本揭示內容關於一種分離及擴增間質幹細胞(mesenchymal stem cell，MSC)及從其衍生細胞群的方法。The present disclosure relates to a method of isolating and expanding mesenchymal stem cells (MSCs) and deriving cell populations therefrom.
可從各種組織分離間質幹細胞，其包含但不限於新生兒組織(例如：人類臍帶的華通氏膠(Wharton’s jelly))、骨髓、周邊血液、胎盤、臍帶血以及脂肪組織。在該些組織中，周邊血液是最容易取得的間質幹細胞來源。然而，周邊血液中間質幹細胞的含量相當低，即便於活體外擴增間質幹細胞，相關領域仍無法取足量之間質幹細胞，進而用於後續分化及相關應用。Mesenchymal stem cells can be isolated from a variety of tissues including, but not limited to, neonatal tissues (eg, Wharton's jelly of the human umbilical cord), bone marrow, peripheral blood, placenta, cord blood, and adipose tissue. In these tissues, peripheral blood is the most readily available source of mesenchymal stem cells. However, the content of peripheral blood mesenchymal stem cells is quite low. Even if the mesenchymal stem cells are expanded in vitro, the relevant fields cannot obtain sufficient amount of interstitial stem cells, which are used for subsequent differentiation and related applications.
因此，相關領域亟需發展一種可自周邊血液取得間質幹細胞的改善方法，以製備足以用於後續治療用途的間質幹細胞群。Accordingly, there is a need in the art to develop an improved method for obtaining mesenchymal stem cells from peripheral blood to prepare a population of mesenchymal stem cells sufficient for subsequent therapeutic use.
為了給讀者提供基本的理解，以下提供本揭示內容的簡要發明內容。此發明內容不是本揭示內容的廣泛概述，同時非用來定義本發明的關鍵/必需元件或勾勒本發明的範圍。其唯一目的是以簡化的形式呈現本揭示內容的一些概念，以作為呈現於後文中更詳細描述的序言。In order to provide the reader with a basic understanding, a brief summary of the disclosure is provided below. This Summary is not an extensive overview of the disclosure, and is not intended to define the key or essential elements of the invention or the scope of the invention. The sole purpose is to present some concepts of the present disclosure in a simplified
本發明發現可從人類個體之周邊血液取得間質幹細胞(mesenchymal stem cells，MSCs)，並於含有甘油的培養液中進行體外擴增，從而製備一經分離之、實質上純的、未分化人類間質幹細胞(human mesenchymal stem cells，hMSCs)群，其適用於進行自體移植。The present invention finds that mesenchymal stem cells (MSCs) can be obtained from the peripheral blood of a human individual and expanded in vitro in a culture medium containing glycerol to prepare an isolated, substantially pure, undifferentiated human. A population of human mesenchymal stem cells (hMSCs) suitable for autologous transplantation.
據此，本揭示內容的第一個態樣是關於一種提供未分化之人類間質幹細胞群的方法。該方法包含以下步驟： (a) 從人類個體取得周邊血液； (b) 將甘油添加至步驟(a)之該周邊血液中； (c) 分離步驟(b)之該周邊血液中，相對於其他成體幹細胞的人類間質幹細胞；以及 (d) 將步驟(c)之經分離的人類間質幹細胞培養於一包含甘油的培養液中，藉以製備該人類間質幹細胞群； 其中該培養液不含選自由顆粒性白血球群落刺激因子(granulocyte colony stimulating factor，G-CSF)、顆粒性白血球-巨噬細胞群落刺激因子(granulocyte-macrophage colony stimulating factor，GM-CSF)以及其組合所組成的群組之任何移動劑(mobilization agent)。Accordingly, a first aspect of the present disclosure is directed to a method of providing an undifferentiated human mesenchymal stem cell population. The method comprises the steps of: (a) obtaining peripheral blood from a human subject; (b) adding glycerol to the peripheral blood of step (a); (c) separating the peripheral blood of step (b), relative to the other Human mesenchymal stem cells of adult stem cells; and (d) cultivating the human mesenchymal stem cells isolated in step (c) in a culture medium containing glycerol to prepare the human mesenchymal stem cell population; wherein the culture solution is not Containing a group consisting of a granulocyte colony stimulating factor (G-CSF), a granulocyte-macrophage colony stimulating factor (GM-CSF), and a combination thereof Any mobilization agent.
根據本揭示內容的實施方式，在步驟(a)中，將該周邊血液收集於不含任何溶血劑的試管中。According to an embodiment of the present disclosure, in step (a), the peripheral blood is collected in a test tube without any hemolytic agent.
根據本揭示內容的實施方式，在步驟(b)中，甘油於周邊血液中的濃度約為每毫升1至10 毫克。According to an embodiment of the present disclosure, in step (b), the concentration of glycerol in the peripheral blood is about 1 to 10 mg per ml.
根據本揭示內容的實施方式，在步驟(d)中，該培養液包含至少20% (體積比)的血清，且甘油於該培養液中的濃度約為每毫升0.1至1.0 毫克，且於37℃下，將該人類間質幹細胞培養於一包含該含甘油培養液的培養盤中至少16小時。According to an embodiment of the present disclosure, in the step (d), the culture solution contains at least 20% by volume of serum, and the concentration of glycerol in the culture solution is about 0.1 to 1.0 mg per ml, and at 37 The human mesenchymal stem cells were cultured in a culture dish containing the glycerol-containing culture solution at ° C for at least 16 hours.
根據本揭示內容的替選實施方式，方法更可包含： (e) 將該人類間質幹細胞轉移至另一培養盤中，其表面積至少為步驟(d)之培養盤表面積的至少16倍大，且持續培養於包含甘油的培養液中，以製備未分化之人類間質幹細胞群。According to an alternative embodiment of the present disclosure, the method may further comprise: (e) transferring the human mesenchymal stem cells to another culture dish having a surface area at least 16 times greater than the surface area of the culture disk of step (d), And continuously cultured in a culture solution containing glycerol to prepare an undifferentiated human mesenchymal stem cell population.
根據本揭示內容之實施方式，在步驟(e)中，將人類間質幹細胞至少培養6天，以製備未分化之人類間質幹細胞群。According to an embodiment of the present disclosure, in step (e), human mesenchymal stem cells are cultured for at least 6 days to prepare an undifferentiated human mesenchymal stem cell population.
根據本揭示內容之實施方式，所製備的未分化之人類間質幹細胞群是對CD34-、CD45-及HLA-DR細胞表面標記呈現陰性反應，而對CD73+、CD90+及CD 105+細胞表面標記呈現陽性反應。According to an embodiment of the present disclosure, the prepared undifferentiated human mesenchymal stem cell population exhibits a negative reaction to surface markers of CD34-, CD45-, and HLA-DR cells, and exhibits surface markers of CD73+, CD90+, and CD 105+ cells. Positive reaction.
據此，本揭示內容是關於一單羣性細胞株(clonal cell line)，該單羣性細胞株包含根據本揭示內容方法製備的實質上純的未分化人類間質幹細胞群。Accordingly, the present disclosure is directed to a clonal cell line comprising a substantially pure population of undifferentiated human mesenchymal stem cells prepared according to the methods of the present disclosure.
此外，本揭示內容也關於未分化之人類間質幹細胞群或其分化子代用途，其可藉由本揭示內容方法進行製備，據以治療一有需要之個體的疾病或病症。Furthermore, the present disclosure also relates to undifferentiated human mesenchymal stem cell populations or their differentiated progeny uses, which can be prepared by the methods of the present disclosure to treat a disease or condition in an individual in need thereof.
根據本揭示內容之實施方式，該人類間質幹細胞群是實質上純的人類間質幹細胞。According to an embodiment of the present disclosure, the human mesenchymal stem cell population is a substantially pure human mesenchymal stem cell.
根據本揭示內容之實施方式，實質上純的人類間質幹細胞可經誘導分化成軟骨細胞、軟骨及脂肪細胞。According to embodiments of the present disclosure, substantially pure human mesenchymal stem cells can be induced to differentiate into chondrocytes, cartilage, and fat cells.
據此，本揭示內容亦提供一種用以治療亟需移植自體人類間質幹細胞之個體的疾病或病症的方法。該方法包含對個體投予有效量之根據本揭示內容方法所製備的未分化之人類間質幹細胞群，據以治療該疾病或病症。Accordingly, the present disclosure also provides a method of treating a disease or condition in an individual in need of transplantation of autologous human mesenchymal stem cells. The method comprises administering to the individual an effective amount of an undifferentiated population of human mesenchymal stem cells prepared according to the methods of the present disclosure, whereby the disease or condition is treated.
根據本揭示內容的實施方式，可藉由自體移植人類間質幹細胞來治療的疾病或病症係選自由一骨疾病或軟骨疾病、一神經變性疾病、 一心臟疾病、一肝臟疾病、一癌症、一自體免疫疾病、移植物抗宿主疾病(graft versus host disease，GvHD)及傷口癒合與組織再生所組成之群組。According to an embodiment of the present disclosure, the disease or condition treatable by autologous transplantation of human mesenchymal stem cells is selected from a bone disease or a cartilage disease, a neurodegenerative disease, a heart disease, a liver disease, a cancer, An autoimmune disease, graft versus host disease (GvHD), and a group of wound healing and tissue regeneration.
根據本揭示內容的實施方式，可於3天或更短的期間、2天或更短的期間、或1天或更短的期間，持續對個體投予有效量之未分化人類間質幹細胞群或其分化子代。According to an embodiment of the present disclosure, an effective amount of undifferentiated human mesenchymal stem cell population can be continuously administered to an individual over a period of 3 days or less, a period of 2 days or less, or a period of one day or less. Or its differentiated progeny.
在參閱下文實施方式後，本發明所屬技術領域中具有通常知識者當可輕易瞭解本發明之基本精神及其他發明目的，以及本發明所採用之技術手段與實施態樣。The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.
為了使本揭示內容的敘述更加詳盡與完備，下文針對了本發明的實施態樣與具體實施例提出了說明性的描述；但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。然而，亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.
1. 定義1. Definition
為了便於說明，此處統整性地說明本說明書、實施例以及後附的申請專利範圍中所記載的特定術語。除非本文另有定義，本文所有的技術及科學術語與本發明所屬技術領域具有通常知識者習知的術語的意思相同。For the convenience of the description, the specific terms described in the specification, the examples, and the appended claims are hereby incorporated by reference. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as the terms
除非上下文另有明確說明，本文所使用的單數形式「一」(a, an)以及「該」(the)均包含複數形式。The singular forms "a", "," and "the"
本文使用的「幹細胞」(stem cell)一詞是以廣義的方式使用，也包含傳統幹細胞群(traditional stem cell)、前驅細胞群(progenitor cell)、前前驅細胞群(pre-progenitor cell)等。「幹細胞」(stem cell)一詞是指能夠增殖並產生更多前驅細胞的未分化細胞，該些前驅細胞具有可大量產生母細胞的能力，進而產生分化的或是具分化能力的子細胞。子細胞本身可被誘導以增殖並產生後續分化成一或多種成熟細胞類型的子代。「幹細胞」(stem cell)一詞也可指在特定情況下，具有能力或有潛力分化成更特化或更分化的表現型，且在某些情況下，仍保留以未分化狀態增殖之能力的細胞。The term "stem cell" as used herein is used in a broad sense, and also includes a traditional stem cell, a progenitor cell, a pre-progenitor cell, and the like. The term "stem cell" refers to undifferentiated cells that are capable of proliferating and producing more precursor cells, which have the ability to produce large numbers of mother cells, thereby producing differentiated or differentiated daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types. The term "stem cell" can also mean a phenotype that has the ability or potential to differentiate into more specialized or differentiated traits under certain circumstances and, in some cases, retains the ability to proliferate in an undifferentiated state. Cell.
術語「間質幹細胞」(mesenchymal stem cells，MSCs)指一種可分化成超過一種特定類型結締組織的多潛能幹細胞，特定類型結締組織可以是脂肪組織、骨組織、基質組織(stroma tissue)、軟骨組織、彈性組織及纖維組織。針對識別目的，可基於表現型標記物CD34-、CD45-、CD73+、CD90+以及CD105+的表現來鑑定人類間質幹細胞，也可基於分化成特定支持生命要素的組織(舉例來說但不限於軟骨細胞、軟骨以及脂肪細胞)的能力來鑑定。The term "mesenchymal stem cells" (MSCs) refers to a pluripotent stem cell that can differentiate into more than one specific type of connective tissue. The specific type of connective tissue can be adipose tissue, bone tissue, stroma tissue, cartilage tissue. , elastic tissue and fibrous tissue. For recognition purposes, human mesenchymal stem cells can be identified based on the expression of the phenotype markers CD34-, CD45-, CD73+, CD90+, and CD105+, or based on tissue that differentiates into specific life-supporting elements (for example, but not limited to, chondrocytes) , cartilage and fat cells) ability to identify.
關於分離的人類間質幹細胞(human mesenchymal stem cells, hMSCs)族群，「族群」、「群體」或「群」(population)一詞是指已經被移除且從一群混合或異質細胞族群中分離的人類間質幹細胞(hMSCs)的群體。在一些實施方式中，相較於從異質細胞族群分離或增殖的細胞而言，人類間質幹細胞群(the population of hMSCs)是實質上純的人類間質幹細胞群。With regard to isolated human mesenchymal stem cells (hMSCs), the term "ethnic", "group" or "population" refers to a term that has been removed and separated from a population of mixed or heterogeneous cell populations. A population of human mesenchymal stem cells (hMSCs). In some embodiments, the human mesenchymal stem cell population (the population of hMSCs) is a substantially pure population of human mesenchymal stem cells compared to cells isolated or proliferating from a heterogeneous population of cells.
本揭示內容中關於特定細胞族群的「實質上純的」(substantially pure)一詞，是指一群體中，相較於總細胞族群的組成，有至少70%、較佳地約80%、更佳地約90%、以及最佳地約95%的細胞是純的。關於根據本揭示內容中描述的方法所製備的人類間質幹細胞族群的術語「實質上純的」，是指含有少於約30%、較佳地少於約20%、15%、10%、8%，更佳地少於約5%、4%、3%、2%、1%或少於1%的非人類間質幹細胞的人類間質幹細胞族群。根據一些實施方式，本揭示內容包含將從周邊血液分離的一群人類間質幹細胞擴增的方法，其中該從周邊血液衍生的人類間質幹細胞擴增族群是一群實質上純的人類間質幹細胞群。The term "substantially pure" in relation to a particular cell population in the present disclosure means that in a population, at least 70%, preferably about 80%, more than the composition of the total cell population. Preferably about 90%, and optimally about 95% of the cells are pure. The term "substantially pure" with respect to the human mesenchymal stem cell population prepared according to the methods described in this disclosure means containing less than about 30%, preferably less than about 20%, 15%, 10%, 8%, more preferably less than about 5%, 4%, 3%, 2%, 1% or less than 1% of the human mesenchymal stem cell population of non-human mesenchymal stem cells. According to some embodiments, the present disclosure includes a method of amplifying a population of human mesenchymal stem cells isolated from peripheral blood, wherein the population of human mesenchymal stem cells derived from peripheral blood is a population of substantially pure human mesenchymal stem cells .
「單羣性細胞株」(clonal cell line)一詞是指可在一培養液中培養且具有無限繼代之潛能的細胞系(cell lineage)。單羣性細胞株可以是幹細胞株(例如：本揭示內容從周邊血衍生的人類間質幹細胞)或是從該周邊血衍生的人類間質幹細胞中衍生的幹細胞株。在單羣性細胞株的背景下所使用的單羣性細胞株包含從本揭示內容之周邊血液衍生的人類間質幹細胞，其在體外條件下培養並允許在不分化至少一個月的情況下增殖。這類的單羣性幹細胞株(例如：從本揭示內容周邊血液獲得的人類間質幹細胞)可具有從原始幹細胞沿著幾個細胞系分化的潛力。The term "clonal cell line" refers to a cell lineage that can be cultured in a culture medium and has the potential for unlimited passage. The single population cell strain may be a stem cell strain (for example, human mesenchymal stem cells derived from peripheral blood in the present disclosure) or a stem cell strain derived from human peripheral mesenchymal stem cells derived from the peripheral blood. The single population cell line used in the context of a single population cell line comprises human mesenchymal stem cells derived from peripheral blood of the present disclosure, which are cultured under in vitro conditions and allowed to proliferate without differentiation for at least one month. . Such single population stem cell lines (e.g., human mesenchymal stem cells obtained from peripheral blood of the present disclosure) may have the potential to differentiate from primitive stem cells along several cell lines.
在細胞個體發育的背景中，形容詞「分化的」(differentiated)是一個相對概念。一「分化的細胞」(differentiated cell)是指一細胞的發育路徑相較於正在與其比較的另一細胞更向前進或更向下游。因此，幹細胞可分化成品系限制的前驅細胞，其依次可分化成更下游的其他前驅細胞類型。接著再分化成最後分化階段的細胞，該些細胞在特定的組織類型中扮演特徵性的角色，並且可能或可能不保留再進一步增殖的能力。In the context of cell somatic development, the adjective "differentiated" is a relative concept. A "differentiated cell" refers to a cell's developmental pathway that is more advanced or downstream than another cell that is being compared to it. Thus, stem cells can differentiate into precursor cells that are restricted by the finished product, which in turn can differentiate into other precursor cell types that are further downstream. The cells are then differentiated into the final stage of differentiation, which play a characteristic role in a particular tissue type and may or may not retain the ability to proliferate further.
本文使用的術語「治療」(treatment)是用於指獲得一想要的藥理作用及/或生理作用，例如組織再生。就完全或部份預防疾病或其症狀而言，前述作用可以是預防性的；及/或就完全或部份治療疾病及/或肇因於該疾病不良影響而言，前述作用可以是治療性的。本文使用的「治療」(Treatment)包含對一哺乳動物之疾病的預防性(例如：預防(prophylactic))、治癒性或是緩解性治療，該哺乳動物特別是人類；以及包含：(1)對可能易患該疾病但尚未被診斷出來患有該疾病的一個體所發生的疾病或病症進行預防(preventative)(例如：預防(prophylactic))、治癒性或是緩解性治療；(2)抑制一疾病(例如：藉由終止該疾病的發展)；或(3)緩解一疾病(例如：減低與該疾病相關的症狀)。The term "treatment" as used herein is used to mean obtaining a desired pharmacological and/or physiological effect, such as tissue regeneration. The foregoing effects may be prophylactic in terms of completely or partially preventing the disease or its symptoms; and/or the therapeutic effect may be therapeutic in terms of completely or partially treating the disease and/or causing adverse effects of the disease. of. "Treatment" as used herein, includes prophylactic (eg, prophylactic), curative or palliative treatment of a mammalian condition, the mammal, particularly a human; and includes: (1) Preventive (eg prophylactic), curative or palliative treatment of a disease or condition that may be predisposed to the disease but has not been diagnosed with the disease; (2) inhibition one A disease (eg, by terminating the development of the disease); or (3) ameliorating a disease (eg, reducing symptoms associated with the disease).
術語「投予、給藥」(administered, administering)、或「移植」(transplanting) 在本文中可互換使用，以指任何適當的遞送途徑，其包含但不限於：靜脈內、肌內、腹腔內、動脈內、顱內、或皮下地將本發明的人類間質幹細胞群投予至該個體的所需部位，其中至少一部份的人類間質幹細胞仍是存活狀態。被投予至個體之後，該細胞存活期間也可短至幾小時，例如24小時，至幾天，長至幾個月。The terms "administered", "administered", or "transplanting" are used interchangeably herein to refer to any suitable route of delivery including, but not limited to, intravenous, intramuscular, intraperitoneal. The human mesenchymal stem cell population of the invention is administered intraarterially, intracranically, or subcutaneously to a desired site of the individual, wherein at least a portion of the human mesenchymal stem cells are still alive. After being administered to an individual, the cells can survive for as little as a few hours, such as 24 hours, up to several days, and up to several months.
本揭示內容使用的「有效量」(an effective amount)是指在特定所需時間內，對治療血小板聚集造成的疾病可達成預期結果的有效人類間質幹細胞之劑量。舉例來說，對傷口的治療上，有效促進皮膚及相關軟組織再生的藥劑(即本揭示內容的人類間質幹細胞)。藥劑的有效量非必須治癒疾病或病症但可提供針對一疾病或病症加以治療，像是延後疾病或病症的發作、限制或阻礙疾病或病症的發作、或是使疾病或病症症狀得到改善。特定有效量或足量可隨著各種因素變化，像是預治療的特定病症、病患的生理狀況(例如病患的體重、年紀或性別)、所治療哺乳類或動物的種類、治療時間、並行療程的性質(如果有的話)、以及使用的具體製劑等。有效量可以合適的形式分成一次、兩次或多次劑量，藉此在指定時段內以一次、兩次或多次給藥。As used herein, "an effective amount" refers to a dose of effective human mesenchymal stem cells that achieve the desired result for treating a disease caused by platelet aggregation for a particular desired period of time. For example, an agent that effectively promotes regeneration of the skin and related soft tissues (ie, human mesenchymal stem cells of the present disclosure) for the treatment of wounds. An effective amount of the agent does not necessarily cure the disease or condition but may provide for treatment of a disease or condition, such as delaying the onset of the disease or condition, limiting or impeding the onset of the disease or condition, or improving the symptoms of the disease or condition. A particular effective amount or sufficient amount may vary depending on various factors, such as a particular condition being pre-treated, the physiological condition of the patient (eg, the patient's weight, age, or sex), the type of mammal or animal being treated, the time of treatment, concurrent The nature of the treatment (if any), and the specific formulation used. The effective amount can be divided into one, two or more doses in a suitable form whereby one, two or more administrations are administered over a specified period of time.
在本文中，術語「個體、受試者」(subject)或「患者」(patient)可互換地使用，且意思是指能夠以本揭示內容的化合物治療的哺乳類動物(包含人類)。術語「哺乳動物」(mammal)是指哺乳綱(class Mammalia)的所有成員，其包含人類、靈長類、馴養動物及家畜(例如兔、豬、羊、牛)以及動物園圈養動物、用於運動的動物或寵物；以及囓齒類(例如小鼠與大鼠)。再者，除非明確指出性別，否則「個體」或「受試者」(subject)或「患者」(patient)一詞均有包含男性(雄性)及女性(雌性)。因此，術語「個體、受試者」或「患者」包含受益於本揭示內容的治療方法的任何哺乳動物。「個體、受試者」」或「患者」的實例包含但不限於，人類、大鼠、小鼠、天竺鼠、猴、豬、山羊、牛、馬、犬、貓、鳥及禽類。在較佳的實施方式中，個體是一人類。As used herein, the terms "subject," or "patient" are used interchangeably and are meant to refer to mammals (including humans) that are capable of being treated with a compound of the present disclosure. The term "mammal" refers to all members of the class Mammalia, which include humans, primates, domesticated animals and livestock (eg rabbits, pigs, sheep, cattle) and zoo captive animals for exercise. Animals or pets; and rodents (eg mice and rats). Furthermore, the term "individual" or "subject" or "patient" includes both male (male) and female (female) unless the gender is explicitly stated. Thus, the term "individual, subject" or "patient" encompasses any mammal that benefits from the methods of treatment of the present disclosure. Examples of "individuals, subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and birds. In a preferred embodiment, the individual is a human.
術語「培養液」(medium)是指含有可維持細胞生存力及支持細胞增殖的營養，以用於維持組織或細胞群、或培養含有細胞群(例如培養基質)的基質。細胞培養液含有任何下列物質的適當組合：鹽類、緩衝液、胺基酸、葡萄糖或其他醣類、抗生素、血清或血清替代物、以及其他成分(像是生長因子)等。本發明所屬技術領域具有通常知識者已知特定細胞類型會使用的細胞培養液。The term "medium" refers to a nutrient that maintains cell viability and supports cell proliferation for maintaining a tissue or cell population, or for culturing a cell population (eg, a culture substrate). The cell culture fluid contains any suitable combination of the following: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other ingredients (such as growth factors). The present invention pertains to cell culture fluids that are commonly used by those skilled in the art to know the particular cell type.
本文使用的術語「移動」、「活動」、「轉移」(mobilization)是指細胞離開原本的常駐區位(niche)(例如骨髓)且進入血液的過程。術語「移動劑」(mobilization agent)是可使停留在周邊血液及骨髓幹細胞區位中的幹細胞(例如MSCs)集合體或群體失去附著力的藥劑。As used herein, the terms "mobile," "active," and "mobilization" refer to the process by which a cell leaves its original resident niche (eg, bone marrow) and enters the bloodstream. The term "mobilization agent" is an agent that can cause adhesion or aggregation of stem cells (e.g., MSCs) that reside in peripheral blood and bone marrow stem cell locations.
2. 分離及培養源自周邊血液之人類間質幹細胞2. Isolation and culture of human mesenchymal stem cells derived from peripheral blood
間質幹細胞(mesenchymal stem cells， MSCs)是一般可從骨髓取得的非造血幹細胞。周邊血液中的間質幹細胞含量較低，且即便該些細胞是在體外進行擴增，從周邊血液獲取的間質幹細胞通常無法產生足夠量的細胞以用於治療用途。本研究的發明人意外地發現從周邊血液分離及擴增間質幹細胞的方法，從而可製備實質上純的人類間質幹細胞群，其可單獨使用或與其他細胞合併使用，以於再生療法中進行自體治療，例如傷口癒合及骨骼修復以及其他矯形適應症。Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells that are generally obtained from bone marrow. The amount of mesenchymal stem cells in the peripheral blood is low, and even if the cells are expanded in vitro, mesenchymal stem cells obtained from peripheral blood are generally unable to produce a sufficient amount of cells for therapeutic use. The inventors of the present study unexpectedly discovered a method of isolating and expanding mesenchymal stem cells from peripheral blood, thereby preparing a substantially pure human mesenchymal stem cell population, which can be used alone or in combination with other cells for regenerative therapy. Autologous treatments such as wound healing and bone repair and other orthopedic indications are performed.
據此，本揭示內容的第一個態樣是關於一種提供未分化人類間質幹細胞群體的方法。該方法包含： (a) 從人類個體取得周邊血液； (b) 將甘油添加至步驟(a)之該周邊血液中； (c) 分離步驟(b)之周邊血液中，相對於其他成體幹細胞的人類間質幹細胞；以及 (d) 將步驟(c)之經分離的人類間質幹細胞培養於一包含甘油的培養液中，藉此製備人類間質幹細胞群； 其中培養液不含任何選自由顆粒性白血球群落刺激因子(granulocyte colony stimulating factor，G-CSF)、顆粒性白血球-巨噬細胞群落刺激因子(granulocyte-macrophage colony stimulating factor，GM-CSF)以及其組合所組成的群組之藥劑。Accordingly, a first aspect of the present disclosure is directed to a method of providing a population of undifferentiated human mesenchymal stem cells. The method comprises: (a) obtaining peripheral blood from a human individual; (b) adding glycerol to the peripheral blood of step (a); (c) separating the peripheral blood of step (b) relative to other adult stem cells Human mesenchymal stem cells; and (d) culturing the isolated human mesenchymal stem cells of step (c) in a culture medium containing glycerol, thereby preparing a human mesenchymal stem cell population; wherein the culture solution does not contain any selected from A medicament consisting of a group consisting of a granulocyte colony stimulating factor (G-CSF), a granulocyte-macrophage colony stimulating factor (GM-CSF), and a combination thereof.
根據一些實施方式，在本發明之步驟(a)中，是從一人類個體抽出周邊血液，且將該周邊血液收集於含有抗凝血劑(例如肝素(heparin))之試管中。根據本揭示內容的一實施方式，個體可以是健康個體或罹患亟需人類間質幹細胞移植之疾病或病症的個體。此外，可用於製備本揭示內容人類間質幹細胞的周邊血液的來源個體不限特定年齡。根據本揭示內容之某些實施方式，周邊血液可以從年齡分別介於20歲至30歲之間的人類個體抽取。根據本揭示內容之其他實施方式，周邊血液可以從年齡分別介於50歲至60歲之間的人類個體抽取。根據本揭示內容之其他實施方式，周邊血液可以從年齡分別介於70歲至80歲之間的人類個體抽取。According to some embodiments, in step (a) of the present invention, peripheral blood is withdrawn from a human subject and the peripheral blood is collected in a test tube containing an anticoagulant such as heparin. According to an embodiment of the present disclosure, the individual may be a healthy individual or an individual suffering from a disease or condition requiring human mesenchymal stem cell transplantation. Furthermore, the individual from which the peripheral blood of human mesenchymal stem cells of the present disclosure can be prepared is not limited to a particular age. According to certain embodiments of the present disclosure, peripheral blood may be drawn from a human subject between the ages of 20 and 30 years. According to other embodiments of the present disclosure, peripheral blood may be drawn from a human subject between the ages of 50 and 60 years. According to other embodiments of the present disclosure, peripheral blood may be drawn from a human subject between the ages of 70 and 80 years.
收集周邊血液之後，隨即將其與甘油混合，並於 37℃至少培養4小時，較佳的是至少培養6小時(本揭示內容方法的步驟(b))。根據本揭示內容的實施方式，周邊血液中所含的甘油量較佳地可以是約每毫升1-10 毫克，例如每毫升1、2、3、4、5、6、7、8、9及10 毫克；更佳的是約每毫升 2-8 毫克(例如每毫升2、3、4、5、6、7及8毫克；更佳的是約每毫升5毫克。After collecting the peripheral blood, it is then mixed with glycerol and cultured for at least 4 hours at 37 ° C, preferably for at least 6 hours (step (b) of the method of the present disclosure). According to an embodiment of the present disclosure, the amount of glycerin contained in the peripheral blood may preferably be about 1-10 mg per ml, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 per ml and 10 mg; more preferably about 2-8 mg per ml (e.g. 2, 3, 4, 5, 6, 7, and 8 mg per ml; more preferably about 5 mg per ml).
培養之後，可藉由合適的方式將人類間質幹細胞從含有甘油之周邊血液中的成體幹細胞分離，合適的方式像是細胞分選儀(cell sorter)或離心淘洗(centrifugal elutriation)(本方法的步驟(c))。After culture, human mesenchymal stem cells can be isolated from adult stem cells in the peripheral blood containing glycerol by a suitable means, such as a cell sorter or centrifugal elutriation (this Step (c)) of the method.
根據本揭示內容之某些實施方式，在本方法之步驟(c)，是藉由細胞分選儀(像是雷射掃描流式細胞儀(laser scanning Flow Cytometer))將周邊血液中的人類間質幹細胞從成體幹細胞分離。典型的流式細胞儀是由雷射光、流體測量室(flow measurement chamber)以及光學系統(由鏡片、濾光鏡與光偵測器所組成)構成。兩個光電倍增管(一與雷射夾角為180度且一為90度)是分別用於測量前向散射光(forward scattered light，FSC)及直角散射(側向散射光(side scattered light，SCC))。在前向管中，分別由濾光鏡及光電倍增管組成的三個螢光偵測器可用於偵測螢光。螢光活性細胞分選儀(fluorescence activated cell sorting，FACS)設置以選擇特定的前向散射及/或側向散射之細胞。FSC與細胞表面積或尺寸成比例。FSC大多測量繞射光且其藉由光二極體在正向方向上正好位於入射雷射光束之軸上來偵測。FSC適合用於偵測大於給定尺寸之目標顆粒，而與顆粒的螢光無關。側向散射光(side-scattered light，SCC)是與粒性或內部複雜性成比例。SSC是用以測量在具有折射率變化的細胞內部任何表面(interface)上發生的光折射及光反射。SSC是在與雷射光束夾角約90度之角度，透過一收集透鏡收集該雷射光束，接著透過光束分離器重新定向至合適的偵測器。藉此，可藉由控制特定FSC及SSC閘道來挑選細胞。根據本揭示內容的實施方式，是基於細胞表面標記之表現量將周邊血液的人類間質幹細胞從成體幹細胞中分離，其中細胞群是以不同螢光強度染色的門閘來挑選。According to some embodiments of the present disclosure, in step (c) of the method, the human blood in the peripheral blood is separated by a cell sorter such as a laser scanning flow cytometer. The stem cells are isolated from adult stem cells. A typical flow cytometer consists of a laser beam, a flow measurement chamber, and an optical system (composed of a lens, a filter, and a photodetector). Two photomultiplier tubes (one with an angle of 180 degrees from the laser and one with a degree of 90 degrees) are used to measure forward scattered light (FSC) and right-angle scattering (side scattered light (SCC), respectively. )). In the forward tube, three fluorescent detectors, each consisting of a filter and a photomultiplier tube, can be used to detect fluorescence. Fluorescence activated cell sorting (FACS) is set up to select specific forward scatter and/or side scatter cells. FSC is proportional to cell surface area or size. The FSC mostly measures diffracted light and is detected by the photodiode in the forward direction just above the axis of the incident laser beam. FSC is suitable for detecting target particles larger than a given size, regardless of the fluorescence of the particles. Side-scattered light (SCC) is proportional to graininess or internal complexity. SSC is used to measure light refraction and light reflection that occurs on any interface inside a cell with a change in refractive index. The SSC collects the laser beam through a collecting lens at an angle of about 90 degrees from the laser beam, and then redirects it through a beam splitter to a suitable detector. Thereby, cells can be selected by controlling specific FSC and SSC gates. According to an embodiment of the present disclosure, human mesenchymal stem cells of peripheral blood are separated from adult stem cells based on the amount of expression of cell surface markers, wherein the cell population is selected by gatekeepers stained with different fluorescence intensities.
在步驟(c)中，非必要性地或另外地，將從步驟(b)取得的含有甘油之周邊血液注入細胞分選儀(例如COBE Spectra Apheresis System)中，且可選擇性地添加抗凝血劑以在該流程中使血液免於凝集。將混合物通過離心機循環以將周邊血液分離成間質幹細胞群和來自其他血液成分及血漿的單核細胞。接著將分離的間質幹細胞泵入收集袋中儲存，而將其他血液成分及血漿丟棄或歸還給個體。In the step (c), the glycerin-containing peripheral blood obtained from the step (b) is optionally injected into a cell sorter (for example, COBE Spectra Apheresis System), and optionally, anticoagulation may be added. The blood agent is to protect the blood from agglutination in the process. The mixture is circulated through a centrifuge to separate peripheral blood into a population of mesenchymal stem cells and monocytes from other blood components and plasma. The separated mesenchymal stem cells are then pumped into a collection bag for storage, while other blood components and plasma are discarded or returned to the individual.
在本發明方法的步驟(c)中，可選擇性地或另外地，藉由離心淘洗將含有甘油之周邊血液中的人類間質幹細胞從其他成體幹細胞分離出，其中較小尺寸的幹細胞可從較大尺寸幹細胞中分離出。在特定實施方式，步驟(b)中含有甘油之周邊血液被置入位在自旋離心機中的常規漏斗形分離室(funnel-shaped separation chamber)中。將液體淘洗緩衝液流引入含有周邊血液的分離室中。當液體淘洗緩衝液溶液通過該分離室的流速增加時，液體會將較小尺寸、較慢沉降的細胞掃向分離室的淘洗邊界，而較大較快沉降的細胞會移動至分離室中離心力與沉降力達成平衡之區域。據此，周邊血液可包含許多不同群體的幹細胞，可再透過淘洗將該些幹細胞區分成獨特的群體，其中較小的幹細胞可從具有較大尺寸的幹細胞中分離出。可使用任何淘洗裝置獲得及/或分離從周邊血液衍生的間質幹細胞。In step (c) of the method of the invention, the human mesenchymal stem cells in the peripheral blood containing glycerol may be selectively or additionally separated from other adult stem cells by centrifugation, wherein the smaller size stem cells Can be isolated from larger size stem cells. In a particular embodiment, the peripheral blood containing glycerol in step (b) is placed in a conventional funnel-shaped separation chamber in a spin centrifuge. The liquid panning buffer stream is introduced into a separation chamber containing peripheral blood. As the flow rate of the liquid panning buffer solution through the separation chamber increases, the liquid sweeps the smaller, slower settled cells toward the panning boundary of the separation chamber, while the larger, faster settled cells move to the separation chamber. The area where the centrifugal force and the settling force reach a balance. Accordingly, the peripheral blood may comprise a plurality of different populations of stem cells, which may be further separated into a unique population by panning, wherein the smaller stem cells may be isolated from stem cells having a larger size. Mesenchymal stem cells derived from peripheral blood can be obtained and/or isolated using any panning device.
在本發明之步驟(d)中，使該些從步驟(c)中製備的人類間質幹細胞於可增強該些分離之人類間質幹細胞增殖之培養條件下接受培養，以進一步擴增該些細胞。根據本揭示內容較佳的實施方式，可在培養皿(表面積約10平方公分)中包含甘油的生長培養液裡培養從步驟(c)獲得的人類間質幹細胞，且該生長培養液中缺乏任何已知的幹細胞移動劑(包括，但不限於：顆粒性白血球群落刺激因子(G-CSF)，顆粒性白血球-巨噬細胞群落刺激因子(GM-CSF)以及其組合)。應當理解的是，當說到培養液缺發特定成分時，本揭示內容也預期培養液仍包含該成分，但其濃度遠低於其具有最小活性的濃度。舉例來說，特定培養液可包含微量的前述幹細胞移動劑(即，G-CSF、GM-CSF或其組合)，然而，本揭示內容的方法是關於一種除了原本存在於市售培養液之配方中或由整體調整培養液成分濃度而產生的生長因子之外，不再外源性地添加生長因子的培養液。In the step (d) of the present invention, the human mesenchymal stem cells prepared in the step (c) are cultured under the culture conditions capable of enhancing the proliferation of the isolated human mesenchymal stem cells to further amplify the human mesenchymal stem cells. cell. According to a preferred embodiment of the present disclosure, the human mesenchymal stem cells obtained from the step (c) can be cultured in a growth culture solution containing glycerin in a culture dish (surface area of about 10 cm 2 ), and the growth medium lacks any Known stem cell migration agents (including, but not limited to, particulate leukocyte community stimulating factor (G-CSF), particulate leukocyte-macrophage colony stimulating factor (GM-CSF), and combinations thereof). It will be understood that when it is said that the culture fluid is deficient in a particular component, the present disclosure also contemplates that the culture fluid still contains the component, but at a concentration that is much lower than its concentration with minimal activity. For example, a particular culture fluid may contain minor amounts of the aforementioned stem cell mobile agents (ie, G-CSF, GM-CSF, or a combination thereof), however, the methods of the present disclosure pertain to a formulation other than that originally present in commercially available culture fluids. In addition to the growth factor produced by adjusting the concentration of the culture solution as a whole, the culture solution of the growth factor is no longer exogenously added.
本揭示內容適合用於培養人類間質幹細胞的典型培養液可為完全培養液(例如：DMEM)，其含有至少20% (體積比)的血清，較佳是至少30% (體積比)的血清，更佳是至少50%(體積比)的血清。完全培養液中的甘油濃度可以是約每毫升0.1-1.0 毫克 (例如每毫升 0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9及1.0 毫克)；更可以是約每毫升 0.2-0.8 毫克(例如每毫升0.2、0.3、0.4、0.5、0.6、0.7及0.8毫克)；最佳的是約每毫升 0.5 毫克。在一些實施方式中，用於培養人類間質幹細胞的生長培養液是使用100% (體積比)之血清，再加入每毫升0.1-1.0 毫克的甘油。在培養過程中，也可在培養液中添加細胞新陳代謝所需的補充劑，像是胺基酸、維他命、礦物質以及有用蛋白質(例如運鐵蛋白)等。培養液也可包含抗生素，用以防止酵母菌、細菌及真菌感染。抗生素可以是青黴素(penicillin)、鏈黴素(streptomycin)、建它黴素(gentamicin)之類。根據本揭示內容之一實施方式，培養液包含腦垂腺萃取物、血漿及胎牛血清。可經一段時間的培養，以有效地使人類間質幹細胞擴增，藉此以製備足量的人類間質幹細胞。根據一特定的實施方式，可以含有30% (體積比)之血清及每毫升0.5毫克甘油之培養液，將步驟(c)取得的人類間質幹細胞在37℃至少培養12小時，較佳是至少16小時，更佳的是至少24小時，從而製備適合用於自體移植的人類間質幹細胞群。A typical culture solution suitable for culturing human mesenchymal stem cells in the present disclosure may be a complete culture solution (for example, DMEM) containing at least 20% by volume of serum, preferably at least 30% by volume of serum. More preferably, it is at least 50% (by volume) of serum. The concentration of glycerol in the complete culture solution may be about 0.1-1.0 mg per ml (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg per ml); more preferably about 0.2-ml. 0.8 mg (eg 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mg per ml); optimally about 0.5 mg per ml. In some embodiments, the growth medium for culturing human mesenchymal stem cells is 100% (by volume) serum, and then 0.1-1.0 mg glycerol per ml. In the culture process, supplements required for cell metabolism such as amino acids, vitamins, minerals, and useful proteins (such as transferrin) may also be added to the culture solution. The culture fluid may also contain antibiotics to prevent infection by yeast, bacteria and fungi. The antibiotic may be penicillin, streptomycin, gentamicin or the like. According to an embodiment of the present disclosure, the culture solution comprises a pituitary gland extract, plasma, and fetal bovine serum. Culture can be carried out for a period of time to effectively amplify human mesenchymal stem cells, thereby preparing a sufficient amount of human mesenchymal stem cells. According to a specific embodiment, the human mesenchymal stem cells obtained in the step (c) may be cultured at 37 ° C for at least 12 hours, preferably at least 30% (by volume) of serum and 0.5 mg of glycerol per ml of the culture solution. 16 hours, more preferably at least 24 hours, to prepare a population of human mesenchymal stem cells suitable for autologous transplantation.
根據本揭示內容的特定的實施方式，本發明製備的人類間質幹細胞群對造血幹細胞標記物CD34及CD45呈現陰性染色，以及已知可介導移植物抗宿主疾病(graft-versus-host disease，GvHD)的人類白血球抗原–抗原D相關(HLA-DR)細胞表面標記也呈現陰性染色；對細胞表面標記CD73、CD90及CD 105則呈現陽性染色。測定細胞表面標記表現的方法是本技術領域的通常知識。該些方法實例包含免疫學方法(例如FACS)以及生物化學方法(例如透過放射性、螢光或卵白素-生物素的細胞表面標記)。另一種可行的方式是，可使用磁激細胞分選(magnetic-activated cell sorting，MACS)或免疫淘洗(immunopanning)方法以鑑別細胞。According to a particular embodiment of the present disclosure, the human mesenchymal stem cell population prepared according to the present invention exhibits negative staining for hematopoietic stem cell markers CD34 and CD45, and is known to mediate graft-versus-host disease (graft-versus-host disease, GvHD) also showed negative staining for human leukocyte antigen-antigen D-associated (HLA-DR) cell surface markers; positive staining for cell surface markers CD73, CD90 and CD 105. Methods for determining the expression of cell surface markers are common knowledge in the art. Examples of such methods include immunological methods (e.g., FACS) as well as biochemical methods (e.g., by cell surface markers that are radioactive, fluorescent, or avidin-biotin). Another possible way is to use magnetic-activated cell sorting (MACS) or immunopanning methods to identify cells.
透過本揭示內容方法所製備的人類間質幹細胞群之細胞表面標記表現模式證明：該些人類間質幹細胞群的確是未分化的成熟人類間質幹細胞之富集群，其可用於需要自體幹細胞移植的再生療法。The cell surface marker expression pattern of the human mesenchymal stem cell population prepared by the method of the present disclosure proves that the human mesenchymal stem cell population is indeed a rich cluster of undifferentiated mature human mesenchymal stem cells, which can be used for autologous stem cell transplantation. Regenerative therapy.
3. 人類間質幹細胞單羣性細胞株3. Human mesenchymal stem cell single cell line
本揭示內容的另一態樣是關於包含實質上純的未分化人類間質幹細胞群之單羣性細胞株，其中可根據任何前述方法製備未分化之人類間質幹細胞群。Another aspect of the present disclosure is directed to a single population of cell lines comprising a substantially pure population of undifferentiated human mesenchymal stem cells, wherein an undifferentiated population of human mesenchymal stem cells can be prepared according to any of the foregoing methods.
根據本揭示內容的實施方式，藉由前述方法製備或分離的新鮮人類間質幹細胞在無分化的條件下(例如缺乏任何分化因子)培養，以使人類間質幹細胞進一步擴增。通常，人類間質幹細胞從其宿主分離或被挑選以製備一人類間質幹細胞群(例如：在本揭示內容方法的步驟(d)中所製備的人類間質幹細胞)之後，該些細胞(可以是一異質細胞群或一純的人類間質幹細胞群)可在具有一預定表面積的培養皿(該預定表面積是步驟(d)培養皿表面積的至少16倍大、至少17倍大、至少18倍大或至少20倍大)中接受後續培養，也可以前述的含甘油培養液(即，步驟(d)的培養液)繼續培養。可選地或可替換地，可在生物反應器中培養人類間質幹細胞，從而生產大量的細胞。根據本揭示內容的實施方式，適合用於本發明方法步驟的培養液是缺乏任何細胞移動劑(例如G-CSF、GM-CSF及其組合)；同時該培養液包含具特定濃度的甘油，特定濃度較佳是約每毫升0.1-1.0 毫克，例如每毫升0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9及1.0 毫克；更佳地是約每毫升0.2-0.8 毫克，諸如每毫升0.2、0.3、0.4、0.5、0.6、0.7及0.8毫克；最佳是約每毫升0.5 毫克。對培養物進行培養一段時間，較佳地是至少培養7天、更佳是至少培養10天，及更佳是至少培養14天，以製備同質未分化之人類間質幹細胞群，其包含較佳至少70%的人類間質幹細胞、更佳是至少80%的人類間質幹細胞，及最佳是至少90%之人類間質幹細胞。According to an embodiment of the present disclosure, fresh human mesenchymal stem cells prepared or isolated by the aforementioned methods are cultured under conditions of no differentiation (for example, lacking any differentiation factor) to further expand human mesenchymal stem cells. Typically, after human mesenchymal stem cells are isolated from their host or selected to produce a population of human mesenchymal stem cells (eg, human mesenchymal stem cells prepared in step (d) of the methods of the present disclosure), the cells Is a heterogeneous cell population or a pure human mesenchymal stem cell population) in a petri dish having a predetermined surface area (the predetermined surface area is at least 16 times larger, at least 17 times larger, at least 18 times larger than the surface area of the step (d) culture dish The subsequent culture is carried out in a large or at least 20 times larger), and the glycerin-containing culture solution (i.e., the culture solution of the step (d)) may be further cultured. Alternatively or alternatively, human mesenchymal stem cells can be cultured in a bioreactor to produce a large number of cells. According to an embodiment of the present disclosure, a culture fluid suitable for use in the method steps of the present invention is devoid of any cell mobile agent (eg, G-CSF, GM-CSF, and combinations thereof); while the culture fluid contains glycerin at a specific concentration, specific The concentration is preferably about 0.1 to 1.0 mg per ml, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg per ml; more preferably about 0.2 to 0.8 mg per ml, such as per ML 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mg; optimally about 0.5 mg per ml. The culture is cultured for a period of time, preferably at least 7 days, more preferably at least 10 days, and more preferably at least 14 days to prepare a homogenous undifferentiated human mesenchymal stem cell population, preferably comprising At least 70% of human mesenchymal stem cells, more preferably at least 80% of human mesenchymal stem cells, and optimally at least 90% of human mesenchymal stem cells.
根據本揭示內容的實施方式，可將人類間質幹細胞擴增成至少兩倍、至少四倍、至少六倍、至少八倍、至少十倍、至少十二倍、至少十五倍、至少二十、至少三十倍、或至少四十倍。According to an embodiment of the present disclosure, human mesenchymal stem cells can be expanded to at least two times, at least four times, at least six times, at least eight times, at least ten times, at least twelve times, at least fifteen times, at least twenty At least thirty times, or at least forty times.
與前述步驟(d)的發現相似的是，所製備的同質人類間質幹細胞群維持未分化狀態，且對造血幹細胞標記物CD34及CD45以及細胞表面標記HLA-DR細胞表面標記呈現陰性染色；另同時對細胞表面標記CD73、CD90及CD 105呈現陽性染色。Similar to the discovery of the foregoing step (d), the prepared homogenous human mesenchymal stem cell population maintains an undifferentiated state, and negative staining for hematopoietic stem cell markers CD34 and CD45 and cell surface marker HLA-DR cell surface markers; At the same time, the cell surface markers CD73, CD90 and CD 105 showed positive staining.
使用發明之方法所製備的人類間質幹細胞群培養物可在新鮮狀態下使用，亦可以儲存以待後續使用，例如以冷凍保存劑(包括但不限於甘油、二甲亞碸(DMSO)等)冷凍保存。The human mesenchymal stem cell population culture prepared by the method of the invention can be used in a fresh state, or can be stored for later use, for example, a cryopreservation agent (including but not limited to glycerin, dimethyl hydrazine (DMSO), etc.) Store frozen.
4. 含有人類間質幹細胞的組合物4. Composition containing human mesenchymal stem cells
本揭示內容的人類間質幹細胞可以自身單獨提供、與培養液一起提供，以及與藥學上可接受的載體及其他添加劑合併提供，其中該添加劑可促進細胞植入及/或器官功能(例如：免疫抑制劑、抗生素及生長因子等)。因此，本揭示內容的人類間質幹細胞可以藥學組合物的形式投予至個體體內，在該藥學組合物中，人類間質幹細胞可與藥學載體或稀釋劑混合，例如與無菌食鹽水及水性緩衝溶液混合。The human mesenchymal stem cells of the present disclosure may be provided by themselves, provided with a culture fluid, and provided in combination with a pharmaceutically acceptable carrier and other additives, wherein the additive may promote cell implantation and/or organ function (eg, immunization) Inhibitors, antibiotics and growth factors, etc.). Thus, human mesenchymal stem cells of the present disclosure can be administered to a subject in the form of a pharmaceutical composition in which human mesenchymal stem cells can be mixed with a pharmaceutical carrier or diluent, for example, with sterile saline and aqueous buffer. The solution is mixed.
合適的投予途徑可包括但不限：口服、直腸、經黏膜(例如經鼻腔)、腸內或非口服遞送(包括：肌內、皮下、心室內、鞘內、靜脈內、腹膜內或眼內注射)。Suitable routes of administration may include, but are not limited to, oral, rectal, transmucosal (eg, nasal), enteral or non-oral delivery (including: intramuscular, subcutaneous, intraventricular, intrathecal, intravenous, intraperitoneal, or intraocular) Injection inside).
或者是，可以局部方式而非全身性方式投予該藥學組合物。舉例來說，可將藥學組合物直接注射至目標位置(例如：一器官)。Alternatively, the pharmaceutical composition can be administered in a local rather than systemic manner. For example, the pharmaceutical composition can be injected directly to a target location (eg, an organ).
根據本揭示內容，可以常規方式利用一或多種藥學上可接受的載體製備藥學組合物。合適的製劑形式取決於投予或給藥途徑。In accordance with the present disclosure, pharmaceutical compositions can be prepared in a conventional manner using one or more pharmaceutically acceptable carriers. A suitable form of preparation will depend on the route of administration or administration.
為了注射，本揭示內容的人類間質幹細胞可製成水性溶液製劑，較佳地是製成生理上可接受緩衝液(例如林格氏液)或生理上可接受的鹽類溶液。For injection, the human mesenchymal stem cells of the present disclosure may be formulated as an aqueous solution, preferably as a physiologically acceptable buffer (e.g., Ringer's solution) or as a physiologically acceptable salt solution.
適合用於本揭示內容的藥學組合物包含含有可達成預期目的之有效量活性成分(例如，人類間質幹細胞)之組合物。有效量的測定完全是本技術領域具有通常知識者的能力範圍內，特別是根據本揭示內容的描述。Pharmaceutical compositions suitable for use in the present disclosure comprise a composition comprising an effective amount of the active ingredient (e.g., human mesenchymal stem cells) that achieves the desired purpose. Determination of an effective amount is well within the capabilities of those of ordinary skill in the art, particularly in light of the description of the present disclosure.
本揭示內容的藥學組合物可以套組形式存在，該套組可含有包含本發明人類間質幹細胞的一或多種單位劑量形式。套組可進一步包含在套組上或是與該套組相連的標籤或包裝附件。標籤或包裝附件可載明該套組是用於治療特定疾病及/或病症。可替換地或可額外地，套組可進一步包括一緩衝液，諸如磷酸鹽緩衝食鹽水或林格氏液。套組可進一步包含關於如何將人類間質幹細胞投予至預定目標位置的指示。The pharmaceutical compositions of the present disclosure may be in the form of a kit, which may contain one or more unit dosage forms comprising human mesenchymal stem cells of the invention. The kit can further include a label or package attachment on the kit or connected to the kit. The label or package attachment may indicate that the kit is for treating a particular disease and/or condition. Alternatively or additionally, the kit may further comprise a buffer such as phosphate buffered saline or Ringer's solution. The kit can further include instructions on how to administer human mesenchymal stem cells to a predetermined target location.
根據本揭示內容之實施方式，套組可包含(或至少包含)：(a)含有本發明人類間質幹細胞的第一容器，以及可選地(b)含有緩衝液的第二容器，以及(c)與套組相關以指示使用者如何使用該套組的圖例。圖例可以是小冊子、磁帶、CD、VCD 或 DVD之形式。According to an embodiment of the present disclosure, the kit may comprise (or at least comprise): (a) a first container containing the human mesenchymal stem cells of the invention, and optionally (b) a second container containing a buffer, and c) A legend associated with the set to indicate how the user is using the set. The illustration can be in the form of a booklet, tape, CD, VCD or DVD.
5. 本發明人類間質幹細胞或其分化子代之用途5. Use of human mesenchymal stem cells or differentiated progeny thereof of the present invention
透過上述本發明方法所製備的未分化人類間質幹細胞群或其分化子代可用於自體移植，藉以治療及/或預防無數可藉由自體移植獲得有益效果的疾病及/或病症。The undifferentiated human mesenchymal stem cell population or its differentiated progeny prepared by the above method of the present invention can be used for autologous transplantation, thereby treating and/or preventing countless diseases and/or conditions which can be beneficially obtained by autologous transplantation.
據此，本揭示內容的其他態樣是關於治療個體的疾病及/或病症的方法，其包含在幾天至幾周的期間內，對個體投予一有效量之依據本發明方法所製備之人類間質幹細胞群。Accordingly, other aspects of the present disclosure are directed to methods of treating a disease and/or condition in an individual comprising administering to the individual an effective amount of the method according to the method of the invention over a period of days to weeks. Human mesenchymal stem cell population.
間質幹細胞可以分化成各種細胞系。可透過在富含具有所需表現型的細胞(例如骨細胞、脂肪細胞等)之生長環境培養間質幹細胞，以達成細胞分化。培養液可包含增強分化至特定細胞系的試劑。根據本揭示內容之一實施方式，將人類間質幹細胞群分化成軟骨細胞，其中細胞在含有地塞米松(dexamethasone)、抗壞血酸(ascorbic acid)、胰島素(insulin)、運鐵蛋白及亞硒酸(selenous acid)之培養液中進行培養直到滿盤(confluence) (請參考Williams et al. (2003) Tissue Engineering. 9(4), 679)。分化後的軟骨細胞可藉由鹼性磷酸酶染色以鑑定。根據本揭示內容的另一實施方式，是透過將人類間質幹細胞群在市售軟骨培養液中進行培養，以使人類間質幹細胞分化成軟骨。以愛爾新藍(Alcian Blue)進行蛋白多醣染色以確認分化的軟骨。根據本揭示內容的再一實施方式，人類間質幹細胞群是分化成脂肪細胞。為了誘導脂肪生成的分化，以脂肪生成培養液處理人類間質幹細胞，其含有氫皮質酮(hydrocortisone)及吲哚美辛(indomethacin)；或者是或此外，也可額外使用包含任何市售間質幹細胞脂肪生成促進補充劑(例如從StemCell Technologies Inc (溫哥華，加拿大)購得的補充劑)。可藉由油紅染色確認脂肪生成分化結果。Mesenchymal stem cells can differentiate into various cell lines. Cell differentiation can be achieved by culturing mesenchymal stem cells in a growth environment rich in cells having a desired phenotype (eg, bone cells, fat cells, etc.). The culture broth may comprise an agent that enhances differentiation into a particular cell line. According to one embodiment of the present disclosure, a human mesenchymal stem cell population is differentiated into chondrocytes, wherein the cells contain dexamethasone, ascorbic acid, insulin, transferrin, and selenite ( Incubate in the culture medium of selenous acid until confluence (please refer to Williams et al. (2003) Tissue Engineering. 9(4), 679). The differentiated chondrocytes can be identified by alkaline phosphatase staining. According to another embodiment of the present disclosure, human mesenchymal stem cells are cultured in a commercially available cartilage culture solution to differentiate human mesenchymal stem cells into cartilage. Proteoglycan staining was performed with Alcian Blue to confirm differentiated cartilage. According to still another embodiment of the present disclosure, the human mesenchymal stem cell population is differentiated into adipocytes. In order to induce differentiation of adipogenesis, human mesenchymal stem cells are treated with an adipogenic culture medium containing hydrocortisone and indomethacin; or alternatively, any commercially available interstitial may be included. Stem cell lipogenesis promoting supplements (e.g., supplements available from StemCell Technologies Inc (Vancouver, Canada)). The result of adipogenic differentiation can be confirmed by oil red staining.
根據本揭示內容之實施方式，人類間質幹細胞群或其分化子代可用於治療選自由骨或軟骨疾病、神經變性疾病、心臟疾病、肝臟疾病、癌症、自體免疫疾病、移植物抗宿主疾病(GvHD)及傷口癒合與組織再生所組成之群組的疾病及/或病症。According to an embodiment of the present disclosure, a human mesenchymal stem cell population or a differentiated progeny thereof can be used to treat a disease selected from the group consisting of bone or cartilage diseases, neurodegenerative diseases, heart diseases, liver diseases, cancer, autoimmune diseases, graft versus host diseases. (GvHD) and diseases and/or conditions of the group consisting of wound healing and tissue regeneration.
利用本發明方法製備的人類間質幹細胞群或其分化子代可適合用於治療骨缺陷，其包含，但不限於：骨發生不全(osteogenesis imperfecta)、骨折(fracture)、先天骨缺損(congenital bone defects)等。The human mesenchymal stem cell population or its differentiated progeny prepared by the method of the present invention may be suitably used for the treatment of bone defects including, but not limited to, osteogenic imperfecta, fracture, congenital bone defect (congenital bone). Defects and so on.
也可將透過前述本發明方法獲得的人類間質幹細胞群或其分化子代植入一個體中以提供骨頭及其他(例如牙齒)人工裝置的骨性及結締性支持，像是關節植入物及/或牙齒植入物。The human mesenchymal stem cell population obtained by the method of the present invention or a differentiated progeny thereof can also be implanted into a body to provide bone and connective support for bone and other (e.g., dental) artificial devices, such as joint implants. And / or dental implants.
由於間質幹細胞可分化成軟骨，因此，以本發明方法製備的人類間質幹細胞群或其分化子代可適合用於治療關節病症，其包含但不限於：骨關節炎(osteoarthritis)、類風溼性關節炎(rheumatoid arthritis)、發炎性關節炎(inflammatory arthritis)、軟骨軟化(chondromalacia)、缺血性壞死(avascular necrosis)、外傷性關節炎(traumatic arthritis)等。Since mesenchymal stem cells can differentiate into cartilage, the human mesenchymal stem cell population prepared by the method of the present invention or a differentiated progeny thereof can be suitably used for treating joint disorders including, but not limited to, osteoarthritis, rheumatoid. Rheumatoid arthritis, inflammatory arthritis, chondromalacia, avascular necrosis, traumatic arthritis, and the like.
本發明方法製備的人類間質幹細胞群也可用於治療中樞神經系統(CNS)疾病。中樞神經系統疾病的實例包含但不限於：一疼痛疾病、一運動障礙、一多重人格障礙(dissociative disorder)、一情感疾病(mood disorder)、一情緒失調(affective disorder)、一神經變性疾病以及一痙攣性障礙(convulsive disorder)。這類的疾患的更多特定實例包含，但不限於：帕金森氏症(Parkinson’s disease)、肌肉萎縮性脊髓側索硬化症(amyotrophic lateral sclerosis，ALS)、多發性硬化症(multiple sclerosis)、亨丁頓氏症(Huntington’s disease)、自體免疫腦脊髓炎(autoimmune encephalomyelitis)、糖尿病性神經病變(diabetic neuropathy)、青光眼型神經病變(glaucomatous neuropathy)、黃斑點退化(macular degeneration)、動作性震顫(action tremors)以及遲發性運動障礙(tardive dyskinesia)、恐慌、焦慮憂鬱、酒精中毒(alcoholism)、失眠(insomnia)、狂躁行為(manic behavior)、阿茲海默症(Alzheimer’s disease)以及癲癇(epilepsy)。The human mesenchymal stem cell population prepared by the method of the invention can also be used to treat central nervous system (CNS) diseases. Examples of central nervous system diseases include, but are not limited to, a pain disorder, a movement disorder, a dissociative disorder, a mood disorder, an affective disorder, a neurodegenerative disorder, and A convulsive disorder. More specific examples of such disorders include, but are not limited to, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, and hen Huntington's disease, autoimmune encephalomyelitis, diabetic neuropathy, glaucomatous neuropathy, macular degeneration, action tremor Action tremors) and tardive dyskinesia, panic, anxiety and depression, alcoholism, insomnia, manic behavior, Alzheimer's disease, and epilepsy ).
已知間質幹細胞可與造血幹細胞與免疫細胞交互作用，並展現可增進自體造血植入及避免移植物抗宿主疾病(GvHD)的細胞療法潛能。據此，透過本發明方法製備的人類間質幹細胞群也可用於治療GvHD。Mesenchymal stem cells are known to interact with hematopoietic stem cells and immune cells and exhibit cellular therapeutic potential that enhances autologous hematopoiesis and avoids graft versus host disease (GvHD). Accordingly, a population of human mesenchymal stem cells prepared by the method of the present invention can also be used to treat GvHD.
本發明方法製備的人類間質幹細胞群或其分化子代也可用於促進組織再生。如此一來，人類間質幹細胞的移植可用於治療自體免疫疾病、發炎性疾病、急性及慢性缺血性病症、組織工程；也可重生新組織並自然地治癒發病器官或受損器官。The human mesenchymal stem cell population or its differentiated progeny prepared by the method of the invention can also be used to promote tissue regeneration. In this way, transplantation of human mesenchymal stem cells can be used to treat autoimmune diseases, inflammatory diseases, acute and chronic ischemic diseases, tissue engineering, or to regenerate new tissues and naturally heal the affected organs or damaged organs.
已知的是當將間質幹細胞導入梗塞心臟時，該些細胞可防止有害的(心室)重構(remodeling)並增進其復原。直接將間質幹細胞注入梗塞的心臟，或將該些細胞經靜脈投予，均可使細胞回到損傷位置。據此，本發明的人類間質幹細胞群或其分化子代也可用於治療心臟疾病，特別是心臟梗塞之形成。It is known that when mesenchymal stem cells are introduced into an infarcted heart, the cells can prevent harmful (ventricular) remodeling and enhance their recovery. Direct injection of mesenchymal stem cells into the infarcted heart, or intravenous administration of these cells, can return the cells to the site of injury. Accordingly, the human mesenchymal stem cell population of the present invention or a differentiated progeny thereof can also be used for the treatment of heart diseases, particularly the formation of cardiac infarction.
本發明人類間質幹細胞群或其分化子代可治療的癌症的實例包括但不限於，乳癌(breast cancer)、腦腫瘤(brain tumor)、黑色素瘤(melanoma)、肺癌(lung cancer)、淋巴瘤(lymphoma)、神經上皮細胞瘤(neuroepithelioma)、腎臟癌(kidney cancer)、前列腺癌(prostate cancer)、胃癌(stomach cancer)、大腸癌(colon cancer)、直腸癌(rectal cancer)、胰臟癌(pancreatic cancer)以及子宮癌(uterus cancer)。在一些實施方式中，癌症是轉移性癌症。Examples of cancers treatable by the human mesenchymal stem cell population or its differentiated progeny of the invention include, but are not limited to, breast cancer, brain tumor, melanoma, lung cancer, lymphoma. (lymphoma), neuroepithelioma, kidney cancer, prostate cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer Pancreatic cancer) and uterus cancer. In some embodiments, the cancer is a metastatic cancer.
可使用各種移植方法對治療的個體投予本發明的人類間質幹細胞群或其分化子代，其性質取決於植入部位。可將細胞移植入組織的損傷或健康區域。將人類間質幹細胞投予至健康區域的情況下，該些細胞將會移動至損傷區域。The human mesenchymal stem cell population of the present invention or a differentiated progeny thereof can be administered to a treated individual using various methods of transplantation, the nature of which depends on the site of implantation. Cells can be transplanted into damaged or healthy areas of the tissue. In the case where human mesenchymal stem cells are administered to a healthy area, the cells will move to the damaged area.
可藉由直接注射至一器官、直接注射至血流、腹腔注射、直接注射至淋巴器官的手段移植本發明人類間質幹細胞群或其分化子代。合適的移植方法可藉由監控植入細胞對預定器官的回溯及植入情況、預定器官特定標記物的表現、以及該個體的預定器官功能來決定。The human mesenchymal stem cell population of the present invention or its differentiated progeny can be transplanted by direct injection into an organ, direct injection into the bloodstream, intraperitoneal injection, or direct injection into the lymphoid organs. A suitable method of transplantation can be determined by monitoring the retrospective and implantation of the implanted cells to the predetermined organ, the performance of the predetermined organ-specific marker, and the predetermined organ function of the individual.
以在個體中可達成預期治療效果的前提投予的本發明有效量之人類間質幹細胞群或其分化子代。在本發明中使用的人類間質幹細胞有效劑量可從體內及/或體外細胞培養測定中初步估計。舉例來說，可在實驗動物中調配可達成期望濃度的劑量，然後使用該些資訊來更準確地確定用於人類的劑量。劑量可取決於投予或給藥途徑。確切的劑量以及給藥/投予途徑可以由主治醫師根據患者的狀況及病史來確定。An effective amount of a human mesenchymal stem cell population or a differentiated progeny thereof of the present invention administered on the premise that an expected therapeutic effect can be achieved in an individual. The effective dose of human mesenchymal stem cells used in the present invention can be initially estimated from in vivo and/or in vitro cell culture assays. For example, a dose that achieves a desired concentration can be formulated in an experimental animal and then used to more accurately determine the dose for humans. The dosage can depend on the route of administration or administration. The exact dose and route of administration/administration can be determined by the attending physician based on the condition and medical history of the patient.
取決待治療的病症，本發明有效量之人類間質幹細胞可以是單一劑量或多劑量，治療過程可持續數天至數周，或直到有效治癒或疾病的症狀減少為止。Depending on the condition to be treated, an effective amount of human mesenchymal stem cells of the invention may be in single or multiple doses, the course of treatment may last from days to weeks, or until the effective cure or symptoms of the disease are reduced.
對本領域具有通常知識者來說，透過以下非限制性的實施例的檢驗，本發明的額外目的、優點及特徵將會顯而易見。Additional objects, advantages and features of the invention will become apparent from the <RTIgt;
實施例Example
材料與方法Materials and Methods
表面抗原分析Surface antigen analysis
使用0.25%的胰蛋白酶(trypsin)-EDTA從細胞培養皿上收取細胞。以含有1%的BSA之磷酸鹽緩衝生理食鹽水(Phosphate buffered saline，PBS)溶液洗滌細胞，並以螢光素異硫氰酸酯(fluorescein isothiocyanide，FITC)或藻紅素(phycoerythrin，PE)-複合抗體於4℃下染色30分鐘。間質幹細胞標記物：抗-CD29 (整聯蛋白β1鏈(integrin β1 chain))、抗-CD105 (SH2，內皮蛋白(endoglin)CD 73 (+))、以及造血幹細胞標記物：抗-CD34(負控制組)；以及白血球標記物：抗-CD45(白血球共同抗原)。接著以PBS洗滌細胞並使用流式細胞儀(產品名：FACS calibur flow cytometer，購自FACSCanto, BD Biosciences, Becton, Dickinson and Company, San Jose, CA)分析。細胞以高達1,000細胞/秒的速率穿過，並使用488 nm的氬氣雷射光束作為激發光源。經FITC與PE-複合的同型控制組抗體染色的細胞可用於計算背景螢光值。Cells were harvested from cell culture dishes using 0.25% trypsin-EDTA. The cells were washed with a solution of 1% BSA in Phosphate buffered saline (PBS), and fluorescein isothiocyanide (FITC) or phycoerythrin (PE)- The composite antibody was stained at 4 ° C for 30 minutes. Mesenchymal stem cell markers: anti-CD29 (integrin β1 chain), anti-CD105 (SH2, endoglin CD 73 (+)), and hematopoietic stem cell marker: anti-CD34 ( Negative control group); and white blood cell marker: anti-CD45 (white blood cell common antigen). The cells were then washed with PBS and analyzed using a flow cytometer (product name: FACS calibur flow cytometer, available from FACS Canto, BD Biosciences, Becton, Dickinson and Company, San Jose, CA). The cells were passed at a rate of up to 1,000 cells/second and an argon laser beam of 488 nm was used as the excitation source. Cells stained with FITC and PE-complexed isotype control antibody can be used to calculate background fluorescence values.
使用成骨分化套組(商品名：STEMPRO osteogenesis differentiation kit， Gibco)誘發人類間質幹細胞以形成骨細胞，以及使用脂肪生成分化套組(商品名：STEMPRO adipogenic differentiation kit，Gibco)以及軟骨誘導培養液(Chondrogenic induction medium, Gibco)誘導以形成脂肪細胞及軟骨細胞。接著在37℃之溫度下，於95%的空氣及5%的CO2加濕培養箱中維持該些細胞生存狀態。14天的期間內，每2至5天更換培養液。根據標準流程以愛爾新藍染色以評估分化的軟骨。根據標準流程以油紅-O染色以評估分化的脂肪細胞。Using human osteogenic differentiation kit (trade name: STEMPRO osteogenesis differentiation kit, Gibco) to induce human mesenchymal stem cells to form bone cells, and using a lipogenic differentiation kit (trade name: STEMPRO adipogenic differentiation kit, Gibco) and cartilage-inducing culture solution (Chondrogenic induction medium, Gibco) induces the formation of adipocytes and chondrocytes. The cell viability was then maintained in a 95% air and 5% CO2 humidified incubator at 37 °C. The medium was changed every 2 to 5 days during the 14-day period. Aerin blue staining was performed according to standard procedures to evaluate differentiated cartilage. Oil red-O staining was performed according to standard procedures to evaluate differentiated adipocytes.
實施例1：製備及培養人類間質幹細胞(human mesenchymal stem cells，hMSCs)Example 1: Preparation and culture of human mesenchymal stem cells (hMSCs)
本研究包含15位人類受試者個體，皆均獲得其書面同意。將該些受試者根據年齡分成不同的組別，分別是：50至60歲一組、70至80歲一組或20至30歲一組，每組五位受試者。對每位受試者抽血(10毫升)，並將血液收集於含有肝素的試管中，接著添加0.5 毫升的甘油 (每毫升100 毫克)，將含有甘油的血液置於37℃培養6小時。接著，以3,500 rpm之轉速離心含有甘油的血液18分鐘。離心之後，萃取試管中層的細胞並於PBS溶液(8 毫升)中再懸浮。以18 × g之轉速再次離心細胞懸浮液13分鐘，丟棄上清液，萃取試管中層的細胞並種植於含10 毫升市售培養液(thermo-life)之細胞培養盤中(表面積約10平方公分)，該市售培養液包含：500 毫升的角質細胞(keratinocyte)-SFM、2.5 毫升的牛腦下垂體萃取液、13%的人類新鮮冷凍血漿(fresh frozen plasma，FFP)、30%的胎牛血清、15 毫升的甘油 (每毫升100 毫克)以及表皮生長因子(epidermal growth factor，EGF，2.5 微克)。將種滿細胞的培養盤置於條件為37℃溫度、相對溼度(RH)70%、5% CO2的培養箱中，培養16至18小時。將細胞收取下來並重新種植至另一個含有前述培養液(thermo-life)的培養盤(表面積約175平方公分)上。每三天更換培養液，培養液的更換至少重複一次。間質幹細胞在第5天時開始出現，最後在約第7天時達到滿盤(confluence)。將獲得的人類間質幹細胞分離並儲存於零下溫度直到後續使用。The study included 15 human subjects with their written consent. The subjects were divided into different groups according to age, namely: a group of 50 to 60 years old, a group of 70 to 80 years old, or a group of 20 to 30 years old, with five subjects in each group. Blood was drawn from each subject (10 ml), and blood was collected in a test tube containing heparin, followed by the addition of 0.5 ml of glycerol (100 mg per ml), and the blood containing glycerol was cultured at 37 ° C for 6 hours. Next, blood containing glycerol was centrifuged at 3,500 rpm for 18 minutes. After centrifugation, the cells in the middle layer of the tube were extracted and resuspended in PBS solution (8 ml). The cell suspension was again centrifuged at 18 × g for 13 minutes, the supernatant was discarded, and the cells in the middle layer of the test tube were extracted and planted in a cell culture dish containing 10 ml of a commercial-thermo-life (surface area of about 10 cm 2 ). The commercially available culture solution comprises: 500 ml of keratinocyte-SFM, 2.5 ml of bovine pituitary extract, 13% of fresh frozen plasma (FFP), 30% of fetal calf Serum, 15 ml of glycerol (100 mg per ml) and epidermal growth factor (EGF, 2.5 μg). The cell-filled culture dish was placed in an incubator at a temperature of 37 ° C, a relative humidity (RH) of 70%, and 5% CO2 , and cultured for 16 to 18 hours. The cells were collected and re-planted onto another plate containing the aforementioned thermo-life (surface area of about 175 square centimeters). The culture solution was changed every three days, and the replacement of the culture solution was repeated at least once. Mesenchymal stem cells began to appear on day 5 and finally reached confluence on about day 7. The obtained human mesenchymal stem cells are separated and stored at sub-zero temperature until subsequent use.
進一步，發現到人類間質幹細胞可成功地從一高齡受試者(諸如介於70至80歲的個體)的周邊血液中離體取得並擴增。Further, it was found that human mesenchymal stem cells can be successfully obtained and expanded ex vivo from peripheral blood of an elderly subject, such as an individual between 70 and 80 years old.
實施例 2：實施例1之人類間質幹細胞的特性分析Example 2: Characterization of human mesenchymal stem cells of Example 1
2.1細胞表面抗原分析2.1 Cell surface antigen analysis
使用胰蛋白酶/EDTA分離實施例1之黏附細胞，並根據「材料與方法」一節所描述的流程，使該些細胞於流式細胞儀中接受表面抗原分析。第1圖繪示該實驗結果。The adherent cells of Example 1 were isolated using trypsin/EDTA and subjected to surface antigen analysis in a flow cytometer according to the procedure described in the section "Materials and Methods". Figure 1 shows the results of this experiment.
實施例1的細胞對表面標記物CD73 (數據未示出)、CD90(第1A圖)以及CD105(第1B圖)呈現陽性反應，而對CD34 (第1C圖)、CD45 (數據未示出)以及HLA-DR (數據未示出)呈現陰性反應；這些結果證實該些細胞的確是間質幹細胞。The cells of Example 1 showed a positive reaction to surface markers CD73 (data not shown), CD90 (Fig. 1A), and CD105 (Fig. 1B), while CD34 (Fig. 1C), CD45 (data not shown). And HLA-DR (data not shown) showed a negative response; these results confirmed that the cells were indeed mesenchymal stem cells.
2.2實施例1之人類間質幹細胞之分化2.2 Differentiation of human mesenchymal stem cells of Example 1
在本實施例中，為了證實實施例1的人類間質幹細胞仍具備分化的能力，將該些細胞分別於含有可誘發後續人類間質幹細胞分化之試劑的培養液中進行培養。可根據標準流程，將從實施例1分化而來的軟骨細胞、軟骨以及脂肪細胞，分別以鹼性磷酸酶染色、以愛爾新藍染色以及油紅-O染色來確認其細胞型態，該結果分別顯示於第2A圖、第2B圖以及第2C圖。In the present example, in order to confirm that the human mesenchymal stem cells of Example 1 still have the ability to differentiate, the cells are cultured in a culture solution containing a reagent capable of inducing differentiation of a subsequent human mesenchymal stem cell. The chondrocytes, cartilage, and adipocytes differentiated from Example 1 can be stained with alkaline phosphatase, stained with Alcian blue, and oil red-O stained according to a standard procedure, and the cell type can be confirmed. The results are shown in Fig. 2A, Fig. 2B, and Fig. 2C, respectively.
應當理解的是，前述對實施方式的描述僅是以實施例的方式給出，且本領域所屬技術領域中具有通常知識者可進行各種修改。以上說明書、實施例及實驗結果提供本發明之例示性實施方式之結構與用途的完整描述。雖然上文實施方式中揭露了本發明的各種具體實施例，然其並非用以限定本發明，本發明所屬技術領域中具有通常知識者，在不悖離本發明之原理與精神的情形下，當可對其進行各種更動與修飾，因此本發明之保護範圍當以附隨申請專利範圍所界定者為準。It is to be understood that the foregoing description of the embodiments has been given by way of example only, and various modifications may be made by those of ordinary skill in the art. The above description, examples and experimental results provide a complete description of the structure and use of the exemplary embodiments of the invention. While the embodiments of the present invention have been disclosed in the foregoing embodiments, the present invention is not intended to be limited by the scope of the invention. The various modifications and variations of the present invention are intended to be defined by the scope of the appended claims.
無no
為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之說明如下：The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.
第1A圖及第1B圖分別根據本揭示內容之一實施方式繪示實施例1的人類間質幹細胞對標記物CD90、CD 105之陽性表現結果；第1C圖則繪示針對標記物CD34之陰性表現結果；1A and 1B respectively show the results of positive expression of the human mesenchymal stem cells of the embodiment 1 against the markers CD90 and CD 105 according to an embodiment of the present disclosure; the 1C diagram shows the negative for the marker CD34 Performance result
第2A圖係根據本揭示內容之一實施方式之照片，其為經鹼性磷酸酶染色確認實施例1之人類間質幹細胞分化成軟骨細胞之結果；2A is a photograph according to an embodiment of the present disclosure, which is a result of confirming differentiation of human mesenchymal stem cells of Example 1 into chondrocytes by alkaline phosphatase staining;
第2B圖係根據本揭示內容之一實施方式之照片，其為經愛爾新藍染色確認實施例1之人類間質幹細胞分化成軟骨之結果；以及2B is a photograph according to an embodiment of the present disclosure, which is a result of confirming differentiation of human mesenchymal stem cells of Example 1 into cartilage by Aier New Blue staining;
第2C圖係根據本揭示內容之一實施方式的照片，其為經油紅O染色確認實施例1的人類間質幹細胞分化成脂肪細胞之結果。2C is a photograph according to an embodiment of the present disclosure, which is a result of confirming differentiation of human mesenchymal stem cells of Example 1 into adipocytes by oil red O staining.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW106141271ATWI656215B (en) | 2017-11-28 | 2017-11-28 | Method for preparing mesenchymal stem cell population from peripheral blood and use thereof |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW106141271ATWI656215B (en) | 2017-11-28 | 2017-11-28 | Method for preparing mesenchymal stem cell population from peripheral blood and use thereof |
| Publication Number | Publication Date |
|---|---|
| TWI656215Btrue TWI656215B (en) | 2019-04-11 |
| TW201925462A TW201925462A (en) | 2019-07-01 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW106141271ATWI656215B (en) | 2017-11-28 | 2017-11-28 | Method for preparing mesenchymal stem cell population from peripheral blood and use thereof |
| Country | Link |
|---|---|
| TW (1) | TWI656215B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7101710B2 (en)* | 2003-12-02 | 2006-09-05 | Ming-Song Tsai | Two-stage culture protocol for isolating mesenchymal stem cells from amniotic fluid |
| CN1281739C (en)* | 2002-02-19 | 2006-10-25 | 美迪宝斯特有限公司 | Method for isolating and expanding the culture of mesenchymal stem/progenitor cells from umbilical cord blood and differentiating the mesenchymal stem/progenitor cells derived from umbilical cord blood into various mesenchymal tissues |
| EP1767617A1 (en)* | 2005-09-26 | 2007-03-28 | Letizia Mazzini | Mesenchymal stem cells isolation and expansion method and uses thereof |
| CN101248171A (en)* | 2005-04-12 | 2008-08-20 | 成血管细胞系统公司 | Isolation of adult pluripotent cells by tissue-nonspecific alkaline phosphatase |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1281739C (en)* | 2002-02-19 | 2006-10-25 | 美迪宝斯特有限公司 | Method for isolating and expanding the culture of mesenchymal stem/progenitor cells from umbilical cord blood and differentiating the mesenchymal stem/progenitor cells derived from umbilical cord blood into various mesenchymal tissues |
| US7101710B2 (en)* | 2003-12-02 | 2006-09-05 | Ming-Song Tsai | Two-stage culture protocol for isolating mesenchymal stem cells from amniotic fluid |
| CN101248171A (en)* | 2005-04-12 | 2008-08-20 | 成血管细胞系统公司 | Isolation of adult pluripotent cells by tissue-nonspecific alkaline phosphatase |
| CN101248171B (en) | 2005-04-12 | 2015-11-25 | 迈索布拉斯特股份有限公司 | Isolation of adult pluripotent cells by tissue-nonspecific alkaline phosphatase |
| EP1767617A1 (en)* | 2005-09-26 | 2007-03-28 | Letizia Mazzini | Mesenchymal stem cells isolation and expansion method and uses thereof |
| Publication number | Publication date |
|---|---|
| TW201925462A (en) | 2019-07-01 |
| Publication | Publication Date | Title |
|---|---|---|
| US8440177B2 (en) | Method of treating graft versus host disease using adipose derived mesenchymal stem cells | |
| JP4180228B2 (en) | Diverse mesodermal lineage differentiation capacity and use of stromal cells derived from adipose tissue | |
| US11447748B2 (en) | Encapsulated adipose-derived stem cells, methods for preparation and theraputic use | |
| US20120201791A1 (en) | Methods of treating diseases or conditions using mesenchymal stem cells | |
| US20090274665A1 (en) | Stem Cells For Treating Lung Diseases | |
| US20140341863A1 (en) | Adult mesenchymal stem cell (msc) compositions and methods for preparing the same | |
| WO2014053418A2 (en) | Method for obtaining mesenchymal stem cells and use thereof | |
| Aljagthmi et al. | Hematopoietic stem cells: Understanding the mechanisms to unleash the therapeutic potential of hematopoietic stem cell transplantation | |
| WO2014015229A1 (en) | Isolation of stromal vascular fraction from adipose tissue obtained from postmortem source using ultrasonic cavitation | |
| CN110402286B (en) | Method for preparing mesenchymal stem cell population from peripheral blood and use thereof | |
| TWI656215B (en) | Method for preparing mesenchymal stem cell population from peripheral blood and use thereof | |
| US20150353897A1 (en) | Method of generating multilineage potential cells | |
| TWI810918B (en) | Methods of producing human mesenchymal stem cells from foreskins and uses thereof | |
| TWM638487U (en) | Sampling tube for isolating human mesenchymal stem cells | |
| KR20070113694A (en) | Composition for promoting hematopoietic stem cell engraftment | |
| Petinati et al. | Multipotent Mesenchymal Stromal Cells from Porcine Bone Marrow, Implanted under the Kidney Capsule, form an Ectopic Focus Containing Bone, Hematopoietic Stromal Microenvironment, and Muscles. Cells 2023, 12, 268 | |
| WO2024128224A1 (en) | Composition for amplifying ability of mesenchymal stem cells to differentiate into adipocytes | |
| US20190111089A1 (en) | Method and System for Repairing Damaged Tissue Using Nucleated Plasma Particles (Nuc-P2s) and Mesodermal Stem Cells (MesoSCs) | |
| Alfaifi | Cell therapy for acute liver injury-in vivo efficacy of mesenchymal stromal cells in toxic and immune-mediated murine hepatitis | |
| WO2022210574A1 (en) | Agent for treating muscular dystrophy | |
| JP2024509023A (en) | Pharmaceutical compositions comprising adipose tissue-derived regenerative cells (ADRCs) for use in the prevention and treatment of liver fibrosis and/or cirrhosis | |
| TW202319059A (en) | Mesenchymal stem cells for use in the treatment of osteoarthritis in animals | |
| Naskou | Equine platelet lysate: unlocking the potentials of a novel biological product with multiple applications | |
| CN103961374A (en) | Application of allogenic stromal vascular fraction cells and allogenic mesenchymal progenitor cells in prevention or treatment of rheumatoid arthritis | |
| Siemionow et al. | The Role of Stem Cells in Plastic Surgery |
| Date | Code | Title | Description |
|---|---|---|---|
| MM4A | Annulment or lapse of patent due to non-payment of fees |