JP2004510434A - Method for clonal expansion of hepatic progenitor cells - Google Patents

Abstract

A progenitor cell, such as a hepatic progenitor cell, derived from a mammalian endoderm, or a progeny thereof, or a progeny thereof, comprising culturing the mammalian progenitor cell, its progeny, or a mixture thereof on a mammalian embryo feeder cell in a medium. A method of growing the mixture is developed. The medium may be supplemented with one or more hormones and other growth agents. These hormones and other proliferating agents can include insulin, dexamethasone, transferrin, nicotinamide, serum albumin, β-mercaptoethanol, free fatty acids, glutamine, CuSO ₄ , and H ₂ SeO ₃ . The medium may also include antibodies. Importantly, the medium does not contain serum. The invention also includes a method of inducing the differentiation of progenitor cells into their adult fate, such as the differentiation of hepatic progenitor cells into hepatocytes or bile duct cells, by adding or removing epidermal growth factor, respectively. Mammalian progenitor cells can then be used in one or more of the following processes, such as identification of growth and differentiation factors, toxicity testing, drug development, antimicrobial testing, or preparation of extracorporeal organs such as artificial livers. The production method is useful.

Description

【００５１】
ＳＴＯ５ｈｌｘ上でのコロニー形成培養は、Ｅ１３の胎児肝の各画分から得られる、示された数の細胞を含む。肝コロニーの数を、３連の染色した培養物から確立した（平均±ＳＤ）。コロニー形成の効率は、培養液に接種した細胞のうち、１６日の培養後に分析したところ、コロニーを形成するようになった割合を示す。
【００５２】
６．５． Ｅ１３肝細胞および成体肝細胞の異なる培養要件
Ｅ１３肝臓から得られたソートされた肝細胞の成長要件は、決められたＳＴＯ５フィーダーおよびＨＤＭを用いて調べられた。ＥＧＦは成体肝細胞の強力な増殖因子であることが以前から知られている。したがって、ソートした肝細胞のコロニー形成に対するＥＧＦの効果を調べる。ＲＴ１Ａ^１− ＯＸ１８^ｄｕｌｌ、ＩＣＡＭ−１^＋肝細胞のコロニーサイズはＥＧＦがないと大きくなるが、成体肝細胞はＥＧＦの存在下でのみ、コロニーを生成した（図６Ｃ）。さらに、成体肝細胞に由来するコロニーの形態は、通常Ｆ型であるが、ＲＴ１Ａ^１−肝細胞はＥＧＦなしでＰ型コロニーを形成する。しかし、コロニー効率はＥＧＦがないとわずかに低下する（図６Ａ）。興味深いことに、ＥＧＦのない培養条件は、２種類のＰコロニー、Ｐ１およびＰ２を際立たせた。培養１２日目にはコロニーの大部分はＰ２型であるが、図６Ａのような典型的な形態を持たないコロニーもあるので、２つのタイプを完全に区別するのは困難である。これらの結果は、胎児肝細胞および成体肝細胞は、その増殖要件ならびにＲＴ１Ａ^１発現（図３および４）およびコロニーの形態において、本質的に異なることを示唆している。
【００５３】
増殖が最大に到達したと思われる培養３週間後に、ＲＴ１Ａ^１− 、ＯＸ１８、およびＩＣＡＭ−１の発現を評価する。図５Ｂ〜５Ｄに示すように、ＲＴ１Ａ^１の発現は誘導されず、ＯＸ１８の発現は低下している。ＩＣＡＭ−１のレベルは変化しない。さらに、単一コロニーの平均細胞数を、回収した細胞数、ラット肝細胞の割合、およびコロニー効率から計算する。見積もられる細胞数は３〜４ ｘ １０^３（表２）に達する。これは、コロニーを形成する単一細胞が、この培養条件では平均で約１１〜１２回分裂したことを示す。
【００５４】
【表２】単一肝コロニーにおける細胞数の計算

【００５５】
図４ＣのＲ４からソートした細胞は、６０ ｍｍまたは１００ ｍｍのディッシュでＳＴＯ５ｈｌｘフィーダー細胞上で培養した。示された期間培養した後、全ての細胞を回収し、総細胞数を計算した。ラット細胞の割合は、ラットＩＣＡＭ−１およびマウスＣＤ９８の発現に基づく、フローサイトメトリー分析による。コロニー効率は、培養に接種された細胞のうち、コロニーを形成したものの割合を示す。３連の染色した培養（平均）からのデータは、平行して行なった実験から得られた。
【００５６】
単一コロニー中の平均細胞数＝（回収した細胞の数 ｘ ラット細胞のパーセンテージ／１００）／接種した細胞の数 ｘ コロニー効率／１００）
【００５７】
６．６ ＲＴ１Ａ^１−肝前駆細胞が両能性である証拠
ラット胎齢Ｅ１３において、肝細胞は成熟した肝実質細胞および胆管上皮細胞を生み出す両能性前駆細胞だと考えられる。しかし、本発明以前では、この２つの運命が単一の細胞から生ずるのかどうかを示す直接の証拠がなかった。ＲＴ１Ａ^１− ＯＸ１８^ｄｕｌｌ ＩＣＡＭ−１^＋の胎児肝細胞がこの培養系で胆管細胞系に分化できるかどうかを知るために、胆管上皮細胞に特異的なマーカーとして抗ＣＫ１９でコロニーの染色を行なう。ＣＫ１９は、胎児肝で１５．５日以降に胆管上皮前駆細胞で発現され、この時点で細胞のアルブミン発現が消失する。ソートされたＲＴ１Ａ^１− ＩＣＡＭ−１＋細胞をＥＧＦの存在下または非存在下で培養し、その運命は培養５日目にＣＫ１９およびアルブミンの発現によってモニターする。最初から５日後、ＥＧＦ処理した培養物ではＣＫ１９^＋のコロニーがほとんどないが、ＥＧＦのない培養物ではＣＫ１９^＋細胞を含むコロニーがいくつか見られる。ＣＫ１９の強度はかなり弱いが、ＣＫ１９^＋細胞ではアルブミン発現が低下している。培養１０日目には、ＣＫ１９のみまたはアルブミンのみを発現するコロニーや、両方が陽性のコロニーが存在する。単一コロニーの中でのＣＫ１９^＋およびアルブミン^＋細胞のパターンは相反している。両方陽性のコロニーおよびＣＫ１９のみ陽性のコロニーの数は、やはりＥＧＦ非存在下のほうが高い（図７Ａ）。ＥＧＦ存在下では、１０日目には多くのコロニーがアルブミン^＋細胞のみからなる（図７Ｂ）。最終的には、両方陽性のコロニーの数は、１５日目にＥＧＦ非存在下では１００％近くになる（図７Ａ）。全体では、ＥＧＦは培養を通してＣＫ１９^＋のコロニーの出現を劇的に抑制する（図７Ｂ）。これらの結果は、Ｅ１３胎児肝から得たＲＴ１Ａ^１−、ＯＸ１８^ｄｕｌｌ、およびＩＣＡＭ−１^＋細胞が、胆管細胞系に分化でき、その運命はインビトロではＥＧＦに影響されることを示唆する（図８）。
【００５８】
６．７ 肝幹細胞および肝前駆細胞のクローン増殖を支持する能力のあるフィーダー細胞の単離およびクローニングのためのプロトコール
ブタ、ビーグル、ウサギ、マウス、またはサルの新鮮な胚組織または凍結組織（例えば、肝臓、肺、腎臓、筋肉、腸）を、カルシウムを含まないリン酸緩衝生理食塩水（ＰＢＳ）中で細かく切り刻む。ＰＢＳで２回すすいだ後、０．２５％トリプシンと３７℃で１０分間、または室温で６０分間電磁攪拌機を用いて撹拌しながら、細胞懸濁液をインキュベートする。残った細胞の塊は、懸濁液をメッシュで濾過して除去する。その後、細胞を血清（例えば、１０％胎児ウシ血清）および任意の種々の増殖補給剤（例えば、２ ｍＭグルタミン、ピルビン酸ナトリウム、およびＭＥＭ非必須アミノ酸）を添加した基礎培地（例えば、イーグルＭＥＭ）を用いて組織培養ディッシュで培養する。プラスチックの基底および血清添加培地は、多くの場合中胚葉由来（例えば、間質細胞）である支持細胞（「フィーダー細胞」）の候補である細胞群の増殖を可能にし、かつ別の種類の細胞（例えば、前駆細胞）の生存、増殖、および／または機能を支持する因子を提供する、一般的な条件である。フィーダー細胞は、コンフルエントまたはそれに近くなったら、０．０５％トリプシンを用いて継代培養する。継代培養を数回行なったら、増殖した細胞を凍結ストックとして調製し、使用まで凍結保存する。フィーダー細胞の別の供給源は、市販の初代培養フィーダー細胞またはフィーダー細胞株である。いずれの場合も、適当なフィーダー細胞を同定するためには、以下の基準が必要である。
【００５９】
フィーダー細胞は以下を支持する：
１） 古典的ＭＨＣクラスＩ抗原陰性、ＩＣＡＭ−１陽性、および／または非古典的ＭＨＣクラスＩ抗原弱陽性という表現型マーカーを持つ肝前駆細胞のクローン増殖；
２） 古典的ＭＨＣクラスＩ抗原陰性、ＩＣＡＭ−１陽性、非古典的ＭＨＣクラスＩ抗原弱陽性、αフェトプロテイン陽性、アルブミン陽性、またはＣＫ１９陽性という表現型マーカーを持つ子孫を持つ前駆細胞のクローン増殖；または
３） 両能性肝前駆細胞の定義に必要な、肝細胞系および胆管細胞系の両方への誘導可能な分化。
【００６０】
当技術分野では、古典的ＭＨＣクラスＩ抗原は、ＭＨＣクラスＩａ抗原としても知られる。非古典的ＭＨＣクラスＩ抗原は、ＭＨＣクラスＩｂ抗原としても知られる。ＭＨＣ抗原は、異なる種では異なる呼び方をされる：例えば、ラットではＲＴ１，マウスではＨ−２、およびヒトではＨＬＡである。
【００６１】
上記のアッセイ法は以下に説明される：
【００６２】
肝前駆細胞のクローン増殖の条件
肝前駆細胞は、増殖を停止した、すなわち増殖を予防するように処理されたフィーダー細胞上に、９．６ ｃｍ^２あたり５００細胞を接種する。フィーダー細胞は、マイトマイシンＣまたは放射線照射（細胞の種類によって３０００〜５０００ラド）で処理して、増殖を停止する。増殖停止したフィーダー細胞および前駆細胞には、無血清ＨＤＭを供給する。例えば、齧歯類細胞のＨＤＭは、ダルベッコ変法イーグル培地とハムＦ１２の１：１混合物に１０ ｎｇ／ｍｌ ＥＧＦ、 ５μｇ／ｍｌインスリン、１０^−６Ｍデキサメタゾン、１０μｇ／ｍｌ鉄飽和トランスフェリン、４．４ ｘ １０^−３Ｍニコチンアミド、０．２％ウシ血清アルブミン、５ ｘ １０^−５Ｍ ２−メルカプトエタノール、７．６ μｅｑ／ｌ遊離脂肪酸混合物、２ ｘ １０^−３Ｍグルタミン、１ ｘ １０^−６Ｍ ＣｕＳＯ_４、３ ｘ １０^−８Ｍ Ｈ_２ＳｅＯ_３および抗生物質を添加したものである。培養液は、２日ごとに培地を交換しながら１０〜１４日間インキュベートする。その後、αフェトプロテイン、アルブミン、および／またはＣＫ１９の２重免疫蛍光染色を行なって子孫の運命を同定する。αフェトプロテインおよびアルブミンの発現について、約１００のコロニーが分析される。さらに、コロニーの形態、Ｐ型かＦ型かも適切な子孫の同定に役に立つ場合がある。
【００６３】
フィーダー細胞と肝前駆細胞の理想的な組み合せは、同一の種から得られたものである。好ましくは、フィーダー細胞は肝前駆細胞と同一の組織および同一の種から得られる。しかし、１つの種からのフィーダーと別の種からの前駆細胞を混合することも可能である。例えば、齧歯類のフィーダーでさえも、ヒト肝前駆細胞に使用できる。可溶性および不溶性の因子（種および／または組織特異的な可能性がある）は、肝幹細胞または肝前駆細胞のクローン増殖を助ける。このような因子の供給源には、次のようなものがある：
１） 最適な種および組織のフィーダー細胞を培養した条件培地。フィーダー細胞は間質細胞のみならず、任意の種類の細胞で良い。
２） 重要な因子が既知の場合には、肝前駆細胞に活性を示す最適なフィーダー細胞に由来する適切な分子（シグナル）を合成するために、それぞれ転写または翻訳のためのｃＤＮＡまたはｍＲＮＡを任意の細胞に導入することによって生物活性のあるフィーダー細胞群を作製できる供給源。
３）肝前駆細胞に活性な最適フィーダー細胞から得られるシグナルが、タンパク質、ペプチド、炭化水素、脂質、糖ペプチド、糖タンパク質、リポタンパク質、糖脂質、またはその組み合せであるかに関わらず、重要な因子が既知の場合には、そのようなシグナルを培地に添加することによってフィーダー細胞を完全に置き換えることもできる供給源。
【００６４】
上述の実施例は、例示のためのみに説明されており、本発明の範囲または態様を制限するものではない。上記に特に説明されていない他の態様も、当業者には明らかであると思われる。しかし、そのような他の態様も、本発明の範囲および精神に含まれると考えられる。したがって、本発明は添付の特許請求の範囲によってのみ、正しく限定される。
【００６５】
本明細書で引用された全ての特許および出版物は、その全体が参照として本明細書に組み入れられる。
【図面の簡単な説明】
【図１】１５日目の胎児ラットの肝臓から得た肝細胞株の特徴付けである。
【図２】線維芽細胞フィーダー細胞上のコロニー形成アッセイ法である。
【図３】成体肝細胞中の様々な細胞株上のラット細胞表面抗原の発現である。
【図４】１３日目の胎児ラットの肝臓の表現型分析を現す。
【図５】ＥＧＦ存在下および非存在下での肝臓コロニーの特徴付けである。
【図６】ＲＴ１Ａ^１−肝細胞上のＣＫ１９発現の誘導を表す。
【図７】ＳＴＯ５フィーダー細胞上の肝臓コロニー形成の模式図である。[0001]
1. Field of the invention
The present invention relates to novel conditions for clonal expansion of mammalian hepatic progenitor cells, including pluripotent cells, stem cells, and other early hepatic progenitor cells. In particular, the invention relates to a method for growing hepatic progenitor cells using feeder cells in a synthetic medium and co-culture. The invention also relates to cells used as feeders and capable of supporting the growth of hepatic progenitor cells.
[0002]
2. Description of related technology
Identification of pluripotent progenitor cells in mammalian tissues is of clinical and commercial importance, as well as for understanding developmental processes and tissue homeostasis. Progenitor cells are ideal targets for gene therapy, cell transplantation, and tissue engineering of bioartificial organs (Millar, AD 1992 Nature 357, 455; Langer, R. and Vacanti, JP 1993). Science 260, 920; Gage, FH 1998 Nature 392, 18).
[0003]
The presence of tissue-specific "fate" stem cells or progenitor cells with high proliferative and / or pluripotent properties was confirmed by hematopoietic stem cells (Spangrde, GJ, et al., 1998 Science 241, 58), neural stem cells (Davis, A.A. and Temple, S. 1994 Nature 372, 263; Temple, DL and Anderson, DJ. 1992 Cell 71, 973), and epithelial stem cells (Jones, PH and Watt, FM 1993 Cell 73, 713). These progenitor cells are thought to be responsible for normal hematopoietic, neuronal, or epithelial tissue homeostasis, and for the regenerative response after severe injury (Hall, PA and Watt, FM). 1989 Development 106, 619).
[0004]
The adult mammalian liver has a very high capacity to recover following severe hepatotoxic injury or partial hepatectomy, despite the normally slow turnover and quiescent tissue (Fishback, F. et al. C. 1929 Arch. Pathol. 7, 955); (Higgins, GM and Anderson, RM 1931 Arch. Pathol. 12, 186). Recent test data in mice have shown that adult parenchymal cells have an almost endless proliferation potential when examined in a series of transplantation experiments (Overturf et al., 1997 Am. J. Pathol. 151). (Rhim, JA, et al., 1994 Science 263, 1149). Because these experiments utilize heterogeneous populations of stem cells, the observed growth potential was derived from adult parenchymal cells, a subpopulation of adult parenchymal cells, and / or The ability to prove that it is from an immature phase (ie, a progenitor cell) is limited. Furthermore, experiments have shown no evidence of biliary epithelial differentiation because the host used had a deficiency in the albumin urokinase transgene or tyrosine degrading enzyme; neither of these hosts had the potential to select a hepatocyte lineage. Has characteristics. Therefore, this assay could not test a population of bipotent cells.
[0005]
Several histological studies have established that fetal early hepatocytes in the second trimester have a bipotential ability to differentiate into biliary epithelium and mature stem cells (Shiojiri, N. 1997 Microscopy Res. Tech.). 39, 328-35). Liver development begins in the ventral foregut endoderm immediately after the endoderm epithelium interacts with the cardiogenic mesoderm (Douarin, NM 1975 Medical Biol. 53, 427); (Houssaint, E., et al. 1980 Cell Differ. 9, 269). This link to the liver occurs at 8 days of fetal age (E) in mice. The first stages of liver development are revealed by induction of serum albumin and alpha-fetoprotein mRNA in the endoderm before morphological changes (Gualdi, R et al., 1996 Genes Dev. 10, 1670). At 9.5 days of mouse embryonic age, certain cells proliferate and invade the mesenchyme of the transverse septum in the form of threads, forming the liver primordium. Thereafter, the mass of the liver increases dramatically, but the increase in mass is mainly due to hematopoietic cells, which colonize the fetal liver at E10 in mice (Houssaint, E. 1981 Cell Differ. 10, 243). ), Causing hepatocytes to take an extremely distorted and irregular shape (Luzzatto, AC 1981 Cell Tissue Res. 215, 133). Interestingly, recent data using gene-targeting mutant mice indicate that deletion of several genes caused fatal liver failure, parenchymal apoptosis, and / or necrosis between E12 and E15 (Gunes, C. et al., 1998 EMBO J. 17, 2846), (Hilberg, F. et al., 1993 Nature 365, 1791), (Motoyama, J. et al., 1997 Mech. Dev. 66, 27), (Schmidt, C et al., 1995 Nature 373, 699). Stress activation cascade (Ganiatsas, S. et al., 1998, Proc. Natl. Acad. Sci. USA 95, 6881), (Nishina, H. et al., 1999 Development 126, 505) or anti-apoptotic cascade (Beg, A. et al.). , 1995 Nature 376, 167), (Li, Q. et al., 1999 Science 284, 321), (Tanaka, M. et al., 1999 Immunity 10, 421) disruption of a gene that is inactivated. Despite its widespread expression, it severely impairs hepatic development but does not impair hematopoiesis. It is not clear whether hepatocytes are inherently sensitive to the stimulation of developmental stress or whether certain microenvironments within the fetal liver itself cause such destructive effects (Doi, TS et al., 1999 Proc. Natl. Acad. Sci. USA 96, 2994). On the other hand, the basic architecture of the adult liver depends on the appearance of the first cast of the bile duct epithelium surrounding the portal vein (Shiojiri, N. 1997 Microscopy Res. Tech. 39, 328). Immunohistologically, the first sign of differentiation of intrahepatic bile duct epithelial cells is the expression of bile-specific cytokeratin (CK). The CK protein, the cytoplasmic intermediate filament (IF) protein of epithelial cells, is encoded by a multigene family and is specifically expressed in tissue and differentiation (Moll, R et al., 1982 Cell 31, 11, 11). Since adult hepatocytes do not express CK19 at all, but adult bile duct epithelial cells do, CK19 is one of the most eye-catching bile markers. Only CK8 and CK18 are expressed from early hepatocytes to adult hepatocytes (Moll, R et al., 1982 Cell 31, 11, 11). At E15.5 in rat expression, which corresponds to mouse E14, bile duct progenitors are strongly stained by both CK18 and CK8 antibodies, and some bile duct progenitors express CK19. As development progresses, the mature bile duct slowly begins to express CK7 in addition to CK19 and loses ALB expression (Shiojiri, N et al., 1991 Cancer Res. 51, 2611). Although the early hepatocytes of rat E13 are considered to be a homogeneous population, whether all early hepatocytes can differentiate into the bile duct epithelial cell lineage and how such a fate is. It is not yet known whether it will be decided. No definitive lineage marking tests, such as those using retroviral vectors, have been performed on hepatocytes, and the clonal culture conditions required to establish bipotent hepatic progenitor cells have not been determined.
[0006]
One major obstacle to clonal proliferation assays is the explosive proliferation of hematopoietic cells, which makes observable ex vivo proliferation of hepatocytes unobservable. Therefore, it is necessary to use a method for enriching hepatocytes. Surface markers that require fractionation of hematopoietic cells in fetal liver have been investigated in detail (Dzierzak, E. et al., 1998 Immunol. Today 19, 228), but studies of markers for hepatic progenitor cells are in the early stages. Has not yet been determined (Sigal, S et al., 1994 Hepatology 19, 999). Furthermore, ex vivo growth conditions commonly used for adult hepatocytes result in differentiation with loss of tissue-specific functions such as ALB expression (Block, GD et al., 1996 J. Cell Biol. 132, 1133). The slightly improved ability to synthesize tissue-specific mRNA and the ability to repair tissue-specific genes completely after translation are defined as serum-free, defined by hormones, growth factors, and / or some extracellular matrix components. Is found only in hepatocytes maintained using the mixture described (Jefferson, DM, et al., 1984. Mol. Cell. Biol. 4, 1929; Enat R et al., 1984 Proc. Natl. Acad. Sci. , 81, 1411). However, proliferating fetal hepatocytes maintain expression of such serum proteins in vivo. What is not clear in the art is how to maintain and grow hepatic progenitor cells in vitro. There is an unmet need for identification of conditions that maintain ex vivo expansion of hepatic progenitor cells. Similarly, there is an unmet need for an in vitro colony formation assay (CFA) method to determine the clonal expansion potential of hepatic progenitor cells isolated from fresh liver tissue; One cell is defined as the ability to generate a population of daughter cells of a clone derived from the inoculated cells. Clonal growth consisting of clumps of cells that are inoculated at high cell density and grow in close proximity in liver culture has also been described by others (Block, GD, et al., J. Cell Biol. 1996, 132, 1133). However, the colonies of cells described in these previous studies cannot be subcultured and are, by definition, not clones and have limited utility.
[0007]
Attempts have also been made to grow hepatocytes in vitro. U.S. Patent No. 5,510,524 to Naughton et al. Claims that culturing hepatocytes relies on a three-dimensional framework of biocompatible but non-living material. There is an unmet need for culture conditions that do not have an artificial framework and allow hepatic progenitor cells to grow and culture. Also, cloned hepatic progenitor cells that have the potential for generating both bile duct cells and hepatocytes, and are particularly suitable for use as a component of an artificial liver, for testing hepatotoxins, and for drug development Has unmet demand.
[0008]
US Pat. No. 5,559,022 to Norton et al. Claims hepatic reserve cells that bind to eosin Y, a dye used to characterize "reserve cells". No established markers are used for cell identification, nor is a method for clonal expansion of spare cells nor a marker used to isolate viable liver spare cells. Methods that disclose methods of isolating and culturing cells having many characteristics essential for hepatic progenitor cells, including the expression of at least one specific marker and the ability to differentiate into either hepatocytes or bile duct cells Have unsatisfied demand. There is also an unmet need for methods of clonal expansion of hepatic progenitor cells. Clonal expansion is essential as a clear and precise method of identifying and identifying pluripotent hepatic progenitor cells.
[0009]
U.S. Pat. No. 5,405,772 to Ponting claims a medium for cell growth. In U.S. Patent No. 5,405,772, 3-30 g / ml cholesterol, 5-30 g / ml nucleoside, and 2-100 g / cm.²Type IV collagen or 0.5-100 μg / cm²Any of the fibronectins is required. This requires a medium that is specific and optimized for the growth of hepatic progenitor cells.
[0010]
U.S. Pat. No. 4,914,032 to Kuri-Haruch et al. Claims a process for culturing hepatocytes. Contrary to the present invention, U.S. Pat. No. 4,914,032 does not disclose any conditions for culturing hepatic progenitor cells or clonal expansion of hepatocytes. Similarly, U.S. Pat. No. 5,030,105 to Kri Herkuch et al. Claims a method of evaluating a drug by treating a hepatocyte cell culture. There is an unmet need for clonal expansion conditions for using defined populations of cells for testing, and methods for culturing hepatic progenitor cells.
[0011]
Norton et al., US Patent No. 5,858,721, claims transfection of stromal cells. However, U.S. Pat. No. 5,858,721 is limited in that it requires a framework of biocompatible non-living materials. In contrast, there is an unmet need in the present invention for growth conditions that do not require synthetic meshwork.
[0012]
The present inventors have recognized that it is inappropriate to culture mature hepatocytes, such as hepatocytes, rather than from much more useful hepatic progenitor cells. The inventors have carefully determined the isolation parameters of hepatic progenitor cells and the requirements for clonal expansion. There are many uses for progenitor cells and methods for selecting and culturing progenitor cells, including the evaluation of drugs and toxicants in patients with liver failure, and the evaluation of drugs.
[0013]
U.S. Patent Nos. 5,576,207 and 5,789,246 to Reid et al. Disclose the need for feeder and hormone-supplemented synthetic media. These prior studies have used embryonic liver stromal cells in combination with a defined extracellular matrix basal and serum-free hormone-supplemented medium to provide conditions for hepatic progenitor cell growth. However, the synthetic medium used was more complex than that used in the present invention; the cells were inoculated on purified matrix basal (type IV collagen and laminin), but here the feeder (matrix) was used. And germ stromal cells were prepared as primary cultures from embryonic liver and were not established as a cell line. By using embryonic stromal cell lines, feeder cells provide a much simpler, practical, and reproducible means of supporting cells. Furthermore, it is reasonable to assume that the STO feeder is not limited to hepatic progenitor cells but can also be used for progenitor cells obtained from multiple types of tissues. In prior patents, hepatic progenitor cell cultures were inoculated at high cell densities and their growth was observed as colony formation, which means that clumps, rather than cell clones, were induced to grow.
[0014]
3. Summary of the Invention
The present invention relates to a method of growing a progenitor cell, its progeny, or a mixture thereof. In particular, the invention relates to a method of expanding endoderm-derived progenitor cells, progeny thereof, or mixtures thereof. Cells are obtained from endoderm tissue. The endoderm-derived progenitor cells, their progeny, or a mixture thereof are then cultured in a medium on a layer containing feeder cells. The precursor cell, its progeny, or a mixture thereof can be a vertebrate cell. The progenitor cells, their progeny, or a mixture thereof, express an ICAM or ICAM-1 positive and classical MHC class I antigen negative phenotype. Classical MHC class I antigens are also called MHC class Ia antigens.
[0015]
The present invention also relates to a serum-free, hormone-supplemented synthetic medium and a method for culturing hepatic stem cells and other progenitor cells using feeder cells. The invention also relates to a method of culturing progeny of progenitor cells, or a combination of progenitor cells and progeny of progenitor cells. Preferably, the progenitor cells are liver progenitor cells. Similarly, the invention relates to a method of cloning hepatic pluripotent progenitor cells using specific culture conditions. Preferably, the invention relates to a method for cloning pluripotent progenitor cells of the liver. Hepatic pluripotent progenitor cells can be derived from any invertebrate or vertebrate species, more preferably mammals. More preferably, the pluripotent progenitor cells of the liver are derived from humans, primates, pigs, dogs, rats, rabbits, or mice. Most preferably, the pluripotent progenitor cells are derived from a human. The present invention discloses the specific culture conditions required for the ex vivo expansion of hepatic progenitor cells and their progeny. The invention also discloses the use of embryonic feeder cells, such as STO mouse embryonic cells, as hepatic progenitor feeder cells. Feeder cells are used in combination with the novel, serum-free, hormone-supplemented synthetic medium (HDM) disclosed in the present invention. By this combination, various rat fetal liver cell lines could be established from E15 rat liver without cells undergoing malignant transformation.
[0016]
Furthermore, the present invention relates to a method for cloning feeder cells capable of supporting the growth of hepatic progenitor cells and their progeny.
[0017]
The invention also relates to certain cell lines that support the growth of hepatic progenitor cells when used as feeders.
[0018]
The present invention also relates to a method for cloning hepatic progenitor cells. The present invention discloses the use of a stem cell line and an HDM-STO co-culture system for the development of an in vitro colony formation assay (CFA) method for determining the clonal expansion potential of newly isolated hepatic progenitor cells. Combining CFA with cells purified by a specific antigen profile reveals bipotent hepatic progenitor cells. For example, highly proliferative progenitor cells obtained from E13 rat liver corresponding to mouse E11.5 are derived from classical MHC class I (RT1A¹)⁻, OX18 (Pan MHC Class I)^dull, And intracellular adhesion molecule 1 (ICAM-1)⁺With the same phenotype.
[0019]
The present invention also relates to a medium that can support clonal hepatocyte proliferation. The medium is characterized by some specific hormones and nutrients and does not contain serum.
[0020]
Furthermore, the present invention relates to the culture of hepatic progenitor cells in a medium containing a biosynthetic product of feeder cells.
[0021]
Further, the present invention relates to a method for inducing hepatocyte differentiation, including the phenotype of hepatocytes and bile duct cells. The present invention discloses that epidermal growth factor (EGF) affects both the growth of progenitor cell colonies and the fate of either hepatocytes or biliary epithelial cells.
[0022]
5. Detailed description of preferred embodiments
The present invention is a process of stem cell proliferation and utilization. A variety of tissues are suitable sources of progenitor cells, including tissues of ectoderm, mesoderm, and endoderm origin. Ectodermal tissue includes skin tissue, brain tissue, and other neural tissue. Mesodermal tissue includes muscle, blood, and the hematopoietic system. Endoderm tissue includes the gut, stomach, pancreas, thyroid, and glands associated with the digestive system. In particular, the invention is a process of proliferating hepatic stem cells and other hepatic progenitor cells. The process involves exposing the isolated population of hepatic stem cells and / or hepatic progenitor cells and / or progeny thereof to growth conditions capable of supporting clonal expansion, ie, growth at very low cell densities. Including. In a preferred embodiment, this process involves the use of serum-free, hormone-supplemented, synthetic media to support the growth of hepatic progenitor cells on the feeder cell layer. Feeder cells have multiple functions, including providing nutrients, providing an adherent surface, and secreting certain growth factors and extracellular matrices into the medium necessary for hepatic progenitor cell survival, growth and / or differentiation. . In another preferred embodiment, the process includes selecting cells capable of supporting the growth of hepatic stem cells and hepatic progenitor cells. The feeder cells may be from reptiles, birds, crustaceans, fish, annelids, mollusks, nematodes, insects, or mammals, preferably humans. Preferably, the feeder cells are derived from embryonic tissue. Also preferably, the feeder cells are derived from embryonic tissue. Also preferably, the feeder cells are derived from embryonic liver tissue. Further, the feeder cells may be genetically modified. In a further preferred embodiment, this process involves cloning feeder cells that optimally support the stem cells.
[0023]
Any method for isolating hepatic stem cells and hepatic progenitor cells can be used, such as affinity-based interactions, such as affinity panning, immunosurgery in combination with complement, flow cytometry, centrifugal elutriation, differential centrifugation, and the like. Isolated hepatic stem cells and progenitor cells have some or all of the phenotypic markers (classical MHC class I⁻, ICAM-1⁺, OX18^dull, Α-fetoprotein⁺Or albumin⁺) With the ability to express In another aspect of the invention, the hepatic progenitor cells develop a proliferation pattern characterized by the formation of cells stacked as clumps, colonies or clusters.
[0024]
In a preferred embodiment of the present invention, the stem cells are selectively grown in serum free hormone-supplemented synthetic medium (HDM).
[0025]
The composition of HDM includes EGF up to about 40 ng / ml, insulin up to about 5-10 μg / ml,^-6 M dexamethasone or other glucocorticoid hormone, up to about 10 μg / ml iron-saturated transferrin, about 5 × 10^-2 Nicotinamide up to M, bovine serum albumin up to about 2%, about 5 x 10^-4 M 2-mercaptoethanol or equivalent reducing agent, free fatty acids up to about 8 μeq / l, about 2 × 10^-2 Glutamine up to M, about 1 x 10^-6 CuSO up to M₄, About 3 x 10^-8 H up to M₂SeO₃And nutrient media, including, but not limited to, a mixture of Dulbecco's modified Eagle's medium and Ham F12, optionally with the addition of antibiotics. Antibiotics include penicillin, streptomycin, gentamicin, and other common antibiotics in the art, and combinations thereof. Those skilled in the art will understand that instead of DMEM / F12, after minimal testing, other nutrient media can also be used, such as HamDB-10, Medium199, or the MCDB series including MCDB151 and MCDB302. I think that the. The worst conditions for cell culture are to use feeders without hormones; the most important of the above hormonal requirements are glucocorticoids, insulin, transferrin, and EGF, which are It constitutes a hormonal mitogen. Other hormonal factors can also be added and may have a secondary proliferative effect, but do not replace the important requirements described above. Similarly, alterations of the hormonal composition that can be made by those skilled in the art are within the scope of the invention.
[0026]
Preferred ranges include 10-50 ng / ml EGF, 2-10 μg / ml insulin, 5 × 10^-7 M-5 x 10^-6 M dexamethasone (9a-fluoro-16a-methyl-prednisolone), 5-20 μg / ml iron-saturated transferrin, 2-8 × 10^-3 M nicotinamide, 0.05-0.5% serum albumin, 2-8 x 10^-5 M 2-mercaptoethanol, 5-10 μeq free fatty acid mixture, 1-3 x 10^-3 M-glutamine, 0.5-2 x 10^-6 M CuSO₄, 1-5 x 10^-8 MH₂SeO₃, 1-5 μM palmitic acid, 0.1-0.4 μM palmitoleic acid, 0.5-1.2 μM stearic acid, 0.5-2 μM oleic acid, 1-5 μM linoleic acid, and 0.2-0.8 μM linolenic Contains acids.
[0027]
The serum-free synthetic medium with hormones of the present invention is suitable for clonal expansion of hepatocytes. The HDM may include any of a number of options, such as Dulbecco's Modified Eagle Medium (DME), Ham F12, RPMI 1640, Williams E Medium, and the like. A preferred embodiment is a 1: 1 mixture of Dulbecco's modified Eagle's medium and Ham F12 (DMEM / F12, such as GIBCO / BRL, Grand Island, NY). The basal medium contains a preferred concentration of 10 ng / ml of epidermal growth factor EGF (eg, from Collaborative Biomedical Products), a preferred concentration of 5 μg / ml of insulin (eg, Sigma),^-6M dexamethasone (eg Sigma), 10 μg / ml iron-saturated transferrin (Sigma), 4.4 × 10^-3M nicotinamide (eg, Sigma), 0.2% bovine serum albumin (eg, Sigma), 5 × 10^-5M 2-mercaptoethanol (eg Sigma), 7.6 μeq / l free fatty acid mixture (2.4 μM palmitic acid, 0.21 μM palmitoleic acid, 0.88 μM stearic acid, 1 μM oleic acid, 2.7 μM linoleic acid, and 0 .43 μM linolenic acid), 2 × 10^-3M glutamine (eg, GIBCO / BRL), 1 × 10^-6M CuSO₄, 3 x 10^-8MH₂SeO₃And add antibiotics. Growth factors secreted from feeder cells include, but are not limited to, insulin-like growth factor (IGF), interleukin (IL) -6 family, hepatocyte growth factor (HGF), and fibroblast growth factor (FGF). Can be added to the medium to enhance the effect of the feeder, and addition alone or in various combinations does not replace the effect of the feeder. This means that feeder cells also produce other signals required alone or in combination with these growth factors.
[0028]
In another aspect of the invention, the hepatic progenitor cells are expanded from a single progenitor cell, ie, the cells are cloned. Growing cells in a colony is not necessarily the same as clonal expansion, which is defined implicitly and explicitly, by growing cells derived from a single cell. Any of a number of cloning methods known in the art are suitable, including a method of diluting progenitor cells to one cell or less per well of a culture plate, referred to as limiting dilution. Similarly, progenitor cells can be cloned using cloning rings, selective exfoliation, dilution culture on microparticles, single cell sorting using flow cytometry, selection of individual cells using micropipette or optical tweezers, and agar. .
[0029]
In another aspect of the invention, many of the cloned progenitor cells are capable of mitosis. Preferably, the progenitor cells are capable of at least one mitosis, more preferably at least ten.
[0030]
In yet another aspect of the invention, the hepatic progenitor cells and their progeny are grown in media supplemented with feeder cell metabolism and biosynthesis products. The supplement may be in the form of a conditioned medium, a medium in which live feeder cells have been previously incubated. Preferably, the addition can be in the form of isolating from the conditioned medium of the feeder cells factors including proteins, peptides, lipids, hydrocarbons, and metabolic regulators that maintain and increase the growth of hepatic progenitor cells and their progeny. Proteins can include soluble and insoluble components of the extracellular matrix, and growth factors, including epidermal growth factor and insulin-like growth factor.
[0031]
More preferably, hepatocytes are selectively grown in culture using a layer of feeder cells, which are embryonic or adult cells or other suitable cells. In one aspect, the feeder cells are stromal cells or fibroblasts. Fibroblasts or other suitable cells may be genetically modified, for example, by transfection. Preferably, the fibroblasts or other suitable cells are human, non-human primate, porcine, dog, rabbit, rat, or mouse mesodermal cells, but also other mammalian and avian mesodermal cells are suitable. ing. In addition, fibroblasts can be cloned and selected for their ability to support hepatic progenitor cells.
[0032]
In a preferred embodiment of the invention, the isolated hepatic progenitor cells are committed to a hepatic parenchymal or cholangiocyte cell line by selective use or lack of epidermal growth factor (EGF) or other differentiation signals.
[0033]
In a further preferred embodiment of the present invention, the isolated stem cells and other hepatic progenitor cells can be used as components of an artificial liver that can be used as an extracorporeal liver assist device. In a further preferred embodiment of the invention, the artificial liver comprising the isolated hepatic progenitor cells and their progeny is used to support the life of a patient suffering from hepatic dysfunction or failure.
[0034]
6. Example
The following examples illustrate the invention, but the invention is not limited to these specific examples. Those skilled in the art will obtain the means for practicing the invention from these examples. Those skilled in the art will recognize many alternative embodiments that fall within the scope of the invention.
[0035]
6.1 Preparation and analysis of hepatic stem cells and hepatic progenitor cells
Rat
Pregnant Fisher 334 rats are obtained from the Charles River Breeding Laboratory (Wilmington, MA). For timed pregnancies, the animals are brought together in the afternoon and the morning when the plug is observed is taken as day 0. Male Fischer 334 rats (200-250 g) are used for adult hepatocytes.
[0036]
Establishment of stem cell line
Fetal liver of 15 days of age is prepared. Single cell suspensions are incubated with the liver at 37 ° C. with 0.05% trypsin and 0.5 mM EDTA or 10 units / ml thermolysin (Sigma, St. Louis, Mo.) and 100 units / ml DNase I (Sigma). Is obtained. Cells are overlaid on Ficoll-Park (Pharmacia Biotech, Uppsala, Sweden) and subjected to density gradient centrifugation at 450 g for 15 minutes. For th1120-3 and rter6 or Rhel4321, the fraction of cells in the bottom, 17 mg / ml IV collagen respectively (Collaborative Biomedical Products, Bedford, MA) or 12 [mu] g / ml laminin (Collaborative Biomedical Products) tissue culture dishes coated with Inoculate. Serum-free hormone-supplemented medium HDM was prepared by adding 20 ng / ml EGF (Collaborative Biomedical Products) to a 1: 1 mixture of Dulbecco's modified Eagle medium and Ham F12 (DMEM / F12, GIBCO / BRL, Grand Island, NY). 5 μg / ml insulin (Sigma), 10^-7M dexamethasone (Sigma), 10 μg / ml iron-saturated transferrin (Sigma), 4.4 × 10^-3M nicotinamide (Sigma), 0.2% bovine serum albumin (Sigma), 5 x 10^-5M 2-mercaptoethanol (Sigma), 7.6 μeq / l free fatty acid, 2 × 10^-3M glutamine (GIBCO / BRL), 1 x 10^-6M CuSO₄, 3 x 10^-8MH₂SeO₃And antibiotics added. Each concentration described is the final concentration in the medium. After 4 weeks of culture, trypsinized cells are cultured on the feeder layer of STO mouse embryo fibroblast cell line (American Type Culture Collection, Rockville MD) treated with mitomycin C. Th1120-3, rter6 and rhel4321 are cloned from three independent preparations of fetal hepatocytes and maintained on STO feeder cells using HDM. After cell line establishment, the concentration of EGF is reduced to 10 ng / ml for all cultures.
[0037]
Cell adhesion assay
Cell adhesion to fibronectin (Collaborative Biomedical Products), laminin, and type IV collagen is assessed using 96-well microtiter plates (Corning, Cambridge, Mass.) Coated with these proteins at 0.3-10 μg / ml. . After removing STO cells by Percoll (Pharmacia Biotech) density gradient centrifugation at 200 g for 15 minutes, 3 × 10 5 in each well.⁴The cell hepatocyte lines th1120-3, rter6, and rhel4321 are cultured for 10 hours using HDM. After rinsing twice to remove floating cells, a fresh medium containing the tetrazolium salt WST-1 (Boehringer Mannheim, Indianapolis, Ind.) Is added and the number of viable adherent cells is determined. After 4 hours, the absorbance is determined according to the manufacturer's protocol.
[0038]
STO substrain
100 parental STO cells obtained from the ATCC are 10% heat inactivated fetal bovine serum, 2 x 10^-3M-glutamine, 5 x 10^-5Cultivate for 7 days in a 100 mm culture dish in DMEM / F12 supplemented with M2-mercaptoethanol and antibiotics. Depending on cell morphology and growth rate, four subclones are selected for further analysis. CFA of rter6 is performed on four subclones, one of which, STO6, does not adhere to the culture plate after mitomycin C treatment. One subclone STO5 is transfected with pEF-Hlx-MC1neo or pEF-MC1neo provided by Dr. Adams (Dr. JM Adams, The Walter and Eliza Hall Institute of Medical Research). Plasmids linearized at the Nde I site are introduced into cells by DOPER liposome transfection reagent (Boehringer Mannheim). After G418 selection, six clones are isolated. Each of the three clones is analyzed by CFA.
[0039]
Colony immunohistochemical staining
Culture plates were fixed with methanol-acetone (1: 1) at room temperature for 2 minutes, rinsed, blocked at 4 ° C. with Hanks Balanced Salt Solution (HBSS) containing 20% goat serum (GIBCO / BRL). I do. For dual immunohistochemistry of alpha fetoprotein and albumin, plates were treated with anti-rat albumin antibody (ICN Biomedicals, Costa Mesa, Calif.) followed by Texas Red-conjugated anti-rabbit IgG (Vector laboratories, Burlingame, Calif.), and FITC binding. Incubate with anti-rat α-fetoprotein polyclonal antibody (Nordic Immunology, Tilburg, Netherlands). For double labeling of albumin and CK19, an anti-CK19 monoclonal antibody (Amersham, Buckinghamshire, England) and a FITC-conjugated anti-mouse IgG (Caltag, Burlingame, CA) are used instead of the anti-α-fetoprotein antibody.
[0040]
Fetal liver dissociation at E13
Fetal liver was 10 mM HEPES, 0.8 mM MgSO₄Dissect into Ca ++-free ice-cold HBSS supplemented with 1 mM EGTA (pH 7.4). Liver was 10 mM HEPES, 0.8 mM MgSO₄, And 1 mM CaCl₂Mill with 0.2% type IV collagenase in HBSS (Sigma) and 16.5 units / ml thermolysin (Sigma) prepared in. After 10 minutes incubation at 37 ° C., the cell suspension is digested with 0.025% trypsin and 2.5 mM EDTA (Sigma) for 10 minutes. Thereafter, trypsin is inhibited by adding 1 mg / ml trypsin inhibitor (Sigma). Finally, the cells are treated with 200 units / ml deoxyribonuclease I (Sigma). In all experiments, 3-5 x 10 per liver⁵Cells are obtained.
[0041]
Isolation of adult hepatocytes
To isolate hepatocytes, a two-stage liver perfusion method is used. After perfusion, the cells are centrifuged twice at 50 g for 1 minute to concentrate large parenchymal cells. Viability is> 90% as measured by trypan blue exclusion.
[0042]
Flow cytometry analysis
Cells are analyzed on a FACScan (Becton-Dickinson, Mountain View, CA) and sorted using a FlowFlow Cytometer (Cytomation, Fort Collins, CO). Cell suspensions obtained from E13 fetal liver are incubated on ice with HBSS containing 20% goat serum (GIBCO / BRL) and 1% teleost gelatin (Sigma) to prevent non-specific antibody binding. . After rinsing, cells were FITC-conjugated anti-rat RT1A^{a, b, l}Suspend with antibody B5 (Pharmingen, San Diego, CA) and PE-conjugated anti-rat ICAM-1 antibody 1A29 (Pharmingen). In some experiments, cells were stained with a biotinylated anti-rat monomorphic MHC class I antibody OX18 (Pharmingen) followed by a second stain with streptavidin-red 670 (GIBCO / BRL) for three-color staining. Do. All stains were 10 mM HEPES, 0.8 mM MgSO.₄, 0.2 mM EGTA, and 0.2% BSA, pH 7.4, using Ca ++ free ice-cold HBSS. The three established cell lines are trypsinized and feeder cells are removed by Percoll density gradient centrifugation. Rat hepatoma cell line FTO-2B and rat liver epithelial cell line WB-F344 and adult hepatocytes are stained for comparison with fetal hepatocyte cell lines. The cell lines were Dr. FEK Fournier, Fred Hutchinson Cancer Research Center, Seattle, WA, respectively, and Dr. Saw (Dr. M.-S. Tsao, University of California, University of California, USA). NC). Cells are blocked and stained with FITC-conjugated B5, OX18, PE-conjugated 1A29, or anti-FITC-conjugated rat integrin β1 antibody Ha2 / 5 (Pharmingen). FITC-conjugated anti-mouse IgG is used for OX18. To eliminate the mouse cell population, cell suspensions of the three fetal liver cell lines are stained with biotinylated anti-mouse CD98, followed by a second staining with streptavidin-red 670 and an anti-rat monoclonal antibody.
[0043]
Different antigens are expressed in different relative numbers for each cell. In practical use, the level of expression of a particular antigen can be no expression, low levels of expression, normal levels of expression for many antigens, and high levels of expression. In this usage, the term "low" is used interchangeably with "weak" or "dull". Although a more detailed description of the expression levels can be made, these four levels are sufficient for many purposes. Obviously, measurement of antigen expression by flow cytometry obviously provides a continuous range for antigen expression.
[0044]
CFA of hepatocyte cell lines, sorted cells, and adult hepatocytes
The hepatocyte line was 9.6 cm above the mitomycin C treated STO feeder layer using the same HDM used to maintain the cell line.²Seed in triplicate at 500 cells per cell. Prior to seeding, cells are trypsinized and feeder cells are removed by Percoll density gradient centrifugation. The culture solution is incubated for 10 to 14 days while changing the medium every two days. Thereafter, double immunofluorescence staining of α-fetoprotein and albumin is performed. One hundred colonies in each well are analyzed for colony morphology, P or F type, and expression of α-fetoprotein and albumin. Colonies are stained using Diff-Quick (Baxter, McGaw Park, IL) and the number of colonies in each well is counted. For CFA of primary sorted cells and adult hepatocytes, change the number of cells seeded as described. As another minor change, the culture period was extended between 14 and 17 days and the concentration of dexamethasone was increased to 10^-6Increase to M. Other procedures are performed as described above. In adult hepatocyte CFA, a small number of hepatocyte clamps are not removed from the cell suspension after preparation. Thus, an indefinite number of colonies can be produced from the clamp. For CFA of bile duct differentiation on sorted cells, double immunofluorescence staining of colonies with albumin and CK19 is performed on day 5 of culture in the presence or absence of EGF. On the fifth day of culture, colonies having more than one CK19 + cell are counted as CK19 + colonies. At 10 and 15 days, colonies containing multiple clusters of two CK19 + cells or colonies containing one cluster of more than three CK19 + cells are counted as CK19 + colonies. About 100 colonies are counted in each well. Each point represents the mean ± SD of triplicate stained cultures.
[0045]
6.2. Production and analysis of fetal rat hepatocyte cell line using mouse embryo cell feeder using hormone-supplemented synthetic medium
A simple long-term culture of rat E15 hepatocytes is attempted to see how long fetal hepatocytes can be maintained, proliferated ex vivo, and produce offspring. After density gradient centrifugation to remove hematopoietic mononuclear cells, fetal hepatocytes are cultured using a type IV collagen or laminin-coated culture dish and HDM (see Example 6.1). Cells survive for more than 4 weeks. However, secondary cultures on dishes newly coated with type IV collagen or laminin do not proliferate further. When the STO embryonic mouse fibroblast cell line treated with mitomycin C is used as a feeder layer for secondary culture, many clumps of cells proliferate. Finally, from four independent experiments, several stable hepatocyte cell lines are established.
[0046]
Immunohistochemical analysis of alpha fetoprotein and albumin is performed on a continuously growing population of cells prior to cloning of the cell line. Both alpha-fetoprotein and albumin proteins are used as markers to confirm that the cell population is of hepatocyte origin. A group of cells that tend to form cell stacks, called P colonies, strongly expressed α-fetoprotein and albumin, while a flat monolayer, called F colonies, had reduced expression of α-fetoprotein and albumin. Did not. Embryonic mouse fibroblast STO shows no reactivity with any antibody. For further analysis, three cloned hepatocyte cell lines are selected in independent experiments by morphological criteria of P-type or F-type colonies. Rhel4321 (FIG. 1A) consists mainly of P-cell colonies packed with small cells, while th1120-3 (FIG. 1C) forms only a flat monolayer of F-type colonies. Rter6 (FIG. 1B) is intermediate between the two phenotypes. Interestingly, the heterogeneity of rter6 is observed even after three consecutive clonings of flat colonies. To see the heterogeneity of the colonies from single cells of rhel4321 and rter6, 9.6 cm²Cells were seeded at a density of 500 cells (one well of a 6-well plate) and cells were cultured on STO fibroblasts for 10-14 days. Thereafter, the morphology of the colonies and the expression of α-fetoprotein and albumin were analyzed. 2A, 2B, 2C, 2D, 2E, and 2F show the results. The rhel4321 (FIG. 2B) and rter6 (FIG. 2C) cell lines and the original cell population before cloning (FIG. 2A) express α-fetoprotein strongly in almost all P-type colonies, whereas F-type colonies Does not appear. Furthermore, strong expression of both α-fetoprotein and albumin is observed only in P-type colonies. Morphological differences in cloned hepatocyte lines correlate with the percentage of P-type colonies (FIGS. 2B and 2C). The percentages of P-type colonies of CFA of rter6 and rhel4321 are 33.3% (± 8.6% SD) and 65.7% (± 4.0% SD), respectively. The total number of colonies per well is counted, and the clonal growth efficiency (colony efficiency) is calculated. The efficiencies of rter6 and rhel4321 are 45.7% (± 1.3% SD) and 36.4% (± 1.1% SD), respectively. The th1120-3 cells are strongly adhered to each other along the lateral border, making the preparation of a single cell suspension very difficult. However, th1120-3 cells do not form overlapping clusters.
[0047]
Since the adhesion of mouse hepatocytes to extracellular matrix (ECM) proteins such as laminin, type IV collagen, and fibronectin varies by developmental stage, we next examine each cell line's preference for adhering to specific ECM components. . Similar to the findings in adult hepatocytes, type IV collagen is most effective for th1120-3 adhesion (FIG. 1C), but not so much for rter6 (FIG. 1B) and rhel4321 (FIG. 1A). Laminin is the most effective substrate for rhel4321 adhesion (FIG. 1A). This preference is similar to the primary culture of fetal mouse hepatocytes. In summary, the conserved expression of α-fetoprotein and albumin in P-type colonies, and preferential adhesion of laminin by rhel4321 indicates that the population of cells producing P-type colonies is more strictly associated with hepatic progenitor cells Suggests.
[0048]
6.3 Isolation of STO subclone for colony formation; Assay for hepatic progenitor cells
To develop a CFA system to identify highly proliferative, bipotent hepatic progenitor cells, culture systems support cell growth at clonal seeding densities and preserve important original liver function. Must-have. Two of the most important markers for early liver development are albumin and alpha-fetoprotein. Culture conditions that optimize P-type colonies are best, as P-type but not F-type colonies maintain α-fetoprotein and albumin expression during clonal expansion. Therefore, the ability of STO subclones to support the rter6 P-type colonies is compared. One clone, STO5, supports P-type colony formation over all other sublines and parental strains (FIG. 2D). The CFA of rhel4321 also confirms that STO5 is a more effective feeder than the parent STO (FIG. 2E). The mouse H1x gene product expressed in the mesenchymal cells lining the gastrointestinal tract of E10.5 is essential for the growth of fetal hepatocytes. When H1x gene mRNA expression was analyzed in STO subclones, there was no significant difference in expression between subclones (data not shown). Furthermore, stable transfectants in mouse H1x STO5 do not improve the colony formation assay (FIG. 2F). However, one clone of the transfectant, at a relatively high density of passages, more stably retains the original form of STO5, so this clone is used for further experiments.
[0049]
6.4 Identification of hepatic progenitor cells from E13 fetal liver using surface antigen markers and colony formation assays
Liver formation and massive hematopoiesis coexist in fetal liver. To date, the antigenic profile of hematopoietic progenitor cells has been extensively analyzed, but studies of early hepatic progenitor cells are still in their infancy. Using the three hepatocyte lines established in this study, an adult hepatoma cell line (FTO-2B), an epithelial cell line derived from adult rat liver (WB-F344), and newly isolated adult hepatocytes, The antigenic profile of the cells is analyzed. Compared to FTO-2B, WB-F344 and adult hepatocytes, the most immature fetal hepatocyte cell line, rhel4321, is a classic MHC class I (RT1A¹) Are very unique in that there is no expression (FIGS. 3A-3X). Cell line th1120-3 (FIGS. 3I-3L) contains RT1A¹, OX18 (pan-MHC class I), and ICAM-1 patterns are similar to rhel4321 (FIGS. 3A-3D), but rter6 (FIGS. 3E-3H) is RT1A.¹And OX18 expression is relatively high (FIG. 3). In addition, another cell line obtained in a different experiment has the same morphology as rhel4321, but also RT1A^1-, OX18^dull, And ICAM-1⁺It is. Integrin b1 expression is similar in all cell lines, but RT1A^{a, b, 1}And the pattern of ICAM-1 is unique in cells. The antigenic profile of adult hepatocytes is RT1A¹⁺, OX18⁺, And ICAM-1⁺It is. In adult rats, fetal hepatocytes can be separated from hematopoietic cells by MHC class I expression, because all bone marrow cells except erythrocytes strongly express MHC class I molecules. Cell suspension obtained from rat E13 liver was used as anti-RT1A¹And staining with ICAM-1 antibody. FIG. 4A shows RT1A.¹2 shows a two-color staining pattern of ICAM-1 and ICAM-1. To determine which fractions contain the hepatocyte population, five fractions are isolated by fluorescence activated cell sorting and screened for clonal expansion potential by CFA. FIG. 4B shows the result of re-sorting the five fractions after sorting. Hepatocyte colonies defined by the expression of albumin and α-fetoprotein are morphologically distinguishable, so that the number of liver colonies in each well can be counted. Most of the liver colonies are RT1A^{1 dul}And ICAM-1⁺(Table 1, FIG. 4B, gate 2), and the frequency of P-type colonies is 75.6% (± 4.9% SD). Gate 1 showed a much lower number of colonies, with only a few cells having the ability to form colonies of the other fractions. Gates 1 and 2 confirm expression of both alpha-fetoprotein and albumin in all liver colonies. Some colonies from gate 2 cells are larger than others. To examine MHC class I expression on hepatocytes in detail, for cell fractionation, RT1A¹, ICAM-1, and OX18, and side scatter (SSC) as another parameter. Since fetal hepatocytes contain lipid droplets even at the fetal age as early as E11 (Luzzatto, 1981), side scatter (SSC), which reflects the granularity of the cells, is a useful parameter for separating hepatocytes from hematopoietic cells. It is. FIG. 4C shows that Gate 2 contains the largest number of colony forming cells. When gated at R2 based on SSC, the cell population corresponding to gate 2 is clearly RT1A^1-And OX18^dullAre shown (FIGS. 4C and 4D). CFA confirms that R4 contains more colony forming cells than Gate 2 (Table 1). These results demonstrate that RT1A obtained from E13 rat liver^1-And OX18^dull, And ICAM-1⁺Most of the cells are alpha-fetoprotein⁺And albumin⁺Suggest that the cells are hepatocytes that produce colonies. This is the same antigenic profile found in rhel4321 cells (FIG. 3).
[0050]
TABLE 1 Frequency of liver colonies from E13 fetal liver sorted based on RT1A and ICAM-1 expression

[0051]
Colony forming cultures on STO5hlx contain the indicated number of cells obtained from each fraction of E13 fetal liver. The number of liver colonies was established from triplicate stained cultures (mean ± SD). The efficiency of colony formation indicates the percentage of cells inoculated into the culture solution, at which the colony was formed after analysis after 16 days of culture.
[0052]
6.5. Different culture requirements for E13 hepatocytes and adult hepatocytes
The growth requirements of sorted hepatocytes obtained from E13 liver were examined using a defined STO5 feeder and HDM. EGF has long been known to be a potent growth factor for adult hepatocytes. Therefore, the effect of EGF on sorted hepatocyte colony formation is examined. RT1A^1- OX18^dull, ICAM-1⁺Hepatocyte colony size increases in the absence of EGF, but adult hepatocytes generated colonies only in the presence of EGF (FIG. 6C). Furthermore, the morphology of colonies derived from adult hepatocytes is usually F-type, but RT1A^1-Hepatocytes form P-type colonies without EGF. However, colony efficiency is slightly reduced in the absence of EGF (FIG. 6A). Interestingly, culture conditions without EGF highlighted two types of P colonies, P1 and P2. At day 12 of culture, most of the colonies are of the P2 type, but it is difficult to completely distinguish the two types, as some colonies do not have the typical morphology as in FIG. 6A. These results indicate that fetal and adult hepatocytes have their growth requirements and RT1A¹It suggests that the expression (FIGS. 3 and 4) and the morphology of the colonies are essentially different.
[0053]
After 3 weeks in culture when growth appears to have reached a maximum, RT1A^1- , OX18 and ICAM-1 are evaluated. As shown in FIGS. 5B-5D, RT1A¹Is not induced and OX18 expression is reduced. The level of ICAM-1 does not change. In addition, the average cell number of a single colony is calculated from the number of cells recovered, the percentage of rat hepatocytes, and the colony efficiency. The estimated cell number is 3-4 x 10³(Table 2) is reached. This indicates that a single cell forming a colony divided about 11 to 12 times on average in this culture condition.
[0054]
Table 2: Calculation of cell number in a single liver colony

[0055]
Cells sorted from R4 in FIG. 4C were cultured on STO5hlx feeder cells in 60 mm or 100 mm dishes. After culturing for the indicated period, all cells were harvested and the total cell number was calculated. The percentage of rat cells is by flow cytometry analysis based on the expression of rat ICAM-1 and mouse CD98. Colony efficiency indicates the percentage of cells that formed a colony among the cells inoculated into the culture. Data from triplicate stained cultures (average) were obtained from experiments performed in parallel.
[0056]
Average number of cells in a single colony = (number of cells recovered x percentage of rat cells / 100) / number of cells inoculated x colony efficiency / 100)
[0057]
6.6 RT1A^1-Evidence that hepatic progenitor cells are bipotent
In rat embryonic age E13, hepatocytes are considered to be bipotent progenitor cells that give rise to mature hepatocytes and bile duct epithelial cells. However, prior to the present invention, there was no direct evidence to indicate whether these two fates originated from a single cell. RT1A^1- OX18^dull ICAM-1⁺Colonies are stained with anti-CK19 as a marker specific for bile duct epithelial cells to determine if the fetal hepatocytes of this can be differentiated into bile duct cell lines in this culture. CK19 is expressed in biliary epithelial progenitor cells in fetal liver after day 15.5, at which point the cells have lost albumin expression. RT1A sorted^1- ICAM-1 + cells are cultured in the presence or absence of EGF, and their fate is monitored on day 5 of culture by expression of CK19 and albumin. Five days after the beginning, EGF-treated cultures contained CK19⁺Cultures without EGF, CK19⁺Some colonies containing cells are seen. Although the strength of CK19 is considerably weak, CK19⁺Cells have reduced albumin expression. On day 10 of culture, there are colonies expressing only CK19 or albumin, and colonies positive for both. CK19 in a single colony⁺And albumin⁺Cell patterns are in conflict. The number of both positive and CK19 only positive colonies is also higher in the absence of EGF (FIG. 7A). In the presence of EGF, many colonies were found to be albumin on day 10.⁺Consists of cells only (FIG. 7B). Ultimately, the number of both positive colonies approaches 100% in the absence of EGF on day 15 (FIG. 7A). Altogether, EGF is present in CK19 throughout culture.⁺(FIG. 7B). These results demonstrate that RT1A obtained from E13 fetal liver^1-, OX18^dull, And ICAM-1⁺The cells can differentiate into a cholangiocyte cell line, suggesting that their fate is affected by EGF in vitro (FIG. 8).
[0058]
6.7 Protocol for isolation and cloning of feeder cells capable of supporting clonal expansion of hepatic stem cells and hepatic progenitor cells
Slice fresh embryo or frozen tissue (eg, liver, lung, kidney, muscle, intestine) of pig, beagle, rabbit, mouse, or monkey in calcium-free phosphate buffered saline (PBS) . After rinsing twice with PBS, the cell suspension is incubated with 0.25% trypsin for 10 minutes at 37 ° C. or 60 minutes at room temperature using a magnetic stirrer. Remaining cell clumps are removed by filtering the suspension through a mesh. The cells are then placed in a basal medium (eg, Eagle MEM) supplemented with serum (eg, 10% fetal bovine serum) and any of a variety of growth supplements (eg, 2 mM glutamine, sodium pyruvate, and MEM non-essential amino acids). Culture in a tissue culture dish using. Plastic basal and serum-supplemented media allow the growth of a population of cells that are candidates for feeder cells (“feeder cells”), often derived from mesoderm (eg, stromal cells), and provide another type of cell General conditions that provide factors that support the survival, proliferation, and / or function of (eg, progenitor cells). Feeder cells are subcultured with 0.05% trypsin when it is at or near confluence. After several passages, the grown cells are prepared as frozen stocks and stored frozen until use. Another source of feeder cells is commercially available primary culture feeder cells or feeder cell lines. In any case, the following criteria are required to identify a suitable feeder cell.
[0059]
Feeder cells support:
1) clonal expansion of hepatic progenitor cells with phenotypic markers of classical MHC class I antigen negative, ICAM-1 positive, and / or non-classical MHC class I antigen weak positive;
2) clonal expansion of progenitor cells having progeny with a phenotypic marker of classical MHC class I antigen negative, ICAM-1 positive, non-classical MHC class I antigen weak positive, α-fetoprotein positive, albumin positive, or CK19 positive; Or
3) Inducible differentiation into both hepatocellular and cholangiocyte cell lines required for the definition of bipotent hepatic progenitor cells.
[0060]
In the art, classical MHC class I antigens are also known as MHC class Ia antigens. Non-classical MHC class I antigens are also known as MHC class Ib antigens. MHC antigens are called differently in different species: for example, RT1 in rats, H-2 in mice, and HLA in humans.
[0061]
The above assay is described below:
[0062]
Conditions for clonal expansion of hepatic progenitor cells
Hepatic progenitor cells were 9.6 cm above feeder cells that stopped growing, ie, were treated to prevent growth.²Inoculate 500 cells per. Feeder cells are treated with mitomycin C or irradiation (3000-5000 rads depending on cell type) to stop growth. Serum-free HDM is supplied to feeder cells and progenitor cells that have stopped growing. For example, the HDM of rodent cells can be obtained by adding 10 ng / ml EGF, 5 μg / ml insulin, 10 μg / ml to a 1: 1 mixture of Dulbecco's modified Eagle's medium and Ham F12.^-6M dexamethasone, 10 μg / ml iron-saturated transferrin, 4.4 × 10^-3M nicotinamide, 0.2% bovine serum albumin, 5 × 10^-5M 2-mercaptoethanol, 7.6 μeq / l free fatty acid mixture, 2 × 10^-3M glutamine, 1 x 10^-6M CuSO₄, 3 x 10^-8MH₂SeO₃And antibiotics added. The culture is incubated for 10 to 14 days, changing the medium every two days. Subsequently, double immunofluorescence staining for α-fetoprotein, albumin, and / or CK19 is performed to identify the fate of the offspring. Approximately 100 colonies are analyzed for expression of alpha fetoprotein and albumin. In addition, the morphology of the colony, P-type or F-type, may also help identify suitable offspring.
[0063]
The ideal combination of feeder cells and hepatic progenitor cells is from the same species. Preferably, the feeder cells are obtained from the same tissue and the same species as the hepatic progenitor cells. However, it is also possible to mix feeders from one species with precursor cells from another species. For example, even rodent feeders can be used for human liver progenitor cells. Soluble and insoluble factors, which may be species and / or tissue specific, assist in clonal expansion of hepatic stem cells or hepatic progenitor cells. Sources of such factors include the following:
1) A conditioned medium in which feeder cells of the optimal species and tissue have been cultured. The feeder cells may be not only stromal cells but also any kind of cells.
2) When important factors are known, cDNA or mRNA for transcription or translation, respectively, is optionally used to synthesize an appropriate molecule (signal) derived from an optimal feeder cell that is active in hepatic progenitor cells. A source from which biologically active feeder cell groups can be produced by introducing the cells into the cells.
3) Regardless of whether the signal obtained from the optimal feeder cell active on hepatic progenitor cells is a protein, peptide, hydrocarbon, lipid, glycopeptide, glycoprotein, lipoprotein, glycolipid, or a combination thereof, A source that, if the factors are known, can also completely replace feeder cells by adding such a signal to the medium.
[0064]
The above examples are described by way of illustration only and do not limit the scope or aspects of the present invention. Other embodiments not specifically described above will be apparent to those skilled in the art. However, such other embodiments are also considered to be within the scope and spirit of the present invention. Therefore, the present invention is properly limited only by the appended claims.
[0065]
All patents and publications cited herein are hereby incorporated by reference in their entirety.
[Brief description of the drawings]
FIG. 1 is a characterization of a hepatocyte cell line obtained from fetal rat liver on day 15.
FIG. 2 is a colony formation assay on fibroblast feeder cells.
FIG. 3. Expression of rat cell surface antigen on various cell lines in adult hepatocytes.
FIG. 4 depicts phenotypic analysis of fetal rat liver on day 13.
FIG. 5 is a characterization of liver colonies in the presence and absence of EGF.
FIG. 6: RT1A^1-FIG. 4 shows induction of CK19 expression on hepatocytes.
FIG. 7 is a schematic diagram of liver colony formation on STO5 feeder cells.

Claims (80)

Culturing endoderm-derived progenitor cells, their progeny, or a mixture thereof that express intercellular adhesion molecule antigens but do not express classical major histocompatibility antigens on a layer containing feeder cells in the presence of medium A method for expanding endoderm-derived progenitor cells, progeny thereof, or a mixture thereof, comprising the steps of: 2. The method of claim 1, wherein the progenitor cells, progeny thereof, or a mixture thereof comprise vertebrate cells. 3. The method of claim 2, wherein the progenitor cells, progeny thereof, or a mixture thereof comprise human cells, non-human primate cells, pig cells, dog cells, rabbit cells, rat cells, mouse cells, or a combination thereof. 4. The method of claim 3, wherein the progenitor cells, progeny thereof, or a mixture thereof comprise liver, lung, intestine, pancreas, thyroid, gonad progenitor cells, or a combination thereof. 5. The method of claim 4, wherein the progenitor cells, progeny thereof, or a mixture thereof are derived from the liver. 2. The method of claim 1, wherein the medium comprises a basal medium. 7. The method of claim 6, wherein the basal medium comprises Dulbecco's modified Eagle's medium and Ham F12. 2. The method of claim 1, wherein the medium contains at least one hormone. 9. The method according to claim 8, wherein the hormone is insulin. 9. The method of claim 8, wherein the medium further comprises a glucocorticoid hormone. 9. The method according to claim 8, wherein the glucocorticoid hormone is dexamethasone. 2. The method of claim 1, wherein the medium further comprises iron-saturated transferrin. 2. The method of claim 1, wherein the medium further comprises nicotinamide. 2. The method of claim 1, wherein the medium further comprises serum albumin. The method of claim 1, wherein the medium further comprises at least one reducing agent. The method according to claim 15, wherein the reducing agent is β-mercaptoethanol. 2. The method of claim 1, wherein the medium comprises at least one lipid supplement. 18. The method of claim 17, wherein the lipid supplement comprises a free fatty acid mixture. 18. The method of claim 17, wherein the free fatty acid mixture comprises palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, or a combination thereof. 2. The method of claim 1, wherein the medium contains glutamic acid. The method of claim 1, wherein the medium further comprises a trace element. Trace elements comprises CuSO _4, The method of claim 21, wherein. Trace elements including _H 2 SeO _3, The method of claim 21, wherein. 2. The method of claim 1, wherein the medium contains an antioxidant. Antioxidants including _H 2 SeO _3, The method of claim 24. 2. The method of claim 1, wherein the medium further comprises an antibiotic. The method of claim 1, wherein the medium further comprises epidermal growth factor. 2. The method of claim 1, wherein the source of feeder cells comprises tissue from at least one vertebrate. 29. The method of claim 28, wherein the vertebrate comprises an embryo. 30. The method of claim 29, wherein the vertebrate comprises a human, non-human primate, pig, dog, rabbit, rat, mouse, or a combination thereof. 2. The method according to claim 1, wherein the feeder cells are clones. 32. The method of claim 31, wherein the clone is STO5. 2. The method of claim 1, wherein the feeder cells comprise stromal cells. 2. The method of claim 1, wherein the feeder cells comprise fibroblasts. 2. The method of claim 1, wherein the hepatic progenitor cells are weakly expressing MHC class Ib, weakly expressing OX18, expressing α-fetoprotein, expressing albumin, expressing cytokeratin 19, or a combination thereof. The method of claim 1, wherein the ICAM is ICAM-1. 2. The liver progeny cell is MHC class Ib, weakly expresses ICAM, weakly expresses OX18, expresses alpha fetoprotein, expresses albumin, expresses cytokeratin 19, or a combination thereof. The described method. 38. The method of claim 37, wherein the ICAM is ICAM-1. 2. The method of claim 1, wherein the hepatic progenitor cells, their progeny, or a combination thereof, grow as stacked colonies. 2. The method of claim 1, further comprising cloning the hepatic progenitor cells. 41. The method of claim 40, wherein cloning utilizes cell number dilution, cloning color, growth in agarose, growth on beads, flow cytometry, or a combination thereof. 2. The method of claim 1, wherein the hepatic progenitor cells undergo at least one mitosis. 43. The method of claim 42, wherein the hepatic progenitor cells undergo at least 10 mitosis. 2. The method of claim 1, wherein the hepatic progenitor cells give rise to daughter cells with another fate. 46. The method of claim 44, wherein the daughter cells differentiate into bile duct cells. 46. The method of claim 44, wherein the daughter cells differentiate into hepatocytes. Providing a sample suspected of containing hepatic progenitor cells, progeny thereof, or a mixture thereof, and at least one liver growth factor, mixing the sample with a progenitor cell, its progeny, or a mixture thereof, and hepatic progenitor cells A method for identifying a liver growth factor, comprising the step of observing a stimulation of growth of hepatic. Providing a sample suspected of containing a hepatic progenitor cell, its progeny, or a mixture thereof, and at least one hepatic differentiation factor, mixing the sample with the progenitor cell, its progeny, or a mixture thereof, and a hepatic progenitor cell A method for identifying a liver differentiation factor, comprising a step of observing differentiation of a liver. Providing a sample suspected of containing a hepatic progenitor cell, its progeny, or a mixture thereof, and at least one hepatotoxin; mixing the sample with the progenitor cell, its progeny, or a mixture thereof; A method for identifying a hepatotoxin, comprising the step of observing death. Providing hepatic progenitor cells, their progeny, or a mixture thereof, and a drug that is considered to be capable of affecting hepatic progenitor cells / metabolism, mixing the progenitor cells, their progeny, or a mixture thereof with a sample, And a step of observing a change in metabolism of hepatic progenitor cells. Providing at least one hepatic progenitor cell, its progeny, or a mixture thereof, and an agent suspected of having an antibacterial effect; mixing the progenitor cell, its progeny, or a mixture thereof, with the agent; A method for identifying a novel antibacterial agent, comprising observing a change in growth. Providing a hepatic progenitor cell, its progeny, or a mixture thereof, and culturing the progenitor cell, its progeny, or a mixture thereof in a bioreactor until a population of cells sufficient to serve as an extracorporeal liver is obtained. And a method for preparing an extracorporeal liver. A method of expanding hepatic progenitor cells, progeny thereof, or a mixture thereof,
(A) providing at least one hepatic progenitor cell that expresses an intercellular adhesion molecule antigen and does not express a major histocompatibility antigen; and (b) in a medium containing a biosynthetic product of at least one feeder cell. A method comprising culturing hepatic progenitor cells. 54. The method of claim 53, wherein the medium further comprises glucocorticoid, insulin, epidermal growth factor, nicotinamide, or a combination thereof. The biosynthesis product of the feeder cell includes a peptide, a glycopeptide, a lipopeptide, a lipid, a glycolipid, a hydrocarbon, a protein, a glycoprotein, a lipoprotein, or a combination thereof, and the biosynthesis product promotes proliferation or differentiation of a hepatic progenitor cell 54. The method of claim 53, wherein promoting. The biosynthesis products of the feeder cells include insulin-like growth factor, interleukin 6 family growth factor, hepatocyte growth factor, fibroblast growth factor, extracellular matrix, or a combination thereof, and the biosynthesis product is a hepatic progenitor cell. 54. The method of claim 53, wherein said method promotes proliferation or differentiation. 54. The method of claim 53, wherein the at least one hepatic progenitor cell comprises at least one cell obtained from a human, non-human primate, pig, dog, rabbit, rat, or mouse. 54. The method of claim 53, further comprising cloning at least one hepatic progenitor cell. 54. The method of claim 53, further comprising adding a growth factor. 60. The method of claim 59, further comprising the addition of a differentiation factor to produce hepatocytes, cholangiocytes, or a combination thereof. 2. The method according to claim 1, wherein the medium is serum-free. Proliferating an endoderm-derived progenitor cell, its progeny, or a mixture thereof, comprising culturing the endoderm-derived progenitor cell, its progeny, or a mixture thereof in the presence of a serum-free medium on a layer containing feeder cells How to let. 63. The method of claim 62, wherein the progenitor cells, progeny thereof, or a mixture thereof comprise liver, lung, intestine, pancreas, thyroid, gonad progenitor cells or a combination thereof. 63. The method of claim 62, wherein the medium comprises Dulbecco's Modified Eagle Medium and Ham F12. 63. The method of claim 62, wherein the medium comprises at least one hormone. 66. The method of claim 65, wherein the hormone is insulin, glucocorticoid hormone, transferrin, epidermal growth factor, or a combination thereof. 63. The method of claim 62, wherein the source of feeder cells comprises tissue from at least one vertebrate. 68. The method of claim 67, wherein the vertebrate comprises a human, non-human primate, pig, dog, rabbit, rat, mouse, or a combination thereof. 63. The method of claim 62, wherein the feeder cells are clones. 63. The method of claim 62, wherein the feeder cells comprise stromal cells. Liver progenitor cells express intercellular adhesion molecule (ICAM) antigen, do not express major histocompatibility complex (MHC) class Ia antigen, weakly express MHC class Ib, weakly express OX18, express α-fetoprotein 63. The method of claim 62, wherein the method expresses, expresses albumin, expresses cytokeratin 19, or a combination thereof. 72. The method of claim 71, wherein the ICAM is ICAM-1. The liver progeny does not express MHC class Ia, expresses MHC class Ib, weakly expresses ICAM, weakly expresses OX18, expresses α-fetoprotein, expresses albumin, expresses cytokeratin 19, or 63. The method of claim 62, which is a combination thereof. 74. The method of claim 73, wherein the ICAM is ICAM-1. 63. The method of claim 62, further comprising cloning the hepatic progenitor cells. 76. The method of claim 75, wherein the hepatic progenitor cells undergo at least one mitosis. 77. The method of claim 75, wherein the hepatic progenitor cells undergo at least 10 mitosis. 76. The method of claim 75, wherein the hepatic progenitor cells give rise to daughter cells with another fate. 79. The method of claim 78, wherein the daughter cells differentiate into bile duct cells. 79. The method of claim 78, wherein the daughter cells differentiate into hepatocytes.

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