. Such cells include human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), human embryonic derived cells (hEDC), and adult derived stem cells. In some embodiments, the pluripotent stem cells are human embryonic stem cells, which may be autologous embryonic stem cells, or may be commercial embryonic stem cells, e.g., H1, H7, H9, H13, or H14 cell lines. The pluripotent stem cells may be genetically modified. In some embodiments, the pluripotent stem cells are not genetically modified. The genetically modified cells may include markers, such as fluorescent proteins, to facilitate their recognition. Pluripotency is also demonstrated by expression of embryonic stem cell markers.
The term "iPSC" or "induced pluripotent stem cell" is used interchangeably and refers to a pluripotent stem cell that is artificially derived (e.g., induced or by complete reversal) from a non-pluripotent stem cell (typically an adult cell), e.g., by inducing forced expression of one or more genes.
The term "hematopoietic stem progenitor cells" (Hematopoietic Stem and Progenitor Cells, HSPCs), i.e., hematopoietic Stem Cells (HSCs) and/or Hematopoietic Progenitor Cells (HPCs), is a group of multipotent stem cells that exist in bone marrow, peripheral blood, and umbilical cord blood, have self-renewal capacity and differentiate into all types of mature blood cells (e.g., erythrocytes, leukocytes, platelets) and immune cells, and are a core cell population that maintains the life-long hematopoietic function of the body. They form progenitor cells of each lineage (e.g., myeloid progenitor cells, lymphoid progenitor cells) by staged, multi-layered proliferation and differentiation, ultimately producing function-specific blood cells. Among them, hematopoietic progenitor cells are progenitor cells which proliferate and differentiate into various blood cells under the regulation of a certain microenvironment and certain factors, and are also a relatively primitive cell with proliferation capability, but have lost the multi-directional differentiation capability, and can only proliferate and differentiate to one or several blood cell lines in a targeted manner, so the hematopoietic progenitor cells are also called targeted stem cells (committed stem cell). Human hematopoietic stem cells and hematopoietic stem progenitor cells typically express the CD34 surface marker.
The term "medium" or "media" or "medium" generally refers to a cell culture medium used to maintain cells or allow cells to grow, which may include salts, amino acids, vitamins, lipids, buffers, growth factors, hormones, cytokines, trace elements, and/or carbohydrates. Examples of salts include magnesium, iron, potassium, sodium and calcium salts. Examples of amino acids include all 20 known protein amino acids such as histidine, glutamine, threonine, serine, methionine. Examples of vitamins include ascorbic acid, biotin, choline, inositol and d-benzoate, riboflavin. Examples of lipids include fatty acids, such as linoleic acid and oleic acid, as well as soy peptides and ethanolamine. Examples of buffers include Hepes. Examples of growth factors/hormones/cytokines include IGF, hydrocortisone and recombinant insulin. Examples of trace elements are Zn, mg and Se. Examples of carbohydrates include glucose, fructose, galactose and pyruvic acid. In some embodiments of the present disclosure, the provided culture medium may be liquid or solid. The solid may be in the form of powder, and may be dissolved at the time of use. In some embodiments, the provided media may be packaged as basal media that has been mixed with the media components described in this disclosure, or packaged as a combination of basal media and the media components, which are mixed in proportion at the time of use. In some embodiments, the medium may be a serum-containing medium (e.g., fetal bovine serum, calf serum, etc.), or a serum-free medium (SFM), preferably a serum-free medium. Any suitable Medium may be selected for purposes of the present disclosure, including, but not limited to, stemfit® Basic04、mTesRTM 1、StemproTM 34、STEMdiff® APEL2 Medium, for example. Typically, the medium may be stored at ambient or refrigerated conditions.
The term "mesodermal progenitor cells (Mesodermal Progenitor Cells)" refers to a population of cells capable of differentiating into various mesodermal lineages, such as vascular cells and cardiac cells. For another example, a population of mesodermal progenitor cells may be capable of differentiating into endothelial cells and/or cardiomyocytes. In another aspect of the present disclosure, there is provided a mesodermal progenitor cell or population of mesodermal progenitor cells obtained by the methods of the present disclosure. Typically, mesodermal progenitor cells express the MIXL1, TBXT and/or Eomes marker genes.
The term "lateral panel mesodermal cells (LATERAL PLATE Mesoderm Cells)" refers to a type of mesodermal cell found at the periphery of an embryo. Lateral mesoderm is a precursor tissue of the vascular lineage that can produce the mesoderm portion of the heart, blood cell system of the vascular and circulatory system. The lateral plate mesoderm is divided into two layers, the somatic lateral plate mesoderm forms the future body wall, and the visceral lateral plate mesoderm forms the circulatory system. In general, lateral mesodermal cells capable of differentiating into endothelial cells and hematopoietic cells express at least the FLI1, KDR, LMO2, SCL marker genes. (see Prummel, K.D., Nieuwenhuize, S., and Mosimann, C. (2020). The lateral plate mesoderm. Development 147. 10.1242/dev.175059.）
The term "hematopoietic endothelial cells" refers to a population of endothelial cells or precursor cells thereof having hematopoietic potential that gradually transform into hematopoietic cells under specific conditions. Hematogenic endothelial cells or hematogenic endothelial progenitor cells have both endothelial cell-related and hematopoietic cell-related molecular characteristics. Hematogenic endothelial cells typically express CD34 and CD144 surface markers, and do not express CD43 and/or CD73. Hematogenic endothelial cells express at least one of the CD34, CDH5, PECAM-1, CXCR4, DLL4, SOX17 genes.
Examples of media compositions for differentiation from pluripotent stem cells to hematopoietic stem progenitor cells in the present disclosure:
medium a comprising:
The effective final concentration of Activin A in the culture medium A can be about 10-100 ng/mL. In some embodiments, the effective final concentration of Activin A in medium A may be about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 26ng/mL, about 27ng/mL, about 28ng/mL, about 29ng/mL, about 30ng/mL, about 31ng/mL, about 32ng/mL, about 33ng/mL, about 34ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of Activin a in medium a may be about 30ng/mL.
The effective final concentration of BMP4 in medium a may be about 10-100 ng/mL. In some embodiments, the effective final concentration of BMP4 in medium a may be about 10ng/mL, about 20ng/mL, about 30ng/mL, about 31ng/mL, about 32ng/mL, about 33ng/mL, about 34ng/mL, about 35ng/mL, about 36ng/mL, about 37ng/mL, about 38ng/mL, about 39ng/mL, about 40ng/mL, about 41ng/mL, about 42ng/mL, about 43ng/mL, about 44ng/mL, about 45ng/mL, about 46ng/mL, about 47ng/mL, about 48ng/mL, about 49ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of BMP4 in medium a may be about 40ng/mL.
GSK-3 inhibitors, GSK3 inhibitors specifically inhibit GSK3 and do not substantially inhibit most other mammalian kinases. Any GSK3 inhibitor may be used in the media compositions described in the present disclosure. Illustrative examples include ,CHIR99021、BIP-135、CP21R7、1-Azakenpaullone、TDZD-8、SB216763、SB415286、BIO-acetoxime、Tideglusib、AR-A014418、 lithium chloride, VP3.15, GSK-3 inhibitor 1 or GSK-3 inhibitor 3, as well as combinations of the foregoing. In some embodiments, the GSK-3 inhibitor is CHIR99021, and its effective final concentration in medium A may be about 2-10. Mu.M. In some embodiments, the effective final concentration of CHIR99021 in medium a can be about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm. Preferably, in one embodiment, the effective final concentration of CHIR99021 in medium a may be about 6 μm.
The effective final concentration of bFGF in medium A may be about 10-100 ng/mL. In some embodiments, the effective final concentration of bFGF in Medium A can be about 10ng/mL, about 11ng/mL, about 12ng/mL, about 13ng/mL, about 14ng/mL, about 15ng/mL, about 16ng/mL, about 17ng/mL, about 18ng/mL, about 19ng/mL, about 20ng/mL, about 21ng/mL, about 22ng/mL, about 23ng/mL, about 24ng/mL, about 25ng/mL, about 26ng/mL, about 27ng/mL, about 28ng/mL, about 29ng/mL, about 30ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of bFGF in medium a may be about 20ng/mL.
Basic Medium the basic medium of medium A may be any suitable serum-free medium for cell differentiation. In some embodiments, the basal medium is SFM medium. The SFM medium can be 40-60% (v/v) Iscove Modified Du's Medium (IMDM), 40-60% (v/v) Ham's F-12K (Kaighn's) medium, 0.5-2 mg/ml polyvinyl alcohol (PVA) final concentration, 0.5-2% (v/v) N-2 additive, 0.5-2% (v/v) B-27 additive, 300-600 mu M Thioglycerol (MTG) final concentration and 0.5-2% (v/v) Glutmax.
In embodiments of the present disclosure, any component may also be added to the above-described medium a as desired, including, for example, but not limited to, buffers (e.g., hepes, sodium bicarbonate), antibiotics (e.g., green streptomycin, gentamicin), antifungal agents (e.g., amphotericin B), stabilizers, amino acids (e.g., L-glutamine, NEAA), vitamins and trace elements (e.g., vitamin B12, biotin, folic acid, se, zn, cu, fe), anticoagulants (e.g., heparin, EDTA), antioxidants (e.g., β -mercaptoethanol, vitamin C), pH indicators (e.g., phenol red), osmotic pressure modifiers (e.g., glucose, sodium chloride), cytoprotective agents (e.g., DMSO, albumin), lipid mixtures, transferrin, insulin, and the like.
Medium B, comprising:
The effective final concentration of BMP4 in medium B may be about 10-100 ng/mL. In some embodiments, the effective final concentration of BMP4 in medium B may be about 10ng/mL, about 20ng/mL, about 30ng/mL, about 31ng/mL, about 32ng/mL, about 33ng/mL, about 34ng/mL, about 35ng/mL, about 36ng/mL, about 37ng/mL, about 38ng/mL, about 39ng/mL, about 40ng/mL, about 41ng/mL, about 42ng/mL, about 43ng/mL, about 44ng/mL, about 45ng/mL, about 46ng/mL, about 47ng/mL, about 48ng/mL, about 49ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of BMP4 in medium B may be about 40ng/mL.
PI3K inhibitors any PI3K inhibitor may be used in the media compositions described in the present disclosure. Illustrative examples include GDC-0941, buparlisib, copanlisib, GSK1059615, alpelisib, enalarisen, idelalisib, duvelisib, IPI-549, dactolisib, omipalisib, taselisib, seletalisib, RP-5264, LY294002, or Wortmannin, and combinations thereof. In some embodiments, the PI3K inhibitor is GDC-0941, which may be present in the medium B at an effective final concentration of about 1-5 μm. In some embodiments, the effective final concentration of GDC-0941 in Medium B can be about 1. Mu.M, about 1.5. Mu.M, about 2. Mu.M, about 2.5. Mu.M, about 3. Mu.M, about 3.5. Mu.M, about 4. Mu.M, about 4.5. Mu.M, about 5. Mu.M. Preferably, in one embodiment, the GDC-0941 can have an effective final concentration in Medium B of about 2.5. Mu.M.
Adenylate cyclase activator any adenylate cyclase activator may be used in the medium composition of the present disclosure. Illustrative examples include Forskolin, epinephrine, norepinephrine, dobutamine, milrinone, levosimendan, corticotropin, or glucagon, and combinations of the foregoing. In some embodiments, the adenylate cyclase activator is Forskolin, which may be present in an effective final concentration of about 5 to 15 μm in medium B. In some embodiments, the effective final concentration of Forskolin in medium B may be about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, about 10 μΜ, about 11 μΜ, about 12 μΜ, about 13 μΜ, about 14 μΜ, about 15 μΜ. Preferably, in one embodiment, the effective final concentration of Forskolin in medium B may be about 10 μm.
TGF-beta inhibitors any TGF-beta inhibitor may be used in the medium compositions described in the present disclosure. Illustrative examples include ,SB431542、Galunisertib、Vactosertib、LY3200882、Fresolimumab、NIS793、AVID200、Sotatercept、Trabedersen、Decorin、BMP-7 or cintigide, as well as combinations of the foregoing molecules. In some embodiments, the TGF- β inhibitor is SB431542, which may be present at an effective final concentration in medium B of about 2-10 μm. In some embodiments, the effective final concentration of SB431542 in culture medium B can be about 2. Mu.M, about 3. Mu.M, about 4. Mu.M, about 5. Mu.M, about 6. Mu.M, about 7. Mu.M, about 8. Mu.M, about 9. Mu.M, about 10. Mu.M. Preferably, in one embodiment, the effective final concentration of SB431542 in culture medium B may be about 6. Mu.M.
The effective final concentration of VEGF in culture medium B may be about 10-200 ng/mL. In some embodiments, the effective final concentration of VEGF in culture medium B may be about 10ng/mL, about 20ng/mL, about 30ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 82ng/mL, about 84ng/mL, about 86ng/mL, about 88ng/mL, about 90ng/mL, about 92ng/mL, about 94ng/mL, about 96ng/mL, about 98ng/mL, about 100ng/mL, about 102ng/mL, about 104ng/mL, about 106ng/mL, about 108ng/mL, about 110ng/mL, about 112ng/mL, about 114ng/mL, about 116ng/mL, about 118ng/mL, about 120ng/mL, about 130ng/mL, about 140ng/mL, about 150ng/mL, about 160ng/mL, about 180ng/mL, about 200ng/mL. Preferably, in one embodiment, the effective final concentration of VEGF in culture Medium B may be about 100ng/mL.
Wnt/β -catenin inhibitors any Wnt/β -catenin inhibitor may be used in the media compositions described in the present disclosure. Illustrative examples include ,XAV939、IWR-1-endo、WIKI4、iCRT3、CWP232228、PRI-724、ICG-001、Nitazoxanide、BC2059、Dictamnine、Longdaysin、Fz7-21 or Zamaporvint, as well as combinations of the foregoing. In some embodiments, the Wnt/β -catenin inhibitor is XAV939, which may be present in medium B at an effective final concentration of about 0.5-2 μm. In some embodiments, the effective final concentration of XAV939 in medium B can be about 0.5 μΜ, about 0.6 μΜ, about 0.7 μΜ, about 0.8 μΜ, about 0.9 μΜ, about 1 μΜ, about 1.1 μΜ, about 1.2 μΜ, about 1.3 μΜ, about 1.4 μΜ, about 1.5 μΜ, about 1.6 μΜ, about 1.7 μΜ, about 1.8 μΜ, about 1.9 μΜ, about 2 μΜ. Preferably, in one embodiment, the effective final concentration of XAV939 in Medium B can be about 1. Mu.M.
The effective final concentration of AA2P in medium B may be about 50-5000 μg/mL. In some embodiments, the effective final concentration of the AA2P in medium B may be about 50 μg/mL, about 100 μg/mL, about 120 μg/mL, about 130 μg/mL, about 140 μg/mL, about 150 μg/mL, about 160 μg/mL, about 170 μg/mL, about 180 μg/mL, about 190 μg/mL, about 200 μg/mL, about 210 μg/mL, about 220 μg/mL, about 230 μg/mL, about 240 μg/mL, about 250 μg/mL, about 300 μg/mL, about 400 μg/mL, about 500 μg/mL, about 600 μg/mL, about 700 μg/mL, about 800 μg/mL, about 900 μg/mL, about 1000 μg/mL, about 2000 μg/mL, about 3000 μg/mL, about 4000 μg/mL, about 5000 μg/mL. Preferably, in one embodiment, the effective final concentration of AA2P in medium B may be about 100 μg/mL.
Basic Medium the basic medium of medium B may be any suitable serum-free medium for cell differentiation. In some embodiments, the basal medium is SFM medium. The SFM medium can be 40-60% (v/v) Iscove Modified Du's Medium (IMDM), 40-60% (v/v) Ham's F-12K (Kaighn's) medium, 0.5-2 mg/ml polyvinyl alcohol (PVA) final concentration, 0.5-2% (v/v) N-2 additive, 0.5-2% (v/v) B-27 additive, 300-600 mu M Thioglycerol (MTG) final concentration and 0.5-2% (v/v) Glutmax.
In embodiments of the present disclosure, any component may also be added to the above-described medium B as needed, including, for example, but not limited to, buffers (e.g., hepes, sodium bicarbonate), antibiotics (e.g., green streptomycin, gentamicin), antifungal agents (e.g., amphotericin B), stabilizers, amino acids (e.g., L-glutamine, NEAA), vitamins and trace elements (e.g., vitamin B12, biotin, folic acid, se, zn, cu, fe), anticoagulants (e.g., heparin, EDTA), antioxidants (e.g., β -mercaptoethanol, vitamin C), pH indicators (e.g., phenol red), osmotic pressure modifiers (e.g., glucose, sodium chloride), cytoprotective agents (e.g., DMSO, albumin), lipid mixtures, transferrin, insulin, and the like.
Medium C, comprising:
The effective final concentration of SCF in medium C may be about 10-200 ng/mL. In some embodiments, the effective final concentration of SCF in medium C may be about 10ng/mL, about 20ng/mL, about 30ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 82ng/mL, about 84ng/mL, about 86ng/mL, about 88ng/mL, about 90ng/mL, about 92ng/mL, about 94ng/mL, about 96ng/mL, about 98ng/mL, about 100ng/mL, about 102ng/mL, about 104ng/mL, about 106ng/mL, about 108ng/mL, about 110ng/mL, about 112ng/mL, about 114ng/mL, about 116ng/mL, about 118ng/mL, about 120ng/mL, about 130ng/mL, about 140ng/mL, about 150ng/mL, about 170ng/mL, about 180ng/mL, about 190 ng/mL. Preferably, in one embodiment, the effective final concentration of SCF in medium C may be about 100ng/mL.
The effective final concentration of bFGF in medium C may be about 10-100 ng/mL. In some embodiments, the effective final concentration of bFGF in Medium C can be about 10ng/mL, about 11ng/mL, about 12ng/mL, about 13ng/mL, about 14ng/mL, about 15ng/mL, about 16ng/mL, about 17ng/mL, about 18ng/mL, about 19ng/mL, about 20ng/mL, about 21ng/mL, about 22ng/mL, about 23ng/mL, about 24ng/mL, about 25ng/mL, about 26ng/mL, about 27ng/mL, about 28ng/mL, about 29ng/mL, about 30ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of bFGF in medium C may be about 20ng/mL.
The effective final concentration of TPO in medium C may be about 10 to 100ng/mL. In some embodiments, the TPO may be present in the culture medium C at an effective final concentration of about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL. Preferably, in one embodiment, the TPO may be present in the culture medium C at an effective final concentration of about 50ng/mL.
The effective final concentration of VEGF in culture medium C may be about 10-100 ng/mL. In some embodiments, the effective final concentration of VEGF in culture medium C may be about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of VEGF in culture medium C may be about 50ng/mL.
The IL-3 may be present in the medium C at an effective final concentration of about 10 to 100ng/mL. In some embodiments, the effective final concentration of IL-3 in medium C can be about 10ng/mL, about 12ng/mL, about 14ng/mL, about 16ng/mL, about 18ng/mL, about 20ng/mL, about 22ng/mL, about 25ng/mL, about 26ng/mL, about 28ng/mL, about 30ng/mL, about 32ng/mL, about 34ng/mL, about 36ng/mL, about 38ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of IL-3 in medium C may be about 25ng/mL.
The effective final concentration of AA2P in medium C may be about 10-200 μg/mL. In some embodiments, the effective final concentration of AA2P in medium C may be about 10ng/mL, about 15ng/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, about 150ng/mL, about 200ng/mL. Preferably, in one embodiment, the effective final concentration of AA2P in medium C may be about 50 μg/mL.
Basic Medium the basic medium of medium C may be any suitable serum-free medium for cell differentiation. In some embodiments, the basal medium is StemproTM medium.
In embodiments of the present disclosure, any component may also be added to the above-described medium C as needed, including, for example, but not limited to, buffers (e.g., hepes, sodium bicarbonate), antibiotics (e.g., green streptomycin, gentamicin), antifungal agents (e.g., amphotericin B), stabilizers, amino acids (e.g., L-glutamine, NEAA), vitamins and trace elements (e.g., vitamin B12, biotin, folic acid, se, zn, cu, fe), anticoagulants (e.g., heparin, EDTA), antioxidants (e.g., β -mercaptoethanol, vitamin C), pH indicators (e.g., phenol red), osmotic pressure modifiers (e.g., glucose, sodium chloride), cytoprotective agents (e.g., DMSO, albumin), lipid mixtures, transferrin, insulin, and the like.
Alternative medium D, comprising:
The effective final concentration of SCF in medium D may be about 10-200 ng/mL. In some embodiments, the effective final concentration of SCF in medium C can be about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 82ng/mL, about 84ng/mL, about 86ng/mL, about 88ng/mL, about 90ng/mL, about 92ng/mL, about 94ng/mL, about 96ng/mL, about 98ng/mL, about 100ng/mL, about 102ng/mL, about 104ng/mL, about 106ng/mL, about 108ng/mL, about 110ng/mL, about 112ng/mL, about 114ng/mL, about 116ng/mL, about 118ng/mL, about 120ng, about 130ng/mL, about 180ng/mL, about 170ng/mL, about 150 ng/mL. Preferably, in one embodiment, the effective final concentration of SCF in medium C may be about 100ng/mL.
The effective final concentration of bFGF in medium D may be about 10-100 ng/mL. In some embodiments, the effective final concentration of bFGF in medium D may be about 10ng/mL, about 11ng/mL, about 12ng/mL, about 13ng/mL, about 14ng/mL, about 15ng/mL, about 16ng/mL, about 17ng/mL, about 18ng/mL, about 19ng/mL, about 20ng/mL, about 21ng/mL, about 22ng/mL, about 23ng/mL, about 24ng/mL, about 25ng/mL, about 26ng/mL, about 27ng/mL, about 28ng/mL, about 29ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of bFGF in medium D may be about 20ng/mL.
The effective final concentration of TPO in medium D may be about 10 to 100ng/mL. In some embodiments, the TPO may be present in the culture medium D at an effective final concentration of about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL. Preferably, in one embodiment, the TPO may be present in the medium D at an effective final concentration of about 50ng/mL.
The effective final concentration of VEGF in culture medium D may be about 5-100 ng/mL. In some embodiments, the effective final concentration of VEGF in culture medium D may be about 5ng/mL, about 8ng/mL, about 10ng/mL, about 11ng/mL, about 12ng/mL, about 13ng/mL, about 14ng/mL, about 15ng/mL, about 16ng/mL, about 17ng/mL, about 18ng/mL, about 19ng/mL, about 20ng/mL, about 21ng/mL, about 22ng/mL, about 23ng/mL, about 24ng/mL, about 25ng/mL, about 26ng/mL, about 27ng/mL, about 28ng/mL, about 29ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 100ng/mL, about 95 ng/mL. Preferably, in one embodiment, the effective final concentration of VEGF in culture medium D may be about 20ng/mL.
The IL-6 may be present in the medium D at an effective final concentration of about 10 to 100ng/mL. In some embodiments, the effective final concentration of IL-6 in medium D can be about 10ng/mL, about 12ng/mL, about 14ng/mL, about 16ng/mL, about 18ng/mL, about 20ng/mL, about 22ng/mL, about 25ng/mL, about 26ng/mL, about 28ng/mL, about 30ng/mL, about 32ng/mL, about 34ng/mL, about 36ng/mL, about 38ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL. Preferably, in one embodiment, the effective final concentration of IL-6 in medium D may be about 25ng/mL.
The effective final concentration of AA2P in medium D may be about 10-200 μg/mL. In some embodiments, the effective final concentration of AA2P in medium D may be about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, about 150ng/mL, about 200ng/mL. Preferably, in one embodiment, the effective final concentration of AA2P in medium D may be about 50 μg/mL.
Basal Medium the basal medium of Medium D may be any suitable serum-free medium for cell differentiation. In some embodiments, the basal Medium is STEMdiff® APEL2 Medium.
In embodiments of the present disclosure, any component may also be added to the above-described medium D as needed, including, for example, but not limited to, buffers (e.g., hepes, sodium bicarbonate), antibiotics (e.g., green streptomycin, gentamicin), antifungal agents (e.g., amphotericin B), stabilizers, amino acids (e.g., L-glutamine, NEAA), vitamins and trace elements (e.g., vitamin B12, biotin, folic acid, se, zn, cu, fe), anticoagulants (e.g., heparin, EDTA), antioxidants (e.g., β -mercaptoethanol, vitamin C), pH indicators (e.g., phenol red), osmotic pressure modifiers (e.g., glucose, sodium chloride), cytoprotective agents (e.g., DMSO, albumin), lipid mixtures, transferrin, insulin, and the like.
The foregoing "effective amount", "effective dose" or "effective final concentration" refers to an amount effective to provide at least one desired biological result.
Examples of methods in the present disclosure:
culture of undifferentiated pluripotent stem cells:
Any suitable pluripotent stem cell may be utilized as a starting cell for the methods of the disclosure, for example, human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), human embryonic derived cells (hEDC), and adult derived stem cells as previously described. In some embodiments, the pluripotent stem cells are human embryonic stem cells, which may be autologous embryonic stem cells, or may be commercial embryonic stem cells, e.g., H1, H7, H9, H13, or H14 cell lines. Human induced pluripotent stem cells (hipscs) can be prepared using methods, techniques well known in the art.
The pluripotent stem cells described above may be cultured and expanded using any suitable method. Reference may be made to the description in ,Paulo A. Marinho, et al. Systematic optimization of human pluripotent stem cells media using Design of Experiments. Scientific Reports volume 5, Article number: 9834 (2015). /Robert T. Schinzel, et al. Efficient Culturing and Genetic Manipulation of Human Pluripotent Stem Cells. PLoS ONE 6(12): e27495. /"Human Pluripotent Stem Cells: Methods and Protocols", for example.
The pluripotent stem cells can be cultured in a culture system with or without serum. In some embodiments, a serum culture system is used as a basal medium to prepare stem cell grade serum, and growth factors such as bFGF and the like are added at the same time, and the assistance of trophoblast cells is needed. The currently common serum-free stem cell culture system is Gibco Essential8 medium and Stemcell Technologies mTesR system. Both systems are serum-free, no additional growth factors are needed, and the cell attachment can be achieved by coating the culture plates without the restriction of trophoblast cells. Thus in other embodiments, a serum-free pluripotent stem cell culture system is used. In some embodiments, the serum-free pluripotent stem cell culture system described above may also be added, for example, including, but not limited to, rock inhibitors (e.g., Y27632), actuase or EDTA (for cell dissociation), PBS (Ca2+/Mg2+) at a final concentration of 10. Mu.M. In the present disclosure, whether a pluripotent stem cell culture system with or without serum is employed, it does not affect the essential contribution of the present invention. In other embodiments, the undifferentiated pluripotent stem cells may also be cultured using 3D suspension culture methods.
Compared to the differentiation mode of traditional Embryoid Body (EB) formation, trophoblast cell co-culture or 3D micro-scaffold culture, in some embodiments of the present disclosure, a method of pluripotent stem cell monolayer culture is adopted, i.e., pluripotent stem cells are digested into single cells, and inoculated onto a solid surface for differentiation culture at a density of 500-5000 cells/cm2. The density may be, for example, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 cells/cm2. The digestion may be performed using a TrypLE digestion to form single cells. In some embodiments, the pluripotent stem cell monolayer culture may be performed on a blank solid surface, or may be performed on a solid surface previously coated with Matrigel®, recombinant Laminin (e.g., laminin-521), vitronectin (Vitronectin), or fibronectin (fibronectin). In some embodiments, the solid surface includes, but is not limited to, standard commercial tissue culture flasks or cell culture plates, such as 6-well, 24-well, 96-well, or 144-well plates. Other solid surfaces include, but are not limited to, microcarriers and discs. Solid surfaces suitable for growing undifferentiated pluripotent stem cells may be made of a variety of materials including, but not limited to, glass or plastic, such as polystyrene, polyvinylchloride, polycarbonate, polytetrafluoroethylene, mylar (melinex), thermanox, or combinations thereof. Suitable solid surfaces may also comprise one or more polymers, such as one or more acrylates. The solid surface may also be three-dimensionally shaped. In some embodiments, the culture conditions for pluripotent stem cells may be 37 ℃,5% co2, and 95% humidity.
Replacement of medium at different stages in the differentiation process:
In some embodiments, differentiation from pluripotent stem cells into hematopoietic stem progenitor cells is accomplished within about 10 days at the fastest rate, greatly reducing cycle time and improving production efficiency compared to the prior art. The differentiation process is divided into four stages.
The first stage is about day 1 or about day 2 after medium a replacement. In some embodiments, the culture endpoint can be determined based on the proportion of cells produced that express mesodermal progenitor cell-related genes (e.g., mix 1, TBXT). For example, the cell proportion expressing the mesodermal progenitor cell-related gene is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%.
The second phase is about day 1 (about day 2 after initiation of differentiation) or about day 2 (about day 3 after initiation of differentiation) after medium B replacement. In some embodiments, the culture endpoint may be determined based on the proportion of cells produced that express a gene associated with a lateral panel mesodermal cell (e.g., FLI1, KDR, LMO2, SCL). For example, the proportion of cells expressing genes associated with lateral panel mesodermal cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%.
The third stage is about day 4 (about day 6 after initiation of differentiation) or about day 5 (about day 7 after initiation of differentiation) after medium C was replaced. In some embodiments, the end of the culture can be determined based on the proportion of cells produced that express a hematopoietic endothelial cell-related gene (e.g., CD34, CDH5, PECAM-1, CXCR4, DLL4, SOX 17). For example, the proportion of cells expressing genes associated with lateral panel mesodermal cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%.
The fourth stage is about day 4 (about day 10 after initiation of differentiation) or about day 5 (about day 11 after initiation of differentiation) after medium D was replaced. The number of hematopoietic progenitor cells (CD 34+CD43+CD45+) and hematopoietic stem cells (CD 34+CD38-CD90+CD45RA-CD49f+) produced were harvested and examined.
The degree of differentiation of the cells at each of the above stages can be determined by methods known in the art, for example, by detecting the expression of a cell marker by techniques such as flow cytometry, immunofluorescence, RT-PCR, etc.
In vitro cell differentiation system in the present disclosure:
in some embodiments, the in vitro cell differentiation system comprises:
a) Pluripotent stem cells;
b) A culture system for pluripotent stem cells;
c) A plurality of medium storage means in which at least the above medium A, medium B and medium C, and optionally the above medium D are stored, and
D) A system or device for replacing said medium a, medium B, medium C or medium D.
In some embodiments, the in vitro cell differentiation system described above may be industrial automated or may be artificial. For an industrial automatic in-vitro cell differentiation system, the automatic culture and automatic liquid exchange of cells can be realized by utilizing a system or a device in the prior art. For example, US20170037357A1 discloses an automated cell culture and harvesting device, and for example CN119020163a discloses an automatic fluid change device and method.
Examples:
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Example 1:
this example uses the protocol shown in FIG. 1to induce the differentiation of multipotent stem cells hiPSC-WTC into hematopoietic stem/progenitor cells. The culture conditions were 37℃and 5% CO2.
Specifically:
(1) The preparation stage (-1~0 days) comprises inoculating hiPSC-WTC in the form of single cell into a culture container coated with extracellular matrix protein, and culturing with zeroth stage culture medium to promote hPSCs adhesion and maintain cell activity and pluripotency, thereby laying foundation for subsequent differentiation step.
(2) And the first stage (0-1 day) is to change the zeroth stage culture medium into the first stage culture medium to induce the differentiation of hPSCs into mesodermal progenitor cells.
(3) And a second stage (1-2 days) of replacing the first stage culture medium with the second stage culture medium to promote differentiation of mesodermal progenitor cells into lateral mesodermal cells, wherein the cells have hematopoietic differentiation potential.
(4) And a third stage (2-6 days), namely replacing the second stage culture medium with the third stage culture medium, and inducing the lateral plate mesoderm cells to differentiate into the blood endothelial cells, which is a key intermediate step of generating the HSPCs.
(5) And a fourth stage (6-10 days) of sterilizing the blood-producing endothelial cells obtained in the third stage into single cells, inoculating the single cells into a culture dish coated with extracellular matrix proteins, and using a fourth stage culture medium to promote differentiation into HSPCs. The resulting hematopoietic stem progenitor cells were harvested on day 10 for functional assays.
The degree of differentiation of cells at each stage can be determined by detecting the expression of cell markers by techniques such as flow cytometry, immunofluorescence, RT-PCR, etc.
Wherein, the specific formulation of the culture medium in the zeroth stage is that Rock inhibitor (such as Y27632) with the final concentration of 10 mu M is added into the commercial culture medium (such as StemfitTMBasic04、mTesRTM) of human pluripotent stem cells.
The specific formulation of the first stage medium was to add Activin A (Activin A) at a final concentration of 30ng/mL, bone morphogenetic protein 4 (BMP 4) at a final concentration of 40ng/mL, GSK-3 inhibitor CHIR99021 at a final concentration of 6. Mu.M and basic fibroblast growth factor at a final concentration of 20ng/mL to serum-free medium SFM. Wherein the serum-free medium SFM has a formulation of 50% (v/v) Iscove's Modified Du's Medium (IMDM), 50% (v/v) Ham's F-12K (Kaighn's) medium, a final concentration of 1mg/ml polyvinyl alcohol (PVA), 1% (v/v) N-2 additive, 1% (v/v) B-27 additive, a final concentration of 450. Mu.M Thioglycerol (MTG) and 1% (v/v) Glutmax.
The specific formulation of the second stage medium was that BMP4 with a final concentration of 40ng/mL, PI3K inhibitor GDC-0941 with a final concentration of 2.5 μm, adenylate cyclase activator Forskolin with a final concentration of 10 μm, TGF- β inhibitor SB431542 with a final concentration of 6 μm, vascular Endothelial Growth Factor (VEGF) with a final concentration of 100ng/mL, wnt/β -catenin inhibitor XAV939 with a final concentration of 1 μm and L-ascorbic acid 2-phosphate (AA 2P) with a final concentration of 200 μg/mL were added to serum-free medium SFM.
The specific formulation of the third stage medium was to add 100ng/mL final concentration of Stem Cell Factor (SCF), 20ng/mL final concentration of bFGF, 50ng/mL final concentration of angiopoietin (TPO), 50ng/mL final concentration of VEGF, 25ng/mL final concentration of interleukin 3 (IL 3) and 50 μg/mL of AA2P to Stempro commercial medium.
The specific formulation of the fourth stage medium was to add SCF at a final concentration of 100ng/mL, bFGF at a final concentration of 20ng/mL, TPO at a final concentration of 50ng/mL, VEGF at a final concentration of 20ng/mL, FMS-like tyrosine kinase 3 ligand (FIT 3L) at a final concentration of 100ng/mL, IL3 at a final concentration of 25ng/mL, interleukin 6 (IL 6) at a final concentration of 25ng/mL and AA2P at a final concentration of 50. Mu.g/mL to APEL2 commercial medium.
The degree of differentiation of cells at each stage can be determined by detecting the expression of cell markers by techniques such as flow cytometry, immunofluorescence, RT-PCR, etc.
Example 2:
the same procedure as in example 1 was followed, except that the human induced pluripotent stem cells hiPSC-SPA were selected as the pluripotent stem cell species.
Example 3:
the same procedure as in example 1 was followed except that the type of pluripotent stem cells was selected from human embryonic pluripotent stem cells H9.
Comparative example 1:
This comparative example uses the method reported in (Tursky et al 2020) (see Melinda L. Tursky et al. Direct Comparison of Four Hematopoietic Differentiation Methods from Human Induced Pluripotent Stem Cells. Stem Cell Reports Vol. 15 735–748 September 8, 2020）, pluripotent stem cell type induced pluripotent stem cell hiPSC-WTC was used as comparative example for example 1).
Cell samples were collected on day 1 (examples 1, 2, 3) and day 2 (comparative example 1) of differentiation, respectively, for real-time fluorescent quantitative PCR analysis. As shown in fig. 3, the mesodermal progenitor marker gene (mix 1, TBXT) expression levels of examples 1, 2, 3 exhibited significant upregulation compared to comparative example 1. Further examination of the cell samples on day 2 (examples 1, 2, 3) and day 4 (comparative example 1) revealed that the expression level of the side panel mesoderm signature genes (FLI 1, KDR, LMO2, SCL) was significantly higher in the example group than in comparative example 1 (see A, B, C, D of fig. 4). In addition, the paraxial mesoderm gene (MSGN 1, TBX 6) and ectodermal marker gene (SOX 2) expression levels of example 1 were significantly reduced as compared to comparative example 1, indicating that this protocol can significantly improve differentiation specificity.
To achieve differentiation of functional hematopoietic stem/progenitor cells, targeted induction of Hematopoietic Endothelial Cells (HECs) is critical. Based on the CD34+CD43-CD144+CD73- phenotype identification criteria, flow cytometry detection showed (see fig. 6) that the ratio of hematopoietic endothelial cells and arterial endothelial cells obtained on day 6 of example 1 was significantly improved over comparative example 1. qPCR analysis (see fig. 5) further confirmed that the endothelial-related genes (CD 34, CDH5, PECAM-1) and arterial signature genes (CXCR 4, DLL4, SOX 17) of example 1 were both significantly higher than control 1, fully verifying the advantage of this protocol in hematogenic endothelial induction.
For the 10 th day differentiated suspension cells (see fig. 2), flow analysis (see fig. 7, 8) showed that the examples successfully obtained hematopoietic progenitor cells (CD 34+CD43+CD45+) and hematopoietic stem cells (CD 34+CD38-CD90+CD45RA-CD49f+), with hematopoietic progenitor cells up to 65% -90%, significantly better than control 1.qPCR assays (see fig. 9) and bulk RNA sequencing (see fig. 11) further showed that example cells had a highly similar gene expression profile as human cord blood CD34+ hematopoietic stem/progenitor cells. Through the H4434 medium colony formation experiment (10,000 cells inoculated, 14 days of culture), the hematopoietic stem cells of example 1 not only formed larger-sized colonies, but also had significantly higher colony formation numbers than comparative example 1 (see FIG. 10), suggesting that the differentiated cells of this protocol had a greater in vitro expansion capacity.
Functional verification shows that under the induction of EPO, the cells in examples 1,2 and 3 can be differentiated into enucleated erythrocytes in 12 days, express adult hemoglobin genes (HBA and HBB), and show characteristic brick red precipitation after centrifugation, and realize 20-70 times of amplification (see FIG. 12). In addition, IL7/IL15 can be directionally differentiated into natural killer cells (NK) (see FIG. 13), and the hematopoietic stem/progenitor cells obtained by the scheme have multi-lineage differentiation potential.
In conclusion, the scheme successfully realizes the efficient directional differentiation of the pluripotent stem cells to the functional hematopoietic stem/progenitor cells.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.