CN101384703A - Method for obtaining xeno-free hBS cell lines - Google Patents

Abstract

Method for obtaining a stable xeno-free hBS cell line, xeno-free hBS cell line obtained according to said method and use thereof. The method comprises the following steps: i) removing zona pellucida from blastocysts by a xeno-free procedure to obtain an inner cell mass surrounded by trophectoderm, ii) removing trophectoderm at least partially by a xeno-free procedure to obtain isolated inner cell mass cells, iii) placing the inner cell mass cells on a xeno-free medium on a layer of human feeder cells, iv) co-culturing the inner cell mass cells with human feeder cells in a xeno-free medium for a period of time of about 5 days to about 10 days, v) if any, releasing the inner cell mass cells or cells derived therefrom from overgrown trophectoderm by a xeno-free procedure, vi) obtaining xeno-free hBS cells by selecting cells from the inner cell mass cells or cells derived therefrom which are transferred to a layer of fresh human feeder cells in a xeno-free medium, vii) proliferating the xeno-free hBS cells by co-culturing with human feeder cells in a xeno-free medium, thereby obtaining the hBS cell line without foreign matters. The xeno-free hBS cell line is suitable for use in medicine and in vitro assays.

Description

Method for obtaining xeno-free hBS cell lines

field of invention

The present invention relates to a method for obtaining stable xeno-free hBS cells, xeno-free hBS cells obtained according to said method and uses thereof.

Background of the invention

Stem cells have the unique ability to self-renew and generate specialized or differentiated cells. While most cells of the body, such as heart cells and skin cells, are responsible for specialized functions, stem cells are not responsible for specialized functions until they are signaled to form specialized cell types. What makes stem cells unique is their ability to proliferate and their ability to become specialized. For years, researchers have focused on discovering ways to use stem cells to replace lost, damaged or diseased cells and tissues. Most research so far has focused on two types of stem cells, embryonic stem cells and adult stem cells. Embryonic stem cells are derived from pre-implanted fertilized eggs, ie, blastocysts, while adult stem cells are found in adult organisms, such as in the bone marrow, epidermis and intestine. Pluripotency tests have shown that although stem cells derived from embryos or blastocysts (hereafter called blastocyst-derived stem cells or BS cells) can give rise to all cells in an organism, including germ cells, Adult stem cells have a more limited repertoire of progeny cell types.

Perhaps the most far-reaching potential application of hBS cells is the production of cells and tissues that can be used in so-called cell therapies. Many diseases or conditions result from disruption of cellular function or destruction of body tissues. Today, donated organs and tissues are often used to replace diseased or damaged tissue. Unfortunately, to date, the number of people suffering from conditions amenable to treatment by these methods exceeds the number of organs available for transplantation. The availability of hBS cells and intensive research on the development of efficient methods for directing these cells to different cell fates (e.g., insulin-producing β-cells, cardiomyocytes, and dopamine-producing neurons) holds promise for future research in degenerative diseases ( degenerative diseases) such as diabetes, myocardial infarction, and Parkinson's disease have increasing hopes for application in cell-based therapies.

However, all currently available human blastocyst-derived stem cell (hBS) lines have been exposed directly or indirectly to animal material at some point during their derivation and/or maintenance. A potential consequence of using xeno-contaminated hBS cells in patients is an increased likelihood of graft rejection [Martin JM et al., 2005]. In addition, xeno-exposure increases the risk of transferring non-human pathogens in any clinical application. Thus, the anticipated hazards associated with the use of xeno-exposed hBS cells in patients make such cells unsuitable for clinical use. In an attempt to overcome these problems, several groups have used feeder-free matrices [xu C et al., 2001] or human-derived feeder cells for the derivation of hBS cell lines [Richards M et al., 2002, Inzunza J et al.] and culture [Richards M et al., 2003]. However, to date it has not been possible to derive and continuously culture hBS cell lines in a completely xeno-free system. In order to obtain and culture hBS cells that are completely xeno-free, 3 major obstacles must be overcome. First, protocols for deriving new hBS cell lines under xeno-free conditions had to be developed. Isolation of the inner cell mass (ICM) is classically performed by immunosurgical methods, a method involving the use of guinea pig serum. We have reported earlier [Heins et al., 2004] that immunosurgery can be ruled out, allowing isolation of ICM cells without exposure to animal components. Second and third, a xeno-free feeder culture system combined with the use of xeno-free media is essential. Since it is not the basal medium that causes xeno contamination, but the commonly used fetal bovine serum (FBS) or serum latent substances containing various animal proteins, we decided to test whether hBS cells can be cultured in human serum for a long time. There are very few previous reports on the use of human serum for hBS cell culture and the culture only showed short-term culture stability, ie stability of less than 10 passages [Richards et al., 2002 and 2004]. The difficulty reported by others to maintain hBS cells in an undifferentiated state using media supplemented with human serum motivated the inventors from the outset to seek to develop well-defined culture conditions, i.e. feeder-free cultures and with perfectly known compositions and concentrations. culture medium (defined medium) instead of developing a hBS cell culture system using human and synthetic components. However, the present inventors have been able to develop a proliferation system for hBS cells based on human fibroblasts and the serum-containing medium described here. The hBS cell line SA121 (established according to the method of WO2003055992) has been successfully cultured in this serum-based medium for more than 50 passages.

Furthermore, the present inventors have developed a system for deriving and culturing human feeder cells that are completely xeno-free. The existence of xeno-free human feeder cells such as human foreskin fibroblasts has been previously reported, although in many cases the feeder cells have been in contact with many animal materials during their actual establishment and culture.

In the present invention is reported the successful development of a completely xeno-free system for the derivation and maintenance culture of hBS cells using only components meeting regulatory requirements, such as components of human or synthetic origin. The methods described here completely avoid direct or indirect exposure to animal components, whereby completely xeno-free hBS cells can be obtained for further use as an unrestricted source for the development of cell therapies and other uses (e.g. For drug discovery and development, toxicity testing), for the manufacture of drugs such as antibodies and vaccines.

Detailed description of the invention

The present invention relates to a method for obtaining a xeno-free hBS cell line, the method comprising the steps of:

i) removal of the zona pellucida from the blastocyst by a xeno-free manipulation to obtain an inner cell mass surrounded by trophectoderm,

ii) at least partially removing the trophectoderm by a xeno-free manipulation to obtain isolated inner cell mass cells,

iii) placing the inner cell mass cells on a layer of human feeder cells in xeno-free medium,

iv) co-cultivating the inner cell mass cells with the human feeder cells in xeno-free medium for a period of about 5 days to about 10 days,

v) release of inner cell mass cells or cells derived therefrom from overgrown trophectoderm by xeno-free manipulation, if any,

vi) optionally, xeno-free hBS cells are obtained by transferring cells of the inner cell mass or cells derived therefrom onto a layer of fresh human feeder cells in a xeno-free medium,

vii) Propagation of xeno-free hBS cells by co-cultivation with human feeder cells in xeno-free medium to obtain xeno-free hBS cell lines.

In a specific embodiment, the present invention provides a method for obtaining the above-mentioned xeno-free hBS cell line, but the starting point in the method can be any of the above-mentioned steps, that is, the method of the present invention can be Including step ii)-step vii), step iii)-step vii), step iv)-step vii), step v)-step vii), step vi)-step vii) or step vii) (or below-mentioned Step viii)).

As used herein, the term "xeno-free" is intended to mean never directly or indirectly exposed to materials of non-human animal origin such as cells, tissues and/or bodily fluids and derivatives thereof.

In order to maintain the xeno-free hBS cell line obtained according to the present invention, the method may further comprise the step

viii) proliferating xeno-free by co-culturing with human feeder cells in xeno-free medium and passaging the cells at suitable time intervals from about 2 days to about 20 days, such as from about 4 days to about 12 days cell line.

The time interval chosen in step viii) depends on cell growth.

Step viii) above is also a special aspect of the invention.

It may be desirable to maintain the obtained xeno-free hBS cell line in a feeder-free culture system, whereby the method of the invention may further comprise the step

ix) Transfer of xeno-free hBS cell lines to xeno-free and feeder-free culture systems.

Step ix) can be performed according to WO2004099394, which is hereby incorporated by reference.

step i)

The zona pellucida is the glycoprotein-rich thick extracellular matrix surrounding the mammalian ova (ova). The zona pellucida can be removed from the zygote to obtain the inner cell mass surrounded by trophectoderm in step i) by using a) acidic solution, b) recombinant enzymes such as hyaluronidase or pronase or c) mechanical methods . The degradation process of the zona pellucida can be observed by visual inspection under a microscope.

As used herein, the term "inner cell mass surrounded by trophectoderm" is intended to mean the material obtained after subjecting a fertilized egg to step I) in which the zona pellucida is removed by acid hydrolysis, enzymatic digestion or mechanical means.

The recombinases used herein have amino acid sequences corresponding to the human amino acid sequences of these enzymes, ie, the recombinases used here have human amino acid sequences but are produced by recombination.

When the zona pellucida is removed by using an acidic solution, the blastocyst is subjected to the acidic solution for about 5 seconds to about 180 seconds, for example about 10 seconds to about 120 seconds, about 15 seconds to about 90 seconds, about 20 seconds to about 60 seconds, about 30 seconds to about 50 seconds.

Importantly, the pH of the acidic solution is low enough to hydrolyze the carbohydrate structures of the zona pellucida, ie the pH of the acidic solution is from about 2 to about 3, such as from about 2.5 to about 2.8, such as 2.5. Any suitable acid can be used. In a preferred embodiment of the invention, the acidic solution is an Acid Tyrodes solution (Sigma) with a pH of 2.5±0.3.

If the zona pellucida is removed by use of one or more recombinases, the blastocyst is subjected to a solution of one or more recombinases. One or more of the recombinases has an amino acid sequence corresponding to the human amino acid sequence of these enzymes.

Examples of suitable enzymes are eg recombinant pronase, recombinant hyaluronidase and recombinant trypsin. Additional examples of suitable cell enzyme digests in step (i) are enzymes that are recombinant or xeno-free and potentially combine proteolytic and collagenolytic activities, such as Accutase ^™ (Chemicon) and are recombinant or xeno-free Enzymatic digests of source material, potentially combining proteolytic, collagenolytic and DNase activities such as Accumax ^™ (InnovativeCell Technologies).

Suitable concentrations and treatment times are given below:

Pronase: 10 U/ml for a time period of about 2 to about 20 minutes, such as about 2 to about 10 minutes, about 2 to about 5 minutes or about 3 to 4 minutes (optimum).

Hyaluronidase: 70.000 U/ml for a time period of about 2 to about 240 minutes.

Human Recombinant Trypsin: 5-10.000 U for a time period of about 0.5 to about 30 minutes.

The zona pellucida can also be removed by mechanical means, for example using glass capillaries under visual inspection with the aid of a microscope.

step ii)

After removal of the zona pellucida, the remainder of the blastocyst, the inner cell mass surrounded by trophectoderm, is subjected to step ii) to at least partially remove the trophectoderm. Alternatively, spontaneously hatched blastocysts can be directly subjected to step ii), thereby skipping step i).

Step ii) can be carried out by using a) an acidic solution, b) one or more recombinant enzymes or c) a mechanical method.

When step ii) is performed by using an acidic solution, the trophectoderm-surrounded inner cell mass obtained in step i) is subjected to the acidic solution for about 5 seconds to about 180 seconds, for example about 10 seconds to about 120 seconds, about 15 seconds to about 90 seconds, about 20 seconds to about 60 seconds, about 30 seconds to about 50 seconds.

Importantly, the pH of the acidic solution is low enough to hydrolyze the proteinaceous structure of the trophectoderm, ie the pH of the acidic solution is from about 2 to about 3, such as from about 2.5 to about 2.8, such as 2.5. Any suitable acid can be used. In a preferred embodiment of the invention, the acidic solution is acidic Tyrode's solution (Sigma) at pH 2.5±0.3.

If the trophectoderm is at least partially removed by using one or more recombinases, the inner cell mass surrounded by the trophectoderm is subjected to a solution of the one or more recombinases. The one or more recombinases are recombinases having amino acid sequences corresponding to the human amino acid sequences of these enzymes.

Suitable enzymes are recombinant proteolytic enzymes, such as serine proteases, including recombinant trypsin and TrypLE ^™ Select. If using TrypLE ^™ Select, typically by subjecting the trophectoderm-surrounded inner cell mass obtained in step i) to an undiluted, ready-to-use concentration of TrypLE ^™ Select for about 0.5 to 10 minutes, for example about 0.5 to about 8 minutes, Step ii) is carried out for about 0.5 to about 5 minutes, about 1 to about 3 minutes, for example 1.5 minutes. If recombinant trypsin is used, step ii) is generally performed by subjecting the trophectoderm-surrounded inner cell mass obtained in step i) to about 5.000 U to about 10.000 U of recombinant trypsin for about 0.5 minutes to about 30 minutes.

Additional examples of suitable cell enzyme digests in step (ii) are enzymes that are recombinant or xeno-free, potentially combining proteolytic and collagenolytic activities, such as Accutase ^™ (Chemicon) and recombinant or xeno-free Enzymatic digests of source material, potentially combining proteolytic, collagenase and DNase activities such as Accumax ^™ (Innovative Cell Technologies).

The trophectoderm can also be at least partially removed by mechanical means, which can be performed by using a glass capillary as a cutting tool. Detection of inner cell mass cells is readily performed visually using a microscope.

Step iii)

塑料培养皿中。如果需要，可用合适的基质材料涂铺培养皿的培养表面，只要该基质材料是无异源物质的。适合用于该背景的材料包括重组人明胶。In step iii), the material obtained after step ii) is placed on the human feeder layer, usually by using a glass capillary under a microscope. Additionally, if desired, human feeder layers can be filled into suitable Petri dishes such as PRIMARIA

in a plastic petri dish. If desired, the culture surface of the culture dish can be coated with a suitable matrix material, provided that the matrix material is free of xenogeneic substances. Materials suitable for use in this context include recombinant human gelatin.

Step iv)

In step iv) of the method of the invention, the inner cell mass cells are co-cultured for about 5 days to about 50 days, for example about 5 days to about 30 days, about 5 days to about 20 days or about 5 days to about 15 days time period, thereby expanding the cell population. In one embodiment of the invention, the inner cell mass cells are co-cultured in step iv) for a period of 10 days.

It may be performed one or more times during the co-cultivation of the inner cell mass cells in step iv) by exchanging about 20% to about 100%, for example about 30% to about 80%, about 40% to about 60% of the medium Culture medium replacement. In one embodiment of the invention, one or more medium changes are performed during co-cultivation of the inner cell mass cells in step iv) by changing approximately 50% of the medium. One or more medium changes may be performed at intervals of about 2 days to about 14 days, eg, about 4 days to about 7 days.

Because residual trophectoderm cells may have been placed on the human feeder cell layer when placing the inner cell mass cells in step iii), visual inspection using a microscope can be performed periodically to see if the trophectoderm interferes with the inner cell mass cells Or the growth of cells derived from the inner cell mass. A suitable time point to bring the cells into step v) is when substantial growth has been observed by inspection under the microscope over a period of several days.

During the propagation of the inner cell mass cells in step iv), some of these cells may start their transformation into blastocyst-derived stem (BS) cells. Thus, the cell population obtained in step iv) may include inner cell mass cells and cells derived therefrom, namely BS cells.

Steps v) and vi)

As mentioned above, inner cell mass cells and cells derived therefrom can be contaminated with residual trophectoderm. If this is the case, then the trophectoderm tends to surround the inner cell mass cells and the cells of their origin. Inner cell mass cells or cells derived therefrom may be released from the overgrown trophectoderm by using a) mechanical means or b) one or more recombinant enzymes.

A suitable mechanical method is to use a glass capillary as the cutting tool. Cell mass cells or cells derived therefrom are selectively dissected based on visual inspection under a microscope and transferred to a layer of fresh human feeder cells in xeno-free medium to obtain xeno-free hBS Cells (step vi).

^{Alternatively} , the overgrown trophectoderm (if (if any) to release inner cell mass cells or cells derived therefrom. If recombinant trypsin is used in step v) to release the inner cell mass cells or cells derived therefrom, typically the inner cell mass cells or cells derived therefrom are incubated with about 5.000 U to about 10.000 U of trypsin for about 0.5 minutes to about 30 minutes. If TrypLE ^™ Select is used to release inner cell mass cells or cells derived therefrom in step v), usually the inner cell mass cells or cells derived therefrom are incubated with undiluted, ready-to-use concentration of TrypLE ^™ Select for about 0.5 to about 15 minutes.

Further examples of suitable cell enzymatic solutions in steps (v) and (vi) are recombinant or xeno-free enzymes potentially combining proteolytic and collagenolytic activities such as Accutase ^™ (Chemicon) And enzymatic digests such as Accumax ^™ (Innovative Cell Technologies) potentially combining proteolytic, collagenolytic and DNase activities that are recombinant or xeno-free.

After the inner cell mass cells or cells derived therefrom have been released from the trophectoderm (if any), the inner cell mass cells or cells derived therefrom are selected based on visual inspection under a microscope and then transferred to the Fresh human feeder cell layer in culture for xenogeneic material to obtain xeno-free hBS cells (step vi).

Step vii)

In step vii), the xeno-free hBS cells are propagated by co-cultivation with human feeder cells, thereby obtaining a xeno-free hBS cell line. During the propagation of the xeno-free hBS in step vii) one or more passages may be performed, wherein hBS cells are selectively passaged according to visual inspection under a microscope. These passaging can be performed using glass capillaries as cutting tools. Alternatively, one or more passages may be performed in step vii) using one or more recombinant enzymes such as TrypLE ^™ Select, recombinant trypsin and/or recombinant collagenase.

If TrypLE ^™ Select is used, step vii) is generally performed by using undiluted, ready-to-use concentration of TrypLE ^™ Select for about 0.5 minutes to about 15 minutes.

If recombinant trypsin is used, step vii) is typically performed by using about 5.000 U to about 10.000 U of recombinant trypsin for about 0.5 minutes to about 30 minutes.

If recombinant collagenase is used, step vii) is typically performed by using about 200 U/ml of recombinant collagenase for about 1 minute to about 40 minutes.

Additional examples of suitable cell enzyme digests in step (vii) are recombinant or xeno-free enzymes that potentially combine proteolytic and collagenolytic activities such as Accutase ^™ (Chemicon) and are recombinant or xeno-free Enzymatic digests of substances and potentially combining proteolytic, collagenolytic and DNase activities such as Accumax ^™ (InnovativeCell Technologies).

The media used for individual passages may be the same or may be different.

step viii)

Proliferation of the xeno-free hBS cell line was consistent with that described in step vii).

In one embodiment of the present invention, cell passaging in step viii) may be performed by mechanical dissection, for example using glass capillaries as cutting tools. Alternatively, cell passaging in step viii) can be performed by using one or more recombinant enzymes such as TrypLE ^™ Select, recombinant trypsin and/or recombinant collagenase. Using TrypLE ^™ Select, concentrations and incubation times of recombinant trypsin and recombinant collagenase, respectively, were as described for step vii).

Additional examples of suitable cell enzyme digests in step (viii) are recombinant or xeno-free enzymes that potentially combine proteolytic and collagenolytic activities such as Accutase ^™ (Chemicon) and recombinant or free Enzymatic digests of heterogeneous material and potentially combining proteolytic, collagenolytic and DNase activities such as Accumax ^™ (Innovative Cell Technologies).

step ix)

In a particular embodiment of the invention, the hBS cell line obtained in step iX) is transferred to a xeno-free, feeder-free culture system comprising a suitable xeno-free support matrix For example, recombinant human gelatin, recombinant human fibronectin, human placental extracellular matrix and a suitable xeno-free medium that can be used for the establishment of hBS cell lines (step iii), iv), vi), vii)) or the xeno-free medium used for maintenance on human feeder cells (step viii)) is the same or different. To maintain undifferentiated growth of hBS cell lines, human feeder cell conditioned xeno-free media can be used or can be supplemented with appropriate factors such as high concentrations of recombinant bFGF and/or activators of the WNT pathway.

Preparation of Human Breeders

The human feeder cells used in any one of steps iii), vi), vii) and viiii) are obtained under xeno-free conditions. Human feeder cells are derived from healthy human tissue and can be obtained by biopsy.

Human tissues from which human feeder cells can be generated include embryonic, fetal, neonatal, juvenile or adult tissues, which also include tissues derived from skin including foreskin, umbilical cord, muscle, lung, epithelium, placenta, fallopian tubes, glandula, The stroma or tissue of the mammary gland. Human feeder cells can be derived from cell types related to human fibroblasts, fibrocytes, myocytes, keratinocytes, endothelial cells, and epithelial cells. Examples of specific cell types that can be used to generate human feeder cells include embryonic fibroblasts, extraembryonic endoderm cells, extraembryonic mesoderm cells, fetal fibroblasts and/or fiber cells, fetal muscle cells, fetal skin cells, fetal lung cells cells, fetal endothelial cells, fetal epithelial cells, umbilical chord mesenchymal cells, placental fibroblasts and/or fibroblasts, placental endothelial cells, postnatal human foreskin fibroblasts and/or fibroblasts, birth Postnatal myocytes, postnatal skin cells, postnatal endothelial cells, adult skin fibroblasts and/or fibroblasts, adult myocytes, adult fallopian tube endothelial cells, adult glandular endometrial cells, adult endometrium adult stromal endometrial cells, adult breast cancer parenchymal cells, adult endothelial cells, adult epithelial cells, or adult keratinocytes.

Feeder cells for use in the present invention can be immortalized or genetically modified. Immortalization of feeder cells means the acquisition of the ability to grow through a theoretically unlimited number of divisions in culture. There are several methods for immortalization, one is transformation of cells with eg viruses, retroviruses and/or by expression of the telomerase reverse transcriptase protein (TERT). TERT is inactive in most cells, but when hTERT is exogenously expressed, cells are able to maintain telomere length sufficient to avoid replicative senescence.

In addition, feeder cells can be genetically engineered to integrate specific genes into the genome. These genes may encode markers of interest or for the synthesis of biomolecules known to be beneficial to hBS cells such as growth factors such as bFGF (Basic Fibroblast Growth Factor).

When the human feeder cells are derived from hBS cells, the cells derived from hBS cells may be fibroblasts.

In one embodiment of the invention, the human feeder cells are derived from neonatal human foreskin.

In a particular embodiment of the invention, the human feeder cells are fibroblasts, preferably derived from human neonatal foreskin fibroblasts.

Human foreskin samples can be obtained from circumcised male infants. Samples can be selected aseptically in a sterile suitable medium, for example in sterile IMDM medium containing 2X gentamicin (Invitrogen). The skin explants were placed in tissue culture flasks such as 25 cm ^primaria tissue culture flasks (Becton Dickinson) containing IMDM medium (Invitrogen), 1% penicillin-streptomycin (Gibco Invitrogen Corporation) and 10% human serum (Tallheden et al., 2005) . After about 10 days, a confluent monolayer was established. Cells were serially passaged using TrypLE ^™ Select (Invitrogen). The inventors have found that a suitable period of time between passages is from about 2 to about 20 days, usually at least 15 passages are suitable eg a maximum of 20 passages. After amplification, the list of human pathogens (including mycoplasma, HIV types 1 and 2, hepatitis B and C, cytomegalovirus, herpes simplex virus types 1 and 2, Epstein-Barr virus, and human papilloma virus) to detect them.

xeno-free medium

The medium can be any minimal medium suitable for proliferation of human inner cell mass cells. A suitable medium is Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1-30% v/v human serum and 2-100 ng/ml recombinant bFGF. In one embodiment, the minimal medium comprises 20% v/v human serum. In another embodiment, the minimal medium comprises 10 ng/ml recombinant bFGF. Other minimal media may be used as long as they provide the inner cell mass cell matrix with nutrients (i.e. inorganic components such as trace elements and organic components such as amino acids, salts, vitamins, energy suppliers, carbohydrates including sugars, etc.) in liquid form. Importantly, the culture medium is xeno-free.

The xeno-free medium used in any of steps iii), iv), vi), vii) and/or viii) comprises a minimal medium suitable for proliferation of human inner cell mass cells. A suitable minimal medium is Dulbecco's Modified Eagle's Medium (DMEM). However, other minimal media will work equally well. In addition to minimal xeno-free medium, xeno-free medium may further contain human serum, recombinant bFGF, L-glutamine or glutamax, non-essential amino acids, β-mercaptoethanol, penicillin, and/or streptavidin white.

The concentration of human serum in the xeno-free medium is preferably about 1% v/v to about 30% v/v human serum, for example about 10% v/v to about 30% v/v human serum, about 15% v/v to about 25% v/v human serum, and more preferably 20% v/v human serum.

The concentration of recombinant bFGF in the xeno-free medium is preferably from about 2 ng/ml to about 100 ng/ml of recombinant bFGF, for example from about 5 ng/ml to about 50 ng/ml of recombinant bFGF, from about 5 ng/ml to about 25 ng/ml Recombinant bFGF of about 5ng/ml to about 15ng/ml of recombinant bFGF, such as 10ng/ml of recombinant bFGF.

的浓度优选是大约0.5mM至大约20mM，例如大约0.75mM至大约10mM，大约1mM至大约5mM例如2mM。L-glutamine or Glutamax in xeno-free medium

The concentration of is preferably from about 0.5 mM to about 20 mM, such as from about 0.75 mM to about 10 mM, from about 1 mM to about 5 mM, such as 2 mM.

The concentration of non-essential amino acids in the xeno-free medium is preferably about 0.01 mM to about 1 mM, for example about 0.03 mM to about 0.8 mM, about 0.05 mM to about 0.6 mM, about 0.07 mM to about 0.4 mM, about 0.09 mM To about 0.2 mM, such as 0.1 mM.

The concentration of β-mercaptoethanol in the xeno-free medium is preferably about 10 μM to about 200 μM, for example about 25 μM to about 175 μM, about 50 μM to about 150 μM, about 75 μM to about 125 μM, for example 100 μM.

The concentration of penicillin in the xeno-free medium is preferably from about 5 units/ml to about 200 units/ml, such as from about 10 units/ml to about 150 units/ml, from about 25 units/ml to about 100 units/ml, eg about 25 units/ml to about 75 units/ml, eg 50 units/ml.

The concentration of streptomycin in the xeno-free medium is preferably about 5 μg/ml to about 200 μg/ml, for example about 10 μg/ml to about 150 μg/ml, about 25 μg/ml to about 100 μg/ml, about 25 μg/ml To about 75 μg/ml, eg 50 μg/ml.

Also other xeno-free media can be used in one or more of the individual steps of the invention, eg for the propagation steps (vii) and (viii). The medium may contain salts, vitamins, energy sources (such as glucose), minerals and amino acids. Suitable growth factors to be added to the medium may be, for example, GABA, pipecolic acid, lithium chloride and transforming growth factor beta (TGF beta) and bFGF. Furthermore, the medium can be chemically defined. Therefore, the xeno-free hBS cell line derived in the present invention can be passaged or propagated in a medium other than a serum-containing medium.

Preparation of human serum

Human serum of good quality is repeatedly produced in our laboratory (Tallheden et al., 2005) . Blood is tested for a number of standard pathogens (hepatitis B, hepatitis C, HIV, HTLV, and syphilis) at the hospital's blood center.

Therefore, the human serum used in any one of steps iii), iv), vi), vii) and viiii) is prepared by the following steps

a) Collect healthy human blood in uncoated heparinized bags,

b) shaking said non-heparinized bag for a period of about 0.5 hour to about 5 hours, such as about 0.5 hour to about 2 hours,

c) Incubate the uncoated heparinized bag at a temperature of up to 5°C for a period of at least 10 hours

d) Optionally, selection based on coagulation quality eg absence of non-clotted fibrin, opacity of liquid phase

e) Separation of serum from clotted material

f) Sterile filtration of serum obtained in step d)

g) Pool sera from at least 15 donors

h) Serum was frozen prior to use.

In a particular embodiment of the invention, the method for obtaining a xeno-free hBS cell line comprises the steps of:

1) removing the zona pellucida and at least part of the trophectoderm from the blastocyst by incubating the blastocyst with an acidic Tyrode solution at room temperature for a time period of about 10 seconds to about 10 minutes, preferably about 30 seconds to about 60 seconds, thereby obtaining isolated inner cell mass cells,

2) Place inner cell mass cells in xeno-free medium containing DMEM, human serum, recombinant bFGF, L-glutamine or glutamax, non-essential amino acids, β-mercaptoethanol, penicillin, and streptomycin human foreskin fibroblast feeder cell layer

3) Inner cell mass cells are co-cultured with human foreskin fibroblast feeder cells in xeno-free medium for a period of about 5 days to about 15 days, wherein at least 50% of the xenobiotics are replaced every 3 to 5 days source material.

4) release of inner cell mass cells or cells derived therefrom from the overgrown trophectoderm, if any, by using TrypLE ^™ Select (Invitrogen) as enzymatic treatment,

5) Optionally, obtaining xeno-free hBS cells by transferring inner cell mass cells or cells derived therefrom to a fresh human foreskin fibroblast feeder cell layer in xeno-free medium,

6) Propagation of xeno-free hBS cells by co-culturing with human foreskin fibroblast feeder cells in a xeno-free medium to obtain a xeno-free hBS cell line.

In a specific embodiment, the present invention provides a method for obtaining the above-mentioned xeno-free hBS cell line, but the starting point in the method can be any of the above steps, that is, the method of the present invention can include step 2 ) to step 6), step 3) to step 6), step 4) to step 6), step 5) to step 6) or step 6) (or step 7 (as mentioned below)).

Maintenance of the xeno-free hBS cell line obtained in step 6) can be performed by a further step as follows, namely

7) By co-culturing with human foreskin fibroblast feeder cells in a xeno-free medium, at least 50% of the xeno-free medium is replaced every 3 to 5 days and at appropriate intervals, for example every 3 Cells were passaged by day 8 to propagate a xeno-free hBS cell line.

The removal of the zona pellucida can be performed in step 1) by visual inspection under a microscope.

For details and details of the preferred concentrations of the individual components in the above-mentioned xeno-free medium, refer to the xeno-free medium used in steps 2), 3), 5), 6) and 7).

In step 3), a visual inspection may be performed periodically to see if the trophectoderm interferes with the growth of inner cell mass cells or cells derived therefrom.

In one embodiment of the present invention, the inner cell mass cells or cells derived therefrom are selectively transferred in step 5) by using a glass capillary as a cutting tool according to visual inspection under a microscope.

The passage interval suitable for the proliferation of the xeno-free hBS cell line used in step 7) depends on the growth of the cells, and passaging is performed by manual transfer using glass capillaries.

other aspects

In other aspects of the invention described below, reference should be made to the details and particulars discussed under the main aspects above for application.

Another aspect of the present invention relates to a method for obtaining a xeno-free hBS cell line comprising the steps of:

i) removing the trophectoderm at least partially from a blastocyst without a zona pellucida by xeno-free manipulation to obtain isolated inner cell mass cells,

ii) placing inner cell mass cells on a layer of human feeder cells in xeno-free medium,

iii) co-cultivating the inner cell mass cells with human feeder cells in a xeno-free medium for a period of time from about 5 days to about 50 days,

iv) release of inner cell mass cells or cells derived therefrom from overgrown trophectoderm by xeno-free manipulation, if any,

v) optionally, xeno-free hBS cells are obtained by transferring inner cell mass cells or cells derived therefrom onto a layer of fresh human feeder cells in xeno-free medium,

vi) Propagation of xeno-free hBS cells by co-cultivation with human feeder cells in xeno-free medium to obtain xeno-free hBS cell lines.

Another aspect of the present invention relates to a method for obtaining xeno-free hBS cells comprising the steps of:

i) placing inner cell mass cells at least partially free of trophectoderm on a layer of human feeder cells in a xeno-free medium,

ii) co-cultivating the inner cell layer cells with the human feeder cells in a xeno-free medium for a period of time from about 5 days to about 50 days,

iii) releasing inner cell mass cells or cells derived therefrom from the overgrown trophectoderm by xeno-free manipulation, if any,

iv) optionally, obtaining xeno-free hBS cells by transferring inner cell mass cells or cells derived therefrom to fresh human feeder cells in xeno-free medium,

v) Propagation of xeno-free hBS by co-cultivation with human feeder cells in xeno-free medium to obtain xeno-free hBS cell lines.

Another aspect of the present invention relates to a method for obtaining a xeno-free hBS cell line comprising the steps of:

i) co-cultivating inner cell mass cells at least partially free of trophectoderm with human feeder cells in the xeno-free environment for a period of time from about 5 days to about 50 days,

ii) releasing inner cell mass cells or cells derived therefrom from the overgrown trophectoderm by xeno-free manipulation, if any,

iii) optionally, xeno-free hBS cells are obtained by transferring inner cell mass cells or cells derived therefrom onto a layer of fresh human feeder cells in xeno-free medium,

iv) Propagation of xeno-free hBS cells by co-cultivation with human feeder cells in xeno-free medium to obtain xeno-free hBS cell lines.

The present invention also relates to the proliferation and maintenance of xeno-free hBS cell lines independent of the methods used to obtain said xeno-free hBS cell lines. Human serum-based media and human feeder cells described here can be adapted mutatis mutandis to the proliferation method. More details are presented in this specification under steps vii) and viii).

Characterization of xeno-free hBS cell lines

The invention also relates to a xeno-free hBS cell line obtained by the method of the invention. Such cell xeno-free hBS cells are not exposed directly or indirectly to any non-human animal material, meaning that all components in all steps of the method are not exposed to any non-human animal material, such as any Mammalian material. Thus, the human feeder cells used in accordance with the present invention have been derived without any exposure to any non-human animal material. Any organic material used during the establishment and maintenance of the xeno-free hBS cell line was of human origin or synthetic, semi-synthetic or recombinant material.

The xeno-free hBS cell line of the present invention maintains self-renewal and pluripotency for an appropriate period of time and is therefore stable for an appropriate period of time. In this specification, the term "stable" is intended to mean that when grown on the human feeder cell layer of the present invention, more than 50 weeks, such as more than 40 weeks, more than 30 weeks, more than 20 weeks, more than 15 weeks in undifferentiated The ability to proliferate in a state. Therefore the xeno-free hBS cell line of the present invention is theoretically immortal.

The xeno-free hBS cell line obtained by the method according to the present invention can be used for the preparation of differentiated cells. The present invention therefore also relates to such differentiated cells.

Furthermore, the xeno-free hBS cells or cell lines of the invention are capable of undergoing freezing and thawing. In a particular embodiment, the xeno-free hBS cell lines obtained in the present invention can be frozen and thawed following the vitrification method previously provided by Cellartis in WO2004098285, which is incorporated herein by reference.

To increase the homogeneity of hBS cell cultures, the cells obtained in the present invention can be subjected to clonal derivation as described in WO2005059116 (which is incorporated herein by reference).

The xeno-free hBS cell lines obtained by the present invention fulfill the general requirements. The cell line has one or more of the following characteristics, in particular at least 4, 5, 6, 7 or 8 of the following characteristics. Therefore, the cell line

i) exhibit the ability to proliferate in an undifferentiated state for more than 15 weeks when grown on mitotically inactivated feeder cells, and/or

ii) exhibit a normal euploid karyotype, and/or

iii) exhibit a stable karyotype and/or during culture

iv) maintain the potential to develop into derivatives of all types of germ layers in vitro and in vivo, and/or

v) Display of the following molecular markers OCT-4, alkaline phosphatase, carbohydrate epitope (carbohydrate epitope) SSEA-3, SSEA-4, TRA 1-60, TRA 1-81 and recognized by monoclonal antibody GCTM-2 Keratan sulfate/chondroitin sulfate at least two molecular markers in the protein core of the pericellular matrix proteoglycan, and/or

vi) does not display the molecular marker SSEA-1 or other markers of differentiation, and/or

vii) maintain its pluripotency and form teratomas in vivo when injected into immunocompromised mice, and/or

viii) Capable of differentiation.

The xeno-free hBS cell line of the invention exhibits the following criteria: positive reaction to Oct-4, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 and negative reaction to SSEA-1 At least 1 of, for example, at least 2, at least 3, at least 4, at least 5, or at least 6 criteria.

In the following, methods for characterizing xeno-free hBS with respect to differentiation stage, pluripotency and karyotype are described. Such methods can be used to investigate whether hBS cells obtained according to the present invention meet the above-mentioned criteria.

immunochemistry

Xeno-free hBS cell lines can be analyzed for the immunohistochemical markers Oct-4, TRA-1-60, TRA-1-81, SSEA-1, SSEA-3, and SSEA-4 of undifferentiated cells to monitor their differentiation state.

alkaline phosphatase

Alkaline phosphatase and telomerase activities are commonly used as markers of undifferentiated hBS cells. Activity can be measured by any commercially available kit.

in vitro pluripotency

Pluripotency of xeno-free hBS cell lines can be tested by allowing colonies to differentiate spontaneously in culture dishes for approximately 3 to 4 weeks, with medium changes every 2 to 7 days. To identify cells from the 3 different germ layers, colonies were analyzed using immunohistochemistry. Suitable antibodies may be β-tubulin for ectoderm, ASMA (alpha smooth muscle actin) for mesoderm and HNF3 β (hepatic nucleofactor 3β) for endoderm.

In vivo pluripotency - teratoma

One approach to analyze whether human BS cell lines retain pluripotency is to xenograft the cells into immunodeficient mice to obtain tumors, teratomas. The different types of tissue found in the tumor should represent all 3 germ layers.

Severe combined immunodeficiency (SCID)-mice (a strain lacking B and T lymphocytes) can be used for the analysis of teratoma formation. Human BS cells can be placed surgically in the testes or under the kidney capsule. In testes or kidneys, BS cells can be transplanted in the range of 10 000-100 000 cells. Tumors are usually palpable after about 1 month. Mice were then sacrificed after 1 to 4 months, tumors were excised, and fixed for paraffin or cryosection analysis. Tumor tissues were then analyzed by immunohistochemistry. Specific markers for all 3 germ layers can be used.

Gene characterization: Karyotyping and fluorescence in situ hybridization (FISH) and telomerase activity

Chromosomes of the tested xeno-free hBS cell lines were visualized using trypsin-Giemsa or DAPI staining. For fluorescence in situ hybridization (FISH) analysis, commercially available reagents containing probes for chromosomes 12, 13, 17, 18, 20, 21 and the sex chromosomes (X and Y) can be used according to the manufacturer's instructions with slight modifications box. Slides are further analyzed in an inverted microscope equipped with appropriate filters and software.

Stem cells and blastocyst-derived stem cells can be further characterized for their telomerase activity, which can be detected, for example, using a kit known as the Telomerase PCR ELISA kit (Roche). The kit exploits the internal activity of telomerase by amplifying the product using polymerase chain reaction (PCR) and detecting it using enzyme-linked immunosorbent assay (ELISA). Telomerase activity can also be measured by QPCR.

Gene Characterization: QPCR

The differentiation state of the cells in the present invention can be further detected by QPCR for specific genes. In the following, how the assay is performed is briefly described: Colonies of undifferentiated or differentiated hBS cells can be isolated mechanically from culture dishes as intact colonies, washed with PBS, and then stored at -80°C. RAN can be further extracted using, for example, the Qiagen RNeasy Mini kit according to the manufacturer's instructions. Reverse transcription and QPCR under appropriate conditions are thus performed using a suitable kit such as the Bio-RadiScript First Strand Synthesis kit in Rotorgene 3000 (Corbett Research) (according to the manufacturer's instructions). If possible, all genes can be quantified in the same reaction and the differentiation status of several samples can be compared by calculating mathematical indices for individual samples based on gene markers. (A more detailed protocol is described in WO2006094798).

Tests against list of human pathogens

Individual components used in the invention herein, such as feeder cells, serum, and blastocysts, can be tested for human pathogens prior to use, as well as immediately after establishment of the xeno-free hBS cell line, which Pathogens are, for example, mycoplasma, human immunodeficiency virus types 1 and 2, hepatitis B and C, cytomegalovirus, herpes simplex virus types 1 and 2, Epstein-Barr virus and human papillomavirus.

The absence of human pathogens is of course very important for any clinical application of xeno-free hBS cell lines and cells or other biological material derived from cell lines.

Sialic acid Neu5Gc detection

Xeno-free hBS cells generated according to the present invention can be further tested for sialic acid Neu5Gc, which is a membrane-bound sugar molecule. A negative result of this test can be considered as no direct or indirect exposure to non-human animal material occurred.

Use of xeno-free hBS cells or cell lines of the present invention

The xeno-free hBS cell line of the present invention can be used for the production of differentiated derivatives thereof such as progenitor cells of all 3 germ layers and further differentiated cells exhibiting characteristics of different differentiated cell types, For example, hepatocyte-like features, cardiomyocyte-like features, neuron-like features.

GMP (Good Manufacturing Procedure) production

The xeno-free hBS cell lines of the present invention can further be used for GMP production, eg clinical GMP production of hBS cells and/or differentiated cells thereof. Furthermore, the methods described here for xeno-free derivatization can be performed under GMP and/or cGMP conditions to provide clinically applicable cell lines and derivatives (Martin et al. Nat. Med 2005).

medical use

The xeno-free hBS cells or differentiated derivatives thereof of the present invention can be used in medicine. For example xeno-free hBS cell lines or differentiated derivatives thereof can be used in the manufacture of medicinal products for the prevention and/or treatment of pathological conditions and/or diseases caused by tissue degeneration. Furthermore, xeno-free hBS cells or differentiated derivatives thereof can be used in the manufacture of medicinal products for the treatment and/or prevention of metabolic pathological conditions and/or diseases.

The hBS cells obtained by the method of the present invention can be used for the manufacture of medicaments for transplanting xeno-free hBS cells into mammals to prevent or treat diseases. A particular aspect is the use of xeno-free hBS cells in autologous transplantation, ie only human material from said particular patient is used for the preparation of xeno-free hBS cells or cell lines.

Many different kinds of diseases are envisaged in which the xeno-free cells or cell lines of the invention may be suitable for use include liver disease, cardiovascular disease (including myocardial infarction), degenerative disease (e.g. neurodegenerative disease, including Parkinson's disease and Alzheimer's disease), diabetes.

Such a medicament may comprise undifferentiated xeno-free hBS cells or differentiated xeno-free hBS cells dispersed in a pharmaceutically acceptable vehicle, such as an aqueous vehicle. The vehicle may contain one or more additives selected from pH regulators, stabilizers, preservatives, osmotic pressure regulators and physiologically acceptable salts; and/or one or more additives selected from therapeutic active substances, prophylactic Sexually active substances, engraftment improving agents, viability improving agents, differentiation improving agents and immunosuppressive agents.

Regenerative medicine and cell therapy

Cells produced by the xeno-free methods provided herein due to their pluripotency and ability to differentiate into fully differentiated tissue cell types and/or progenitor cell types (cell types that proliferate toward specific tissue types) could prove extremely useful in regenerative medicine. Treatment of heart-related diseases is conceivable after differentiating cells along a certain biological pathway towards e.g. multipotent cardiac progenitors, or e.g. Treatment of liver-related diseases, or after differentiation of cells towards neural progenitors, neurological diseases such as multiple sclerosis, hypoxic injury, ischemic injury, traumatic injury, Parkinson's treatment of demyelination and demyelination disorders.

Additional progenitor cell types derived from xeno-free hBS cell lines obtained as described herein may be mesodermal cells (with the potential to generate bone and cartilage in addition to cardiac cell types), or may be endoderm cells (with the potential to also give rise to pancreatic cell types such as beta cells).

In order to restore function in a heart that has suffered a cardiac infarction, replacement of cardiomyocytes and new blood vessels is necessary. When clinically compliant hBS cell lines are available, such as hBS cell lines produced according to the methods provided herein, it is possible to use such cells to derive progenitor cells that can then be transplanted and evaluated for potential use in humans. Such progenitor cells may have the potential to further differentiate in situ into cell types of degenerative tissues, such as the site of a cardiac infarction.

Cells differentiated from the xeno-free hBS cell line may additionally be used in the treatment of conditions associated with, for example, necrotic, apoptotic, damaged, dysfunctional or morphologically abnormal myocardium. Such conditions include, but are not limited to, ischemic heart disease, cardiac infarction, rheumatic heart disease, endocarditis, autoimmune heart disease, valvular heart disease, congenital heart disease, cardiac arrhythmia, and cardiac insufficiency ( cardiac insufficiency). Such differentiated cells capable of proliferating and having the potential to differentiate into cardiac cell types, including cardiomyocytes, endothelial cells, and smooth muscle cells, may therefore be suitable for use in reversing, inhibiting, or preventing myocardial damage caused by ischemia resulting from myocardial infarction. damage to treat most heart disorders and diseases.

In other aspects, cells differentiated from the xeno-free cells provided herein are useful in the treatment of cardiac disorders characterized by abnormal heart rhythms, such as arrhythmias.

The treatment is preferably performed by administering to the heart of the subject, preferably by injection into the heart, a therapeutically effective dose of the cells. A therapeutically effective dose is an amount sufficient to produce a beneficial or desired clinical result, which may be administered in 1 or more administrations. Depending on the type of heart tissue that needs to be repaired, injections can be administered to different areas of the heart. Administration may be performed using catheter-based methods following opening of the chest cavity or access through any suitable vessel. An effective dose of cells can be based on factors such as body weight, age, physiological condition, medical history, infarct size, and time elapsed since onset of ischemia. Administration of the cells includes treating the subject with an immunosuppressive regimen, preferably prior to such administration, to suppress the rejection.

Other uses

The xeno-free hBS cell lines or differentiated derivatives thereof obtained by the method of the present invention are also useful in medical research, since they are very suitable in vitro models for the study of human diseases, such as human degenerative diseases.

Potential applications of xeno-free hBS cells themselves and cell lines and cell populations derived therefrom are found in drug discovery and drug development eg in the pharmaceutical industry and in toxicity testing of all kinds of chemical pharmaceutical products. Today, large-scale and high-throughput screening of drug candidates often relies on biochemical assays that provide information on compound binding affinity and specificity but little or no information on function. Functional screens rely on cell-based screens and typically use organisms of less clinical relevance, such as bacteria or yeast, which can be produced economically and rapidly at high volumes. Successive rounds of screening use more clinically relevant model species, but these are more expensive and the screening process is not time-consuming. Screening tools exist based on human primary cells or immortalized cell types, but these cells are limited in supply or usefulness due to loss of vital functions through in vitro culture and transformation. The availability of xeno-free hBS cells and hBS cells differentiated under engineered conditions provides a new and unique ability to perform human cell-based assays with high capacity without compromising clinical relevance.

Xeno-free hBS cells can be used in high-throughput screening by combining high capacity with improved clinical significance. The ability to use gene targeting to precisely engineer the genome in hBS cells (differentiation or non-differentiation of genetically engineered cells into various cell types) allows the use of this technology to identify new therapeutically active substances through primary and secondary screening.

Accordingly, the present invention relates in other aspects to determinations obtained by the method of the present invention for

i) Production of monoclonal antibodies

ii) in vitro toxicity screening,

iii) in vitro screening of potential drug substances, or

iv) Users of identified hBS cells for potential drug substances.

Description of drawings

Figure 1 shows a blastocyst at 40x magnification before treatment with acidic Tyrode's solution to remove the zona pellucida and trophectoderm fraction.

Figure 2 shows a blastocyst at 40x magnification after treatment with acidic Tyrode's solution to remove the zona pellucida and trophectoderm fraction.

Figure 3 shows A) hBS cell line SA611 at passage 7, day 4 at 10x magnification.

Figure 4 shows a positive reaction to the marker A) ALP in undifferentiated hBS cells. .

Figure 5 shows a positive reaction to the undifferentiated hBS cell marker SSEA-4 and a negative reaction to the differentiated hBS cell marker SSEA-1, both at passage 6 and at 20X magnification.

Figure 6 shows a positive reaction for the markers Tra 1-60 and Tra 1-81 in undifferentiated hBS cells, both at passage 6 and at 20X magnification

Figure 7 shows positive responses for the endoderm marker HNF-3β and the neuroectoderm marker β-tubulin in passage 6 after spontaneous differentiation over a course of 21 to 28 days at 20X magnification.

Figure 8 shows additional morphological and immunohistological features of the xeno-free hBS cell line SA611. (A) shows blastocysts (scale-bar 25um). (B) shows the morphology of SA611 after 12 passages in the xeno-free condition (scale bar = 100um). (C-H) show the use of Oct-4 (C), SSEA-1 (D), Tra1-60 (E), Tra1-81 (F), SSEA-3 (G), SSEA-4 (H) after 12 passages ) for immunofluorescence of undifferentiated SA611. (Please note that the images in (C) and (E) are double-stained images generated using different secondary antibodies.) In (C), (E) and (G) the scale bar is 50um, in (F) and 100um in (H).

Figure 9 shows the genetic characteristics of the xeno-free hBS cell line SA611 at passage 9. (A) shows that the chromosomes are diploid and normal. (B) and (C) show the in situ hybridization fluorescence of selected chromosomes from SA611, which proves that the cells are XY and that normal chromosomes 12 and 17 are diploid. (C) Further, the X chromosome is shown in blue, the Y chromosome in golden yellow, chromosome 13 in red, chromosome 18 in light green and chromosome 21 in green (which is almost impossible to visualize in black and white).

Figure 10 shows the pluripotency of the xeno-free hBSC line SA611 in vivo) (in (A), (C) and (E)) and in vitro (in (B), (D) and (F)) Confirmation of sex: Histological analysis of teratomas from SA611 after 11 passages under xeno-free conditions: (A) neuroectoderm (ectoderm), (C) cartilage (mesoderm), (E ) secretory epithelium (endoderm). In vitro differentiated SA611 was analyzed using immunofluorescence 2 to 4 weeks after passaging: (B) β-III-tubulin-positive neurons (ectoderm), (D) ASMA-positive smooth muscle actin (mesoderm), and ( F) HNF3β (Foxa2) positive cells (endoderm). Scale bar 50 μm (A, B, D, E, F), scale bar 100 μm (C).

Example

Example 1 - Obtaining and culturing xeno-free hBS cells

in obtaining Surplus human embryos from clinical in vitro fertilization (IVF) treatments were donated with the consent and approval of the University's local ethics committee. Donated embryos were cultured into 5-day-old blastocysts in media routinely used for IVF treatments. Blastocysts were graded as 4AA according to Gardner and quality A according to WO2003055992 (extended blastocyst with many different tightly packed ICM cells and adherent trophectoderm with many cells). Before placing the inner cell mass cells on human foreskin fibroblast feeder cells inactivated by mitomycin C in xeno-free serum, they were treated with acidic Tyrode's solution (Medicult) at room temperature (i.e. concentration) to treat blastocysts for 15 to 30 seconds to remove parts of the zona pellucida and trophectoderm, said xeno-free serum contains an osmolarity of approximately 270, supplemented with 20% (v/v) human serum, 4ng/ mL of human recombinant bFGF, 1% penicillin-streptomycin, 1% Glutamax, 0.5mmol/l β-mercaptoethanol and 1% non-essential amino acids (GibcoInvitrogen Corporation) in DMEM. (See Figures 1 and 2.) The blastocysts were then incubated at 37°C in air containing 5% _CO2 . 50% of the medium was changed every 2 to 3 days and after 10 days the cells were passaged mechanically to fresh hFF feeders. From passage 2, hBS cells (cell line SA611) were passaged mechanically using glass capillaries as cutting and transfer tools. They were subcultured approximately once a week and were cultured for more than 11 passages at the time of priority filing (October, 2005). (See Figure 3). The xeno-free SA611 hBS cell line at the time of PCT entry (October, 2006) was cultured for a total of more than 30 passages.

Example 2 - Establishment of a human foreskin fibroblast feeder cell line (eg cell line hFF003)

Human foreskin samples from circumcised 8-week-old boys were aseptically collected in sterile IMDM (Invitrogen) containing 2X gentamicin. Skin explants were placed in ²⁵ Gm primariatissue culture flasks (Becton Dickinson) containing IMDM medium (Invitrogen), 1% penicillin-streptomycin (Gibco Invitrogen Corporation) and 10% human serum middle. After about 10 days, a confluent monolayer had established. Cells were serially passaged using TrypLE ^™ Select (Invitrogen). After amplification, the list of human pathogens (mycoplasma, HIV types 1 and 2, hepatitis B and C, cytomegalovirus, herpes simplex virus types 1 and 2, Epstein-Barr virus, and human papillomavirus ) to detect it.

The preparation of embodiment 3-feeder layer

Tissue culture wells were coated with 0.1% recombinant human gelatin (Fibrogen) for a minimum of 1 hour at room temperature prior to plating xeno-free human fibroblast feeders. Confluent monolayers of xeno-free hFF003 (passages 5 to 8) grown in IMDM, 10% human serum, and 1% penicillin-streptomycin were then treated with mitomycin C (Sigma) for 2.5 Hours. The medium (the medium based on supplemented with 10% (v/v) human serum, 1% penicillin-streptomycin, 1% Glutamax, 0.5mmol/l β-mercaptoethanol and 1% non-essential amino acids (Gibco Invitrogen Corporation) in DMEM (supra)), Mitomycin C-treated feeders were plated onto IVF wells (Becton Dickinson) at 200,000 cells/2.89 cm ² . Before placing blastocysts with their inner cell mass cells and cells derived therefrom or hBS cells, the medium was changed to supplemented now with 20% (v/v) human serum, 10 ng/mL human recombinant bFGF, 1 % Penicillin-Streptomycin, 1% Glutamax, 0.5mmol/l β-mercaptoethanol and 1% non-essential amino acids (Gibco Invitrogen Corporation) in DMEM (as above). (Same medium as described in Example 1).

Example 4 - Preparation of serum-based media

Human serum was obtained from blood samples of at least 15 healthy individuals (Blodcentralen, Sahlgrenska University Hospital) by collecting blood in heparin-free plastic bags overnight at approximately 8°C, where the serum could be separated from clotted material. The sera were further sterile filtered, pooled and frozen in appropriate proportions. (At Blodcentralen, Sahlgrenska University_Hospital tests the blood before use for a standard panel of pathogens including hepatitis B, hepatitis C, HIV, HTLV and syphilis.)

The medium was further prepared by adding 20% (v/v) of thawed serum and other components described in Example 1 to DMEM (as above).

Example 5 - Freezing and Thawing by Vitrification of Xeno-free hBS Cells

Cells of the hBS line SA611 were frozen and thawed at several passages, for example at passage 25, following the method described in WO2004098285. Prepare two solutions A and B (Solution A: sterile-filtered 10% ethylene glycol, 10% DMSO in Cryo-PBS; solution B: sterile-filtered 0.3M trehalose, 20% ethylene glycol, 10% DMSO in Cryo-PBS). Colonies of the selected hBS cell line SA611 were chipped in the same manner as when cells were chipped for routine passage using a stem cell chipping tool (Swemed Labs International, Billdal, Sweden). First, incubate the cell fragments in 500ml of preheated (37°C) solution A for 1 minute, then transfer to 25ml of solution B, incubate for 30 seconds, and then transfer to fresh droplet of solution B, incubate for 20 to 30 seconds. The volume is approximately 40-50 μl. The cell debris was aspirated into the pipette prepared for vitrification, and the pipette was closed with a bond. Then immerse the pipette in liquid nitrogen.

After a few days, thaw the pipettes containing frozen SA611 as follows:

Two solutions C and D were prepared (solution C: sterile filtered 0.2M trehalose in Cryo-PBS; solution D: sterile filtered 0.1M trehalose in Cryo-PBS). Solutions C and D and hBS-medium were prewarmed at 37 °C. Remove the closed pipette containing the vitrified SA611 from the liquid nitrogen tank. Hold the pipette at room temperature for 10 seconds, then thaw quickly (within seconds) in a 40°C water bath. Use a pair of autoclaved scissors to cut open the pipette at the clogged end and use a syringe to push the contents out of the pipette into Solution C. hBS cells were incubated in 500 μl solution C for 1 min, then transferred to 500 μl solution D and incubated for 5 min. Under a stereomicroscope, hBS cell fragments were quickly rinsed in xeno-free serum-based medium and then plated in culture dishes on xeno-free human fibroblast feeders in serum-based medium on the cell. The cells were then cultured (incubated at 37°C), the number of new colonies established were counted, and passaged to verify the viability of the hBS cells after vitrification.

Example 6 Characterization of xeno-free hBS cell lines

Immunohistochemical and Histochemical Analysis

Colony cultures of hBS cells were fixed in 4% paraformaldehyde and then permeabilized. After successive washing and blocking steps, cells were incubated with primary antibodies overnight at 4°C. Primary antibodies used were specific for Oct-4, TRA-1-60, TRA-1-81, SSEA-1, SSEA-3 and SSEA-4 (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.southernbiotech.com ) is specific. FITC- or Cy3-conjugated secondary antibodies were used for detection. Nuclei were counterstained with DAPI (Vectashield; Vector Laboratories, Burlingame, CA; http://www.vectorlabs.com ). Alkaline phosphatase (ALP) activity was determined according to the manufacturer's protocol (Sigma-Aldrich Stockholm, Sweden; http://www.sigmaaldrich.com ).

To identify cells from the 3 different germ layers, immunohistochemical analysis was performed as described above using the following antibodies: HNF3β for endoderm cells (Santa Cruz Biotechnology, Santa Cruz; http://www.southernbiotech.com ). Mesodermal cells were detected using ASMA (company), and neuroectodermal cells were identified using β-tubulin-III mAb (Sigma-Aldrich).

Alkaline phosphatase (ALP) reactions were detected using commercially available kits and following the manufacturer's protocol (Sigma-Aldrich).

In undifferentiated colonies, positive reactions were detected for ALP (see Figure 4), Oct-4, Tra1-60, Tra1-81, SSEA-3 and SSEA-4, as well as for SSEA-1 (see Figure 5). , 6, 8) are negative reactions. (see Figure 4-6).

Karyotyping and FISH

hBS cells engineered for genetic characterization were retransferred to mouse embryonic fibroblasts for two passages or transferred to Matrigel plates (Becton Dickinson) and further cultured for approximately 10 days. Cells were then incubated in the presence of Calyculin A, harvested by centrifugation, lysed by hypotonic treatment, and fixed using ethanol and cold acetic acid. Chromosomes were visualized using trypsin-Giemsa or DAPI staining. For fluorescence in situ hybridization (FISH) analysis, commercially available reagents containing probes for chromosomes 12, 13, 17, 18, 20, 21 and sex chromosomes (X and Y) were used according to the manufacturer's instructions with slight modifications box. Slides were analyzed under an inverted microscope equipped with appropriate filters and software (CytoVision; Applied Imaging; Santa Clara CA; http://www.appliedimagingcorp.com ). The hBS cell line SA611 was genetically characterized at passages 9 to 10. The hBS cell line SA611 has a diploid normal karyotype demonstrated by karyotype analysis. The karyotype was 46 XY. FISH analysis of selected chromosomes confirmed this finding.

The karyotype of SA611 was confirmed to be normal. (See Figure 9).

in vitro pluripotency

The pluripotency of SA611 was initially tested by allowing hBS cell colonies to differentiate spontaneously on feeders or by transferring undifferentiated hBS cell colonies onto Matrigel ^™ coated plates (Becton Dickinson) on which they were allowed to differentiate spontaneously. In both cases, when differentiation was induced, the medium was switched to VitroHESTM (Vitrolife, Kungsbacka, Sweden). After 3 to 4 weeks of culture, colonies were analyzed by immunohistochemistry to identify cells from the 3 different germ layers.

Positive reactions were identified for the early endoderm marker HN3β (sometimes also referred to as Foxal), the ectoderm markers β-tubulin and ASMA (α-smooth muscle actin). (See Figure 7 for HNF3β and β-tubulin responses, and Figure 10 for all 3 markers (B, D, F)).

In vivo pluripotency

To probe the pluripotent properties of the xeno-free hBS cell line SA611 in vivo, clusters of undifferentiated hBS cells were transplanted under the kidney capsule of SCID mice. The appearance of ectodermal (neuroectoderm, Figure 10A ), mesoderm (cartilage, Figure 10C ) and endoderm (gut-like epithelium, Figure 10E ) tissue within terodermomas demonstrates that SA611 exhibits characteristic in vivo characteristics of pluripotent hBS cells. differentiation ability.

In conclusion, the xeno-free hBS cell line SA611 stably expresses the genetic and phenotypic characteristics of undifferentiated pluripotent human BS cells.

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Claims (74)

1. obtain the method for the hBS clone of no allos material, described method comprises step:

I) remove the inner cell mass that zona pellucida surrounds with the acquisition trophectoderm by the operation of no allos material from blastocyst,

Ii) remove trophectoderm at least in part obtaining isolating inner cell mass cell by the operation of no allos material,

Iii) the inner cell mass cell is placed on the people's feeder layer in the substratum of no allos material,

Iv) inner cell mass cell and people's feeder cell are cultivated about 5 days altogether to about 50 days time bar in the substratum of no allos material,

V) if any, the operation by no allos material discharges the inner cell mass cell or derives from its cell from the trophectoderm of hypertrophy,

Vi) optionally, with inner cell mass cell or the cell transfer that derives from it to the Freshman feeder layer in the substratum of no allos material, thereby the hBS cell of the no allos material of acquisition,

Vii) by in the substratum of no allos material, cultivating the hBS cell of breeding no allos material altogether, thereby obtain the hBS clone of no allos material with people's feeder cell.

2. the method for claim 1, it also comprises step

Viii) breed the clone of no allos material: in the substratum of no allos material, cultivate altogether and with about 2 days to about 20 days, cell was gone down to posterity to about 12 days suitable timed interval in for example about 4 days with people's feeder cell by following manner.

3. claim 1 or 2 method, it also comprises step

Ix) hBS clone that will not have an allos material is transferred to no allos material and no raiser's culture systems of the substratum of the supported matrix that comprises suitable no allos material and no allos material.

4. each method in the aforementioned claim, wherein by using a) acidic solution, b) for example Unidasa or PRONASE A of one or more recombinases, or c) mechanical means carries out step I).

5. the method for claim 4 is wherein carried out step I by the use acidic solution).

6. the method for claim 5 wherein by blastocyst being experienced acidic solution about 5 seconds to about 180 seconds, for example about 10 seconds to about 120 seconds, about 15 seconds to about 90 seconds, about 20 seconds to about 60 seconds, was carried out step I in about 30 seconds to about 50 seconds).

7. claim 5 or 6 method, wherein the pH of acidic solution is about 2 to about 3, for example about 2.5 to about 2.8, for example 2.5.

8. each method in the claim 5 to 7, wherein acidic solution is acid tyrode's solution.

9. each method in the aforementioned claim is wherein by using a) acidic solution, b) one or more recombinases, or c) mechanical means carries out step I i).

10. the method for claim 9 is wherein carried out step I i by the use acidic solution).

11. the method for claim 10, wherein by with step I) in the inner cell mass experience acidic solution that surrounds of the trophectoderm that obtains carry out step I i), the time of wherein experiencing acidic solution is about 5 seconds to about 180 seconds, for example about 10 seconds to about 120 seconds, about 15 seconds to about 90 seconds, about 20 seconds to about 60 seconds, about 30 seconds to about 50 seconds.

12. the method for claim 10 or 11, wherein the pH of acidic solution is about 2 to about 3, for example about 2.5 to about 2.8, for example 2.5.

13. each method in the claim 10 to 12, wherein acidic solution is acid tyrode's solution.

14. the method for claim 9 is wherein by using one or more recombinases to carry out step I i).

15. the method for claim 14, wherein said one or more recombinases are recombinant protein lytic enzymes.

16. the method for claim 15, wherein said one or more recombinant protein lytic enzymes are selected from recombinant trypsin and TrypLE ^TMSelect.

17. each method among the claim 14-16 is wherein by with step I) in the inner cell mass that surrounds of the trophectoderm that obtains experience about 5.000U and carried out step I i in about 0.5 minute to about 30 minutes to the about recombinant trypsin of 10.000U).

18. each method among the claim 14-17 is wherein by with step I) in the undiluted TrypLE that promptly uses concentration of inner cell mass experience that surrounds of the trophectoderm that obtains ^TMSelect about 0.5 for example about 0.5 to about 8 minutes, about 0.5 to about 5 minutes, about 1 to about 3 minutes, for example carried out step I i in 1.5 minutes to about 10 minutes).

19. each method in the aforementioned claim, wherein the time bar of step I in v) be step I v) in about 5 days to about 30 days, for example about 5 days to about 20 days or about 5 days to about 15 days.

20. each method in the aforementioned claim, wherein between the common incubation period of the inner cell mass cell of step I in v) by changing about 20% to about 100%, for example about 30% carries out once or the replacing of more times substratum to about substratum of 80%, about 40% to about 60%.

21. each method in the aforementioned claim, wherein about 50% substratum carries out once or the replacing of more times substratum by changing between the common incubation period of the inner cell mass cell of step I in v).

22. the method for claim 20 or 21 wherein with about 2 days to about 14 days, was carried out once or the replacing of more times substratum to about 7 days timed interval in for example about 4 days.

23. each method in the aforementioned claim, wherein, if any, step v) in by using mechanical means from the trophectoderm of hypertrophy, to discharge the inner cell mass cell or deriving from its cell.

24. the method for claim 23, wherein mechanical means comprises that the use glass capillary is as cutting tool.

25. each method in the aforementioned claim, wherein, if any, step v) in by using one or more recombinases from the trophectoderm of hypertrophy, to discharge the inner cell mass cell or deriving from its cell.

26. the method for claim 25, wherein said one or more recombinases are recombinant protein lytic enzymes.

27. the method for claim 26, wherein said one or more recombinant protein lytic enzymes are selected from recombinant trypsin and TrypLE ^TMSelect.

28. each method in the claim 25 to 27 is wherein by carrying out step v) in about 0.5 minute to about 30 minutes with inner cell mass cell or the cell that derives from it and about 5.000U to the about recombinant trypsin incubation of 10.000U.

29. each method in the claim 25 to 28 is wherein by with the inner cell mass cell or derive from its cell and the undiluted TrypLE that promptly uses concentration ^TMSelect incubation together carried out step v) in about 0.5 minute to about 15 minutes.

30. each method in the aforementioned claim is wherein carried out once during the propagation of the hBS cell of the no allos material of step in vii) or more times goes down to posterity.

31. the method for claim 30 is wherein carried out selectivity to the hBS cell and is gone down to posterity.

32. the method for claim 30 or 31 is wherein by using mechanical means to go down to posterity.

33. the method for claim 30 or 31 is wherein by using one or more recombinases to go down to posterity.

34. the method for claim 33, wherein said one or more recombinases are selected from TrypLE ^TMSelect, recombinant trypsin and recombinant collagen enzyme.

35. each method in the claim 33 to 34 was wherein carried out step vii) in about 0.5 minute to about 30 minutes by the recombinant trypsin incubation that uses the extremely about 10.000U of about 5.000U.

36. each method in the claim 33 to 34 is wherein by using the undiluted TrypLE that promptly uses concentration ^TMSelect incubation about 0.5 carried out step vii) to about 15 minutes.

37. each method in the claim 33 to 34 is wherein by using about 200U/ml recombinant collagen enzyme incubation to carry out step vii) in about 1 minute to about 40 minutes.

38. each method in the aforementioned claim is wherein cut apart by machinery and is carried out going down to posterity of the cell of step in viii).

39. the method for claim 38 is wherein cut apart by using glass capillary to carry out machinery as cutting tool.

40. each method in the aforementioned claim is wherein by using one or more recombinant proteins to carry out the passage of step in viii).

41. the method for claim 40, wherein said one or more recombinases are selected from TrypLE ^TMSelect, recombinant trypsin and recombinant collagen enzyme.

42. each method in the claim 40 to 41 was wherein carried out step viii) in about 0.5 minute to about 30 minutes by the recombinant trypsin incubation that uses the extremely about 10.000U of about 5.000U.

43. each method in the claim 40 to 41 is wherein by using the undiluted TrypLE that promptly uses concentration ^TMSelect incubation about 0.5 carried out step viii) to about 15 minutes.

44. each method in the claim 40 to 41 is wherein by using about 200U/ml recombinant collagen enzyme incubation to carry out step viii) in about 1 minute to about 40 minutes.

45. each method in the aforementioned claim is wherein at step I x) in the supported matrix of the no allos material that uses optional from recombinant human gelatin, recombinant human fibronectin polypeptide, people's placenta cells epimatrix.

46. each method in the aforementioned claim, wherein step I x) in the no allos material that uses substratum can with step I ii), iv), vi), vii) and the substratum of the no allos material that uses in the arbitrary step viii) identical or different.

47. each method in the aforementioned claim is wherein at step I x) in the substratum of the no allos material that the uses feeder cell of can choosing carry out conditioning, or it can replenish the suitable factor to keep undifferentiated growth.

48. the method for claim 47, the optional activator of the wherein suitable factor from recombinate bFGF and WNT approach.

49. each method in the aforementioned claim wherein obtains under the condition of no allos material in step I ii), iv), vi), vii) and the people's feeder cell that use in the arbitrary step viii).

50. the method for claim 49, wherein people's feeder cell derive from healthy people's tissue.

51. the method for claim 49 or 50, wherein people's feeder cell are the skin flbroblast that derive from newborn infant people's foreskin.

52. each method in the aforementioned claim, wherein step I ii), iv), vi), vii) and/or the substratum of the no allos material that uses in the arbitrary step viii) comprise the minimum medium that is suitable for the cell proliferation of people's inner cell mass.

53. the method for claim 52, wherein minimum medium is DMEM.

54. the method for claim 52 or 53, the substratum that does not wherein have the allos material also comprises about 2ng/ml to about 100ng/ml recombinant bfgf, for example about 5ng/ml is to about 50ng/ml recombinant bfgf, approximately 5ng/ml is to about 25ng/ml recombinant bfgf, and about 5ng/ml is the 15ng/ml recombinant bfgf extremely approximately.

55. each method in the claim 52 to 54, the substratum that does not wherein have the allos material also comprises the 10ng/ml recombinant bfgf.

56. each method in the claim 52 to 55, the substratum that does not wherein have the allos material also comprises about 1%v/v to about 30%v/v human serum, and for example about 10%v/v is the 30%v/v human serum extremely approximately, approximately the extremely about 25%v/v human serum of 15%v/v.

57. each method in the claim 52 to 56, the substratum that does not wherein have the allos material also comprises the 20%v/v human serum.

58. each method in the aforementioned claim wherein prepares in step I ii), iv), vi), vii) and the human serum that uses in the arbitrary step viii) through the following steps

A) in the sack that is not coated with the shop heparin, collect the healthy human blood,

B) shake the described sack that is not coated with the shop heparin, carried out about 0.5 hour to about 5 hours, for example about 0.5 hour about 2 hours time bar extremely,

C) the described sack that is not coated with the shop heparin of incubation under 5 ℃ temperature at the most carries out at least 10 hours time bar,

D) randomly, for example do not exist the opaqueness of not solidifying fibrin, liquid phase to select based on solidifying quality,

E) with serum and the material separation of solidifying,

F) serum that obtains in the sterile filtration step d),

G) compile serum from least 15 donors,

H) use preceding freezing serum.

59. obtain the method for the hBS clone of no allos material, the method comprising the steps of:

1) by at room temperature using acid tyrode's solution incubation blastocyst about 10 seconds to about 10 minutes, preferably approximately 30 seconds to about 60 seconds time bar removes zona pellucida and to the small part trophectoderm from blastocyst, thereby obtains isolating inner cell mass cell,

2) the inner cell mass cell is placed on the human foreskin fibroblast feeder layer in the substratum of the no allos material that comprises DMEM, human serum, recombinant bfgf, L-glutaminate or glutamax, non-essential amino acid, beta-mercaptoethanol, penicillin and Streptomycin sulphate

3) in the substratum of no allos material, inner cell mass cell and human foreskin fibroblast feeder cell are cultivated altogether, carried out about 5 days to about 15 days time bar, the substratum of the no allos material of replacing at least 50% in per 3 to 5 days,

4) if any, by using TrypLE ^TMSelect (Invitrogen) handles the cell that discharges the inner cell mass cell or derive from it from the trophectoderm of hypertrophy as enzyme,

5) optionally, on inner cell mass cell or the cell transfer that derives from it the fresh human foreskin fibroblast feeder layer to the substratum that does not have the allos material, thereby obtain the hBS cell of no allos material,

6), thereby obtain the hBS clone of no allos material by in the substratum of no allos material, cultivating the hBS cell of breeding no allos material altogether with the human foreskin fibroblast feeder cell.

60. the method for claim 59, it further comprises step

7) breed the hBS clone of no allos material by following manner: in the substratum of no allos material, cultivate altogether with the human foreskin fibroblast feeder cell, the substratum of the no allos material of replacing at least 50% in per 3 to 5 days, with with reasonable time at interval, pair cell went down to posterity in for example per 3 to 8 days.

61. hBS clone by the no allos material of each method acquisition in the aforementioned claim.

62. the hBS clone of the no allos material of claim 61, it is not exposed to any non-human animal's material directly or indirectly.

63. the hBS clone of the no allos material of claim 61 or 62, use therein any organic materials is that the people originates or synthetic, semisynthetic or the reorganization material.

64. the hBS clone of each no allos material in the claim 61 to 63, wherein animal is a Mammals.

65. the hBS clone of each no allos material in the claim 61 to 64, it shows in the following standard at least 1, for example at least 2, at least 3, at least 4, at least 5 or at least 6 standards: for the positive reaction of 0ct-4, TRA-1-60, TRA-1-81, SSEA-3 and/or SSEA-4 with for the negative reaction of SSEA-1.

66. the hBS clone of each no allos material is used to prepare the purposes of the derivative of its differentiation in the claim 61 to 65.

67. by the hBS clone of the no allos material of each method acquisition or the purposes of derivative in medical science of its differentiation in the claim 1 to 60.

68. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used for the purposes of the manufacturing of pharmaceutical prod, described pharmaceutical prod is used to prevent and/or treat pathological conditions and/or the disease that is caused by tissue degeneratiaon.

69. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used for the purposes of the manufacturing of pharmaceutical prod, described pharmaceutical prod is used for the treatment of and/or prevents metabolic pathological conditions and/or disease.

70. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used for the purposes of medical research.

71. be used for studying for example purposes of the external model of people's degenerative disease of human disease by the hBS clone of the no allos material of each method acquisition or the derivative of its differentiation in the claim 1 to 60.

72. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used for the purposes of drug discovery method.

73. be used for the purposes that in vitro toxicity is tested by the hBS clone of the no allos material of each method acquisition or the derivative of its differentiation in the claim 1 to 60.

74. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used to screen the purposes of purpose.

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