BRPI0711055A2 - process of liver stem cell derivation and induction of hgp expression - Google Patents

Abstract

PROCESS OF DERIVING HEPATIC STEM CELLS AND INDUCING HGP EXPRESSION. The present invention provides a process for deriving liver stem cells from stem cells derived from non-hepatic tissue. In one embodiment of the invention, liver stem cells are derived from adipose stem cells. The invention further provides a process for increasing the production of liver cytokines (e.g., HGF) starting from ASCs, which can be useful in the regeneration of liver tissue when transplanted in vivo. Tissue culture conditions, including media conditions, are provided.

Description

Report of the Invention Patent for "HEP CELL DERIVATION AND HGP EXPRESSION INDUCTION PROCESS".

CROSS REFERENCE TO RELATED PATENTS

This patent application claims priority to Provisional US Patent Application No. 60 / 794,508, filed April 25, 2006 and Provisional US Patent Application No. 60 / 894,128, filed March 9, 2007, the disclosures of which are incorporated. here by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of therapeutics. More specifically, the present invention relates to the derivation of hepatic stem cells from stem cells derived from non-liver cells.

Liver failure secondary to hepatitis, exposure to hepatotoxins and cirrhosis endangers the livers of thousands of people in the United States alone. Estimated annual direct costs for liver disease health care in the US range from $ 60 billion to over $ 100 billion. An additional $ 20-40 billion for physical disability is paid annually to these patients and their families by the Social Security Administration. Unfortunately, at this time, liver transplantation is the only therapeutic option available to patients other than palliative measures.

However, due to limited donor organs, less than a third of all patients on liver waiting lists will actually receive one. According to the United Network of Organ Sharing (UNOS), 15,700 patients are currently awaiting liver transplantation in the United States, but only 4,000 to 5,000 donor livers that can be transplanted are available annually. Since, with some exceptions, a donor agency only helps one recipient, over the past decade a growing gap has emerged between available donors and waiting transplant candidates. Therefore, new therapies are required.

Stem cell therapies offer an alternative therapeutic option for those who need full or partial liver function reconstitution. In many ways, liver cell therapies are a desirable alternative to whole-organ transplantation because a liver can be used to treat multiple patients and because surgical procedures involved in cell therapies are safer, easier, and less expensive. for the patient. Stem cell transplantation offers a treatment modality that safely and efficiently improves the biochemical function within the failing liver.

Thus, there is a need for methods to derive liver stem cells from non-hepatic tissue.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, a method of deriving liver stem cells from non-hepatic stem cells is provided comprising: (a) providing non-hepatic tissue derived stem cells; and (b) culturing stem cells on an extracellular matrix comprising at least one fibrillar matrix protein and in serum-free culture medium comprising hepatic growth factor (HGF), wherein the stem cells Non-hepatic tissue derivatives differentiate into hepatic stem cells. Examples of nonhepatic tissue include, but are not limited to, the spleen, intestine, and adipose tissue. In a preferred embodiment, adipose stem cells are derived from adipose tissue to the source of nonhepatic stem cells; however, nonhepatic tissue may be from any adult mammal. The at least one fibrillar matrix protein is preferably selected from the group consisting of collagen I and collagen III. In some embodiments of the invention the culture medium may further comprise Oncostatin M, DMSO or both and the extracellular matrix may further comprise fibronectin, matrigel, vitrogen or combinations thereof.

In some embodiments, more than approximately 70% of non-hepatic tissue-derived stem cells differentiate into hepatic stem cells.

In another embodiment of the present invention, there is provided a method of inducing HGF expression from nonhepatic stem cells comprising: (a) providing non-hepatic tissue derived stem cells; and (b) culturing stem cells on an extracellular matrix comprising at least one fibrillar matrix protein and in serum-free culture medium comprising EGF, bFGF or both, wherein the tissue-derived stem cells are not liver cells produce HGF. The culture medium may further comprise sodium ascorbic diphosphate.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a schematic representation of the derivation of hepatic stem cells from non-hepatic stem cells (e.g. ASCs) according to the present invention.

Figure 2 shows the expression of relative albumin (ALB) and alpha-fetoprotein (AFP) mRNA in ASC-derived liver stem cells.

Figure 3 shows glycogen storage in ASC-derived liver stem cells.

Figure 4 shows the morphology of ASC-derived liver stem cells.

Figure 5 provides a graph showing that EGF and sodium ascorbic diphosphate have the combined ability to induce HGF expression in human ASCs.

Figure 6 provides a graph comparing the ability of EGF and bFGF with or without ascorbic diphosphate to induce HGF expression in ASCs.

Figure 7 is an RT-PCR analysis of receptors expressed in ASCs.

Figure 8 shows the secretion of selected cytokines (n = 6 to 8 ASC donors) at varying times after exposure to Iipopolysaccharide over periods of 0 to 24 hours.

DETAILED DESCRIPTION OF MODALITIES

The present invention is directed to methods for the derivation of hepatic stem cells from non-hepatic tissue. The invention is not limited to the tissue of any of the life stages. That is, the methods of the present invention may comprise adult, fetal or embryonic tissues. In addition, the tissue source is largely directed to any non-hepatic tissue. Non-limiting examples of "non-hepatic" tissue include adipose tissue, intestine, brain, cord blood, and bone marrow. Thus, while much of the invention is described herein with reference to adult fat tissue, such reference is merely an example and should not be construed as limiting.

Derivation of nonhepatic stem cells from nonhepatic tissue

Nonhepatic stem cells may be derived from adipose tissue (ie adipose stem cells, "ASCs"), from human or other mammalian sources. Human ASCs may be isolated from subcutaneous adipose tissue preferably obtained from healthy male or female patients, for example, from those undergoing elective liposuction procedures. Murine ASCs may also be isolated from subcutaneous adipose tissue preferably obtained from male C57BL76 mice and / or male ROSA-26 mice. From these sources, within a period of 5 days of culture, it is typical to recover approximately 250,000 ASCs from one milliliter of liposuction aspirate. The average human liposuction sample is larger than approximately 500 mL. Even larger numbers of cells are recovered from equivalent volumes of murine adipose tissue, presumably due to the young age of donor animals.

Methods of deriving adipose stem cells from adipose tissue are set forth in USP Nos. 6153432, 6391297, 6429013, 6555374, 6841150, 7001746 and 7033587, the disclosures of which are incorporated herein by reference in their entirety. Briefly, in one example, once the tissue is obtained, it is subjected to differential centrifugation and expanded in culture. A single gram of tissue typically produces between 50,000 to 100,000 stem cells within 24 hours of culture. The expansion media preferably comprises 60% DMEM (low glucose) and 40% MCDB-201 supplemented with 10% fetal bovine serum (FBS); 5 pg / ml insulin, 5 pg / ml transferrin and 5 ng / ml selenium; 10-9M dexamethasone; 10 ng / mL epidermal growth factor (EGF) and / or 10 ng / mL basic fibroblast growth factor (bFGF); 10-4 M 2-ascorbic acid phosphate; 100 U / ml penicillin; and 100 U / ml streptomycin. Without being bound or bound by theory, it is believed that the addition of EGF, bFGF or both to the medium increases the ability of ASCs to later differentiate into liver stem cells. Ascorbic acid 2-phosphate may have a similar effect on ASCs particularly in relation to their ability to express HGF (discussed below).

HGF promotes differentiation of stem cells toward the liver line. The addition of HGF to the present culture conditions, where indicated, is believed to be able to complement the differentiation process. Thus, in one embodiment of the present invention, HGF production from the cell populations described herein may be used in vitro and in vivo (e.g., after transplantation) to aid differentiation of otherwise "nascent" stem cells in functional hepatocytes.

Means of differentiation

In the presence of dexamethasone, insulin, isobutyl methyl xanthine and a thiazolidinedione, ASCs undergo adipogenesis. Cells accumulate lipid vacuoles, which can be stained relative to the neutral lipid, and express adipocyte specific markers, including secreted cytokine Ieptin and aP2 protein-binding fatty acid. However, the differentiation potential of ASCs is not limited to adipocyte lineage. For differentiation into liver stem cells and hepatocytes, serum-free expansion medium is additionally supplemented with 10 ng / mL EGF and / or 10 ng / mL bFGF, 10 ng / mL HGF, 10 ng / mL Oncostatin M (OSM), 1% DMSO or combinations thereof.

Conditions of differentiation

ASCs are initially plated with expansion media at a density of approximately 5-15 X 10 3 cells / cm2 in a collagen I pre-coated tissue plate or well. Collagen I-coated tissue plates are commercially available. (e.g., BectonDickinson's MATRIGEL, PureGeI's VITROGEN), any of which may be used with the present invention. In addition, without being bound by or bound by theory, the present inventors have learned that the use of fibrillar hepatic matrix proteins (e.g. collagen, preferably collagen I and collagen III) is preferred in the method of the invention since their use seems to better reproduce the in vivo environment of stem cells found in adult liver tissue. In other words, fibrillar matrix proteins appear to provide differentiating signals that are not available or not as strong with other matrix proteins.

Fibronectin, for example, is a non-fibrillar molecule; It is a glycoprotein (collagen is not glycoprotein), which primarily allows cells to bind to other matrix molecules or tissue culture plate. That is, fibronectin is believed to be a bridge to bind integrins on the cell surface to the organ-coating collagen matrix (in vivo) or to the bottom of a tissue culture plate (in vitro). Thus, the signals that are activated by fibronectin binding are probably quite different from those activated by collagen binding. Although the use of non - fibrillary proteins such as fibronectin is not necessary, they may be used in combination with the fibrillar proteins according to the invention. In some embodiments, the extracellular matrix does not comprise fibronectin.

Thus, it is believed that the present method directs the "conversion" of nonhepatic stem cells into hepatic stem cells, which in some cases may then differentiate into mature hepatocytes rather than a direct differentiation of the stem cells. nonhepatic stem cells directly into hepatocytes, skipping the hepatic stem cell stage at once. This notion is shown schematically in Figure 1. Examples of other matrix proteins that may be suitable in the present invention include embryonic liver matrix proteins.

During this initial period, cells are allowed to reach more than approximately 80% confluence, which may take up to 3 days and are incubated in the expansion medium described above. After reaching more than approximately 80% confluence, cells are washed two or three times with phosphate buffered saline (PBS) alone and subsequently plated in serum-free differentiation medium. ASCs are maintained under these culture conditions for an additional period of 4 to 20 days for differentiation to occur. Representative cultures can be collected at regular intervals for analysis. EXAMPLE 1

Human ASCs were plated on expansion medium (ie

wheezing). After 2-3 passages, the cell population was trypsin-dispersed and inoculated at a density of 5,000 to 10,000 cells per cm 2 in collagen coated plates. The cells were propagated until approximately 80% confluence was reached, at which time. 15 medium was exchanged for hepatic differentiation medium [comprised of serum-free expansion medium supplemented with 10 ng / ml HGF, 10 ng / ml Oncostatin M (OSM) and 0.1% DMSO]. The medium was changed every three days.

Cell lysates were collected at various time points after differentiation for RT-PCR analysis or fixed for histological analysis of liver biomarkers. Some of the biomarkers used to assess in vitro hepatogenesis included: (a) the expression of albumin (ALB) and α-fetoprotein (AFP) by RT-PCR; and (b) glycogen storage (a late-stage hepatic marker analyzed by positive PAS staining).

Figure 2 shows the AFP and ALB expression of ASCs incubated for 10 days in mean differentiation. Total RNA was purified from cells using commercially available kits. 2 pg of total RNA were reverse transcribed using MMLV-RT with Oligo dT at 30 42 ° C for 1 hour in a 20 pL reaction. 40 cycles of PCR were performed using 2 µl diluted cDNA (1:10). 5 µl of the amplified products were separated on a 2% agarose gel and visualized with EtBr. (Cyclophilin B (CycIo) was used as a control for incoming cDNA).

The data show that although albumin is expressed in the population, little to no AFP is present in the same population, which is indicative of a cell population that comprises substantially liver stem cells. In fact, AFP expression is believed to indicate the presence of more mature liver cells that may have been differentiated from liver stem cells. Further analysis confirmed that these cells exhibited other liver stem cell markers, including positive expression of Ep-CAM and ICAM-1 and negative expression of MHC class Ia.

Figure 3 shows initial passage [p3] ASCs inoculated into 6-well plates, cultured to approximately 80% confluence and transferred to hepatic differentiation medium. The individual wells were fixed on day O (panel A) and day 17 (panel B) and stained for glycogen (dark shading). Figure 4 further shows that cells, which were incubated for 18 days under differentiation conditions, have hepatocyte morphology.

Taken together, these data indicate that ASCs can be differentiated into liver stem cells. Some of these stem cells may then mature into hepatocytes or biliary cells, (Figure 1) but may also self-regenerate.

In another embodiment of the invention, a method is provided for inducing expression of cytokines that may play a role in liver regeneration from ASCs. Such cytokines may include HGF, VEGF, IL-6, LIF, TNFα Ieptin and combinations thereof. The present invention provides culture conditions for increasing the production of one or all of these cytokines. Again, without being bound or bound by theory, the present inventors have learned that the use of fibrillar matrix proteins (e.g. collagen I) is generally preferred in the method of invention since their use seems to better reproduce the in vivo environment. of liver stem cells in adult tissue. Furthermore, incorporation of EGF alone or with sodium ascorbate appears to substantially increase cytokine production under these conditions. Additional factors / conditions that may contribute to the production of liver regenerating cytokines include phorbol esters, FGFs, heparin, hypoxia (defined as <5% of 02) or combinations thereof.

EXAMPLE 2

Human ASCs were propagated in DMEM / F12 medium supplemented with 3% serum and the effect of EGF and sodium ascorbate diphosphate on cytokine production was determined. Triplicate wells inoculated at 5-10 x 10 3 cells per cm 2 were incubated with EGF at concentrations of 0, 0.1, 1 or 10 ng / mL sodium +/- ascorbate diphosphate. After 72 hours, the medium was collected for HGF ELISA detection. Figure 5 is representative of the data, normalized to pg HGF / 10 6 cells (n = 3). The medium alone did not contain HGF. The data demonstrate that in the absence of EGF and sodium ascorbate, HGF is synthesized in more than 10,680 pg / 106 cells. However, this production is increased almost 5 times in the presence of both factors.

EXAMPLE 3

Undifferentiated human ASCs were propagated in DMEM / F12 medium supplemented with 3% serum and 10 ng / mL of either EGF or bFGF. Figure 6A shows that in the presence of EGF1 undifferentiated ASCs expressed HGF at levels greater than 3000 ng / million cells, reflecting a 2-fold increase from baseline levels. The presence of ascorbic 2-phosphate further increased the EGF inducing effect up to a 25-fold induction. Under the same conditions, adipocyte-differentiated ASCs expressed HGF levels by an additional 2.0 and 6.3 times, in the absence or presence of ascorbic 2-phosphate, respectively. (Figure 6B) In the presence of bFGF, HGF expression by undifferentiated huASCs was also statistically higher. (Figure 6C) However, bFGF failed to affect HGF expression from differentiated ASCs from adipocytes. (Figure 6D).

Subsequent analysis of total RNA by RT-PCR demonstrated that both undifferentiated and differentiated adipocyte huASCs expressed the bFGF receptor and EGF receptor as well as the HGF receptor (c-met) (Figure 7). ASC Cytokine Expression

In addition to HGF, human ASCs secrete a wide range of hematopoietic and other functional cytokines. When induced with lipopolysaccharide (LPS, 100 ng / mL), otherwise hot ASC cultures exhibit a higher steady-state level of mRNAs for interleukins 6, 7, 8, and 11 (IL-6, -7, - 8, 11); leukemia inhibiting factor (LIF); macrophage colony stimulating factor (M-CSF); granulocyte-macrophage colony stimulating factor (GM-CSF); granulocyte colony stimulating factor (G-CSF); the flt-3 ligand (Flt3L) as shown in Figure 8 as well as the kit ligand (KL, also known as stem cell factor); tumor necrosis factor α (TNFα); and bone morphogenic proteins 2 and 4 (BMP-2, -4). Both murine and human bone marrow-derived stem cells also express these same cytokines. As shown in Table 1 below and Figure 8, ASCs significantly increase their secretion of IL-6, IL-7, IL-8, GM-CSF, and M-CSF within 24 hours of stimulation with LPS Table 1. LPS induction of secreted cytokines (ELISA, pg / mL)

<table> table see original document page 11 </column> </row> <table>

Thus, ASCs transplanted immediately or soon after liver damage can improve liver regeneration, in part through local release of HGF and VEGF. The introduction of ASCs into a mammal may be accomplished by any of the methods known and available in the art, including injection. In another method of cell delivery, ASCs are transplanted into the recipient mammal by intraparenchymal or intrahepatic injection after encapsulation. Transplanting cells embedded, for example, in an alginate matrix, offers several advantages, including: (a) providing cells with a "3-D environment" that enhances viability after implantation; (b) allow a larger number of cells to be implanted; (c) create a microenvironment that supports growth and differentiation; and (d) allow for co-encapsulation of factors (eg growth factors or various cell types as described above) prior to transplantation.

In addition, since cells are soaked in a hydrogel, they are less prone to dispersal to other body sites as is often observed when cell suspensions are used for transplants.

Briefly, cell encapsulation is performed using an electrostatic bead production apparatus after mixing the cells in a sodium alginate solution. The beads are resuspended in PBS at a cell concentration equivalent to approximately 107 cells / ml. An incision for minilaparotomy is performed using a sterilization technique after adequate receiver anesthesia and sedation have been achieved. Cell suspension is provided by direct injection with serine into the tissue periphery (delivery of 0.1 ml is common in mice) with hemostasis achieved by topical compression. This technique may also apply to humans and mouse models.

In a second transplantation methodology, up to 106 cells can be injected intrasplenically following adhesion to Cytodex 3TM microbeads (Amersham Pharmacia). After transplantation, cells rapidly translocate to liver sinuosities and can be detected in the liver within two hours of transplantation.

Dosage

For humans, a "dose" of approximately 3 χ 109 cells or 1% liver mass when injected into the hepatic circulatory system is preferred based on the experience of this dose in mouse studies. In fact, in mice, a dose of 2 million cells is preferred. Medium and high dose levels of 4 and 6 million cells, respectively, were based on the results of an acute pilot pilot study in which groups of 3 male NOD-SCID mice each received doses of O, 2, 5, 5, 10, 20 and 40 million human liver cells in a volume of one milliliter. Transplantation of 10, 20 and 40 million cells resulted in 100% mortality and transplantation of 5 million cells produced mortality in 1 of 3 mice within the 20 hour period, while 2.5 million cells per mice were well tolerated. The high dose of 6 million cells, which would be administered in a 0.2 mL dose volume, was selected because it was considered to be the maximum dose that could be administered without rupturing the spleen and it was expected that most animals tolerated the dose (ie one BAT). The average dose of 4 million cells is preferred as a midpoint between low and high dose levels.

Particularly, when humans are treated, all processing steps should be conducted according to cGMP standards with appropriate SOPs. Human ASCs are preferably isolated by trypsin digestion. The cells could be used immediately or cryopreserved prior to use. Human ASCs, if cryopreserved, would be thawed and suspended in a serum-free isoosmotic medium suitable for infusion at a concentration of not less than approximately 0.1 and no more than 10 million cells per mL. Cell viability can be assessed by typhan blue exclusion or an equivalent assay. The cells would be stored at room temperature or on wet ice and evaluated by microscopic examination for evidence of any lump formation. If observed, the cells would be screened through a 40 micron nylon mesh filter and the final cell concentration would be determined again. Cells would be infused intravenously into a recipient suffering from liver damage (eg, acute exposure to toxins) at a dose of 1 to 10 million cells per kg body weight. Recipients would be monitored for evidence of any acute reactions, such as fever, chills, or change in mental condition, and if observed, would be treated symptomatically through standard medical practice. Subjects would then be monitored over the next 2 months through weekly serum chemistry to assess liver function recovery.

As such, this invention may benefit the treatment of various forms of both acute and chronic liver failure. The availability of autologous or allogeneic human ASCs in the form of an adult stem cell to improve liver function is of value to patients suffering from liver damage secondary to exposure to toxins (carbon tetrachloride, acetaminophen), an infectious agent ( hepatitis, cytomegalovirus) or surgical excision due to tumor metastasis or trauma. The ability of human ASCs to differentiate into functional hepatocytes and / or to disrupt growth factors that underpin progenitor proliferation and differentiation of endogenous hepatocytes may provide a mechanism for improving and accelerating the recovery of liver function under these conditions.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification and it is intended that this patent application cover any variations, uses or alterations of the following invention. In general, the principles of the invention and even such variations of the present disclosure as with respect to known or customary practice within the technique to which the invention belongs and how they may be applied to the essential characteristics set forth hereinbefore and as follows in scope of the appended claims.

Claims (13)

Process for deriving hepatic stem cells from non-hepatic stem cells characterized by the fact that it comprises: (a) the provision of non-hepatic tissue-derived stem cells; and (b) culturing stem cells on an extracellular matrix comprising at least one fibrillar matrix protein and in serum-free culture medium comprising hepatic growth factor (HGF), wherein the tissue-derived stem cells non-hepatic differentiate into hepatic stem cells. Process according to Claim 1, characterized in that the non-hepatic tissue is adipose tissue. Process according to Claim 1, characterized in that the non-hepatic tissue is tissue from an adult mammal. Process according to claim 1, characterized in that the at least one fibrillar matrix protein is selected from the group consisting of collagen I and collagen III. A process according to claim 4 wherein at least one fibrillar matrix protein is collagen I. Process according to claim 1, characterized in that the culture medium further comprises Oncostatin M, DMSO or both. Process according to Claim 1, characterized in that the extracellular matrix further comprises fibronectin, matrigel, vitrogen or combinations thereof. Process according to claim 1, characterized in that more than approximately 70% of non-hepatic tissue-derived stem cells differentiate into hepatic stem cells. Process according to claim 1, characterized in that the stem cells derived from non-hepatic tissue were additionally derived from the culture passage. 10. Process for inducing HGF expression from nonhepatic stem cells characterized by the fact that it comprises: (a) the provision of non-hepatic tissue-derived stem cells; and (b) culturing stem cells on an extracellular matrix comprising at least one fibrillar matrix protein and in serum-free culture medium comprising EGF, bFGF or both, wherein the tissue-derived stem cells are not liver cells produce HGF. Process according to Claim 10, characterized in that the culture medium comprises EGF and bFGF. Process according to Claim 10, characterized in that the culture medium further comprises sodium ascorbic diphosphate. Process according to Claim 11, characterized in that the culture medium further comprises sodium ascorbic diphosphate.