METHODS FOR TREATMENT AND PREVENTION OF ACUTE LIVER FAILURE

Field of the Invention

[0001] The present invention is generally related to methods of treating acute liver failure (ALF). Particularly, the invention is related to methods for the creation of preclinical models of ALF and potential prevention, early stage treatment and treatment for ALF.

Background of the Invention

[0002] ALF is a rapid and severe hepatic injury resulting in massive hepatocyte necrosis and liver function deterioration, which can be induced by exogenous drug/toxin, virus infections, Wilson's disease or genetic predisposition. Clinically, serum liver biomarkers such as albumin (Alb), alkaline-phosphatase (Alk-P), aspartate transaminase (AST), alanine transaminase (ALT), ammonia, international normalized ratio (INR), prothrombin time (PT), and creatinine are used to determine the severity of ALF. The liver is an organ responsible for metabolism and detoxification, ALF leads to metabolic impairment and toxic metabolism associated multiple organ failures, encompassed with neuropsychiatric disturbance from hepatic encephalopathy. Currently, liver transplantation is the only option for liver function restoration. Unfortunately, limited available organs, donor immunogenicity, surgical invasiveness of the patients and requirement of long-term intensive care reduce the survival rate of ALF. Conservative treatment to improve the liver function is critical for ALF patients in prolonging the time window waiting for liver transplantation.

[0003] Cell-based therapy, including mesenchymal stem cells (MSCs), have been recognized as a promising therapeutic option for those unmet need conditions due to their regenerative capability. US 20080241246 relates to cell-based methods for the treatment of liver disease in a subject, wherein a composition comprising AC133+ cells is administered. US 20080311094 relates to isolated liver progenitor or stem cells, originating from adult liver, and to their use in medicine, hepatology, inborn errors of liver metabolism, transplantation, infectious diseases, liver failure. US 20110218143 provides a method for tissue repair or regeneration, the method involving contacting a cell with an effective amount of an agent having at least two, three, or four activities that is any one or more of i) inhibition of hsp-90 biological activity; ii) mobilization of a bone marrow derived stem cell; iii) inhibition of apoptosis; and iv) modulation of an immune response. US 20120189702 provides methods for treating liver failure in a subject comprising transplanting hepatocytes or stem or progenitor cells in an extrahepatic site in the subject in an amount sufficient to provide liver support and/or induce liver regeneration. US 20140219952 provides methods of treating a subject with acute liver injury comprising administering to the subject at least one stem cell mobilizer. US 20160024468 relates to somatic stem cells that are CD 10+, CXCR4+, and CD31+ and another somatic stem cells that are CD105+, CD44+, and nestin+ for treating acute liver failure. In addition, a previous study demonstrates that MSCs are capable of differentiating into functional hepatocyte in vitro and in vivo in a mouse model with CC14-induced ALF (Lee KD, et al, Hepatology. 2004 Dec;40(6): 1275-84; Tom K. Kuo et al, Gastroenterology 2008; 134: 2111-2121, 2121.el-3).

[0004] However, speed and mechanism of postnatal hepatocyte regeneration capacity in a rodent is much better than in other Mammalia (Michalopoulos GK1. Liver regeneration. J Cell Physiol. 2007 Nov; 213(2): 286-300; Miyaoka Yl, Miyajima A. To divide or not to divide: revisiting liver regeneration. Cell Div. 2013 Jun 20;8(1):8), and intensive clinical monitoring is infeasible in mice, a small animal, during the critical period. Therefore, the therapeutic benefit as well as the clinical pathway of MSCs in ALF in a rodent Mammalia cannot be before clinical study. Summary of the Invention

[0005] The invention provides a method for prevention or treatment of an acute liver dysfunction with an elevation of serum ammonia, serum creatinine, or INR in a subject, comprising infusion administering a single unit dose of a mesenchymal stem cell population in an effective amount of about 0.5 x 10 ⁴ to about 5 x 10 ⁶ cells/kg body weight to the subject, wherein the serum ammonia, serum creatinine and INR is greater than about 70 μg/dL, about 1.3 mg/dL and 1.2, respectively. Accordingly, the invention provides a use of a single unit dose of a mesenchymal stem cell population in the manufacture of a medicament for prevention or treatment of an acute liver dysfunction with an elevation of serum ammonia, serum creatinine, or INR in a subject, wherein the mesenchymal stem cell population is in an effective amount of about 0.5 x 10 ⁴ to about 5 x 10 ⁶ cells/kg body weight, and wherein the serum ammonia, serum creatinine and INR are greater than about 70 /g/dL, about 1.3 mg/dL and 1.2, respectively.

[0006] The method and use of the invention can prevent alkalosis in the ALF subject. The method and use also maintains homeostasis of circulation oxygen and the electrolytes and sugar balance, improves tissue oxygen supply, or reconstructs platelet in the ALF subject.

[0007] The reference value indicating the ALF includes for example, serum ammonia, serum creatinine and INR, prothrombin time, Na ⁺ , Ca ²⁺ , K ⁺ , glucose, p0 ₂ , S02c, pC0 ₂ , TC0 ₂ , HC03-, pH, BE(B), AST, ALT and ALP.

[0008] The mesenchymal stem cells used in the invention are preferably mesenchymal stem cell population (MSC), adipose tissue-derived stem cell (ADMSC) population, orbital fat-derived stem cell (OFSC) population or quadri -positive stromal cell (QPSC) population.

[0009] In one embodiment, the single unit dose of the mesenchymal stem cell population used in the method and use of the invention ranges from about 0.5 x 10 ⁴ to about 5.0 x 10 ⁸ cells/kg body weight. [0010] The method and use of the invention prolongs the survival of the subject from ALF and rescues the subject from liver injury, prevents hepatocyte loss in the first three days and stays in the diseased liver up to one month in the subject, reduces one or more the following serum level: aspartate aminotransferase, alanine aminotransferase, albumin and alkaline-phosphatase after 3 days, replenishes platelet count in around 1 week, or prolongs the survival and prevention of lethal sequelae from ALF in the acute stage and supports liver function by hepatocyte differentiation in the subacute stage.

[0011] In the method and use of the invention, liver tissue regeneration is promoted by prevention of extensive necrosis in the first 2 weeks and differentiation into functional hepatocytes in the liver after 2 weeks or hepatocyte growth factor (HGF) is produced and responsible for the first-week therapeutic benefits, while vascular endothelial growth factor is observed when hepatocyte differentiation occurs.

Brief Description of the Drawing

[0012] Figures 1 A to C show survival curve of ALF miniature pigs. A, In the control group, the survival rate was 33.3% at 2 days (2/6) and 16.7% at 4 days (1/6). In the MSC group, the survival rate was 90% at 2 days (9/10), 60% at 4 days (6/10), and 1 animal was still alive on Day 28. B, In the control group during autopsy, a necrotic appearance on both left and right lobe liver was observed. C, In the MSC group, atrophic change on the left lobe and pinkish on the right lobe liver after MSC transplantation for 2 weeks.

[0013] Figures 2 A to J show histopathological analysis and cell fate determination in the liver. Right liver sections stained with hematoxylin and eosin (H&E) showed pictures of (A) normal liver tissue, (B) liver tissue from autopsy without MSC treatment, (C) post operation 30 minutes with MSC infusion, (D) post operation 2 weeks with MSC infusion and (E) post operation 4 weeks with MSC infusion (200x). Right liver sections immunostained with human/porcine albumin (blue) and human nucleus (brown) demonstrated (F) strong albumin signals in normal liver tissue and (G) marked loss of albumin signals during autopsy without MSC treatment. After MSC transplantation, (H) transplanted cells clustered near the central vein area at post operation 30 minutes, and (I) moved to the liver parenchyma 2 weeks after MSC transplantation. (J) Albumin-expressing human cells in liver parenchyma could be easily found at 4 weeks after MSC transplantation.

[0014] Figures 3 A to D show impact of MSCs on complete blood count during ALF. Under intensive care, surgical ligation did not alter the white count level of mini-pigs (A) but slightly increased red cell count (B) and hemoglobin (C) on Days 2 and 3. (D) Surgical induced ALF significantly decreased platelet count from Day 1, and MSCs corrected thrombocytopenia 1 week after transplantation.

[0015] Figures 4 A to C show impact of MSCs on hepatocyte loss after ALF. Surgical ligation resulted in extensive hepatocyte loss bile duct cell loss evidenced by sharply elevation of serum (A) aspartate transaminase (AST), (B) alanine transaminase (ALT), and (C) alkaline-phosphatase (ALP) levels, and MSCs reduced serum ALP on Day 1 and 2 (C) and prevented continuous hepatocyte loss by reducing serum AST (A), ALT (B) and ALP (C) after Day 3 (t-test, p < 0.05 between control and MSC group at the same time point (*), between each time point and before op in the control group (#) and between each time point and before op in the MSC group ($), n=6 in control group, n=10 in MSC group at baseline).

[0016] Figures 5 A to D show impact of MSCs on ALF lethal complications. In the control group, surgical ligation resulted in (A) prolonged prothrombin time (PT) and (B) increased international normalized ratio (INR) from Day 1 of ALF, and MSCs kept a normal PT and INR during the follow-up period. C, Serum creatinine (Cre) was gradually increased from Day 1, and MSCs inhibited the elevation of serum creatinine by ALF. D, Serum ammonia levels flared up from Day 1 after surgical ligation, and MSCs maintained a normal ammonia level during this study (t-test, /? < 0.05 between control and MSC group at the same time point (*), between each time point and before op in the control group (#) and between each time point and before op in the MSC group ($), n=6 in control group, n=10 in MSC group at baseline).

[0017] Figures 6 A to K show impact of MSCs on blood gas and electrolytes. MSCs kept a constant and stable electrolyte levels in the first 24 hours after surgical ligation and prevented (A) hypernatremia, (B) hypokalemia, (C) hypocalcemia, and (D) unstable blood sugar levels induced by ALF. MSCs maintained the blood gas homeostasis in the first 24 hours after surgical ligation and prevented (E) hypoxemia and (F) low oxygen saturation induced by ALF. On Day 1, (G) paC02, (H) total C02, (I) HC03- levels and (J) pH value in the venous circulation were relatively insensitive to ALF. (K) Increased BE was observed in the control group after surgery (t-test, p < 0.05 between control and MSC group at the same time point (*), between each time point and before op in the control group (#) and between each time point and before op in the MSC group ($). n=6 in control group, n=10 in MSC group at baseline).

[0018] Figures 7 A to K show paracrine liver regeneration potential of MSCs in vitro. Microarray analysis revealed that MSCs expressed multiple liver regenerative factors, including (A) hepatocyte growth factor (HGF), (B) epithelial growth factor (EGF), (C) vascular endothelial growth factor (VEGF)-A, (D) VEGF-B, (E) insulin growth factor (IGF)- 1, (F) IGF-2, (G) fibroblast growth factor (FGF)-7, (H) tissue growth factor-alpha (TGF-a), (I) TGF-beta(p)l, (J) TGF-p2 and (K) TGF-p3.

[0019] Figures 8 A and B show paracrine liver regeneration of MSCs in vivo. Quantitative-PCR on specimens from liver in each time point revealed that A, human HGF was detectable in a damaged liver from Day 1 to 2 weeks after MSC transplantation. B, Human VEGF was strongly detectable in a regenerative liver after 3 weeks of MSC transplantation. (n=3 at each time points.) Detailed Description of the Invention

[0020] The invention investigates the safety and efficacy of MSCs to treat and prevent ALF.

[0021] The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0022] As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise.

[0023] As used herein, the terms "and" and "or" may be used to mean either the conjunctive or disjunctive. That is, both terms should be understood as equivalent to "and/or" unless otherwise stated.

[0024] As used herein, the term "cell population" means a population of cells that is substantially homogenous. A population of cells are at least 70%, 75%, 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells are homogenous in cell marker expression.

[0025] As used herein, the term "differentiation" means the formation of cells expressing functional markers known to be associated with cells that are more specialized and closer to becoming terminally differentiated cells incapable of further division or differentiation.

[0026] As used herein, the term "acute liver failure" is defined as the rapid development of hepatocellular dysfunction, specifically coagulopathy and mental status changes (encephalopathy) in a patient without known prior liver disease.

[0027] As used herein, the terms "treating" or "treatment" or "alleviation" refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, and amelioration and palliation of the disease state.

[0028] As used herein, "prevention" or "preventing" of a disorder or condition refers to a compound that reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0029] The term "molecular marker" as used herein designates a gene product, i.e. a protein or a ribonucleic acid including mRNA expressed from said gene.

[0030] The term "therapeutic effect" as used herein refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. For example, a therapeutic effect may include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect may also include, directly or indirectly, the arrest, reduction or elimination of the progression of a disease manifestation.

[0031] As used herein, the term "effective amount" of an agent, e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result.

[0032] As used herein, the term "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment.

[0033] As used herein, the term "subject" is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition prior to administration. [0034] In one aspect, the invention provides a method for prevention or treatment of an acute liver dysfunction with an elevation of serum ammonia, serum creatinine or INR in a subject, comprising infusion administering a single unit dose of a mesenchymal stem cell population in an effective amount of about 0.5 x 10 ⁴ to about 5 x 10 ⁸ cells/kg body weight to the subject, wherein the serum ammonia, serum creatinine and INR are greater than about 70/g/dL, about 1.3 mg/dL and 1.2, respectively. Accordingly, the invention provides a use of a mesenchymal stem cell population in the manufacture of a medicament for prevention or treatment of an acute liver dysfunction with an elevation of serum ammonia, serum creatinine, or INR in a subject, wherein the mesenchymal stem cell population is in a single unit dose of about 0.5 x 10 ⁴ to about 5 x 10 ⁸ cells/kg body weight and wherein the serum ammonia, serum creatinine and INR are greater than about 70 μg/dL, about 1.3 mg/dL and 1.2, respectively. The invention also provides a mesenchymal stem cell population for use in prevention or treatment of an acute liver dysfunction with an elevation of serum ammonia, serum creatinine, or INR in a subject, wherein the mesenchymal stem cell population is in a single unit dose of about 0.5 x 10 ⁴ to about 5 x 10 ⁸ cells/kg body weight and wherein the serum ammonia, serum creatinine and INR are greateer than about 70 μg/dL, about 1.3 mg/dL and 1.2, respectively.

[0035] In another aspect, the invention provides a method for preventing alkalosis in a subject with an acute liver dysfunction with an elevation of serum ammonia serum, creatinine or INR, comprising infusion administering a single unit dose of a mesenchymal stem cell population in an effective amount of 0.5 x 10 ⁴ to 5 x 10 ⁸ cells/kg body weight to the subject, wherein the serum ammonia, serum creatinine and INR are greater than about 70 /g/dL, about 1.3 mg/dL and 1.2, respectively. Accordingly, the invention provides a use of a single unit dose of a mesenchymal stem cell population in the manufacture of a medicament for prevention or treatment of an acute liver dysfunction with an elevation of serum ammonia, serum creatinine, or INR in a subject, wherein the mesenchymal stem cell population is in an effective amount of about 0.5 x 10 ⁴ to about 5 x 10 ⁶ cells/kg body weight, and wherein the serum ammonia, serum creatinine and INR are greater than about 70 /g/dL, about 1.3 mg/dL and 1.2, respectively.

[0036] In a further aspect, the invention provides a method for maintenance of homeostasis of circulation oxygen, the electrolytes and/or sugar balance, improvement of tissue oxygen supply, or reconstruction of platelet in a subject with an acute liver dysfunction with an elevation of serum ammonia, serum creatinine or INR, comprising infusion administering a single unit dose of a mesenchymal stem cell population in an effective amount of 0.5 x 10 ⁴ to 5 x 10 ⁸ cells/kg body weight to the subject, wherein the serum ammonia, serum creatinine and INR are greater than about 70 /g/dL, about 1.3 mg/dL and 1.2, respectively. Accordingly, the invention provides a use of a single unit dose of a mesenchymal stem cell population in the manufacture of a medicament for maintenance of homeostasis of circulation oxygen, the electrolytes and/or sugar balance, improvement of tissue oxygen supply, or reconstruction of platelet in a subject with an acute liver dysfunction with an elevation of serum ammonia, serum creatinine or INR, wherein the mesenchymal stem cell population is in an effective amount of 0.5 x 10 ⁴ to 5 x 10 ⁸ cells/kg body weight to the subject, and wherein the serum ammonia, serum creatinine and INR are greater than about 70 /g/dL, about 1.3 mg/dL and 1.2, respectively.

[0037] In one embodiment, homeostasis of one or more of circulation oxygen, the electrolytes and sugar balance can be maintained.

[0038] In some embodiments, in addition to elevation of serum ammonia, serum creatinine, or INR, the subject with the acute liver dysfunction has one or more the following reference values:

Prothrombin time higher than about 10 seconds;

Na ⁺ lower than about 125 mmol/L (hyponatremia); Ca ²⁺ lower than about 0.8 mmol/L;

K ⁺ lower than about 3 mmol/L;

One touch glucose higher than about 200 mg/dL;

p0 ₂ lower than about 50 mmHg;

S02c lower than about 80 %;

pC0 ₂ higher than about 55 mmHg

TC0 ₂ higher than about 20 mmol/L;

HC03 ^" higher than about 20 mmol/L;

pH lower than about 7;

BE(B) out of the range of +-3 mmol/L;

AST 2-fold higher than upper normal limited;

ALT 2-fold higher than upper normal limited;

ALP 1.5 -fold higher than upper normal limit.

(The normal limit of ALP varies depending on gender, age and other factors. The normal value for alkaline phosphatase is 53 to 128 U/L for a 20- to 50-year-old man and 42 to 98 U/L for a 20- to 50-year-old woman. Pregnant women typically have higher alkaline phosphatase values due to contributions from the placenta. Normal values are slightly different if a subject is older than 60— 56 to 119 U/L if the subject is a man or 53 to 141 U/L if the subject is a woman. Normal values can vary slightly among testing laboratories. Normal values from the laboratory performing the test should be used when interpreting the test results.)

[0039] The reference values (such as blood gas, liver function, CBC and renal function) described herein for a subject with an acute liver dysfunction in a mini pig are similar to those in human. The reference values include, but are not limited to, those listed in the table below. Therefore, the pig model in evaluation of the above values in ALF can be used as references of human ALF.

[0040] Reference value

In a mini pig In a human

Bi rand eas f ven on si

pH 7.38-7.42 ¹ 7.31-7.41 ⁵

pC02 (mmHg) 44-47.5 ¹ 40-52 ⁵

p02 (mmHg) 36.7-45.3 ¹ 30-50 ⁵

Na+ (mmol/L) 139-149 ² 135-147 ⁵

K+ (mmol/L) 5.1-8.8 ² 3.4-4.7 ⁵

Ca++ (mmol/L) 10.7-12.4 ² 1.13-1.31 ⁵

Glucose (mg/dL) 53-173 ² 65-115 ⁵

Hct (%) 26.3-51.7 ² 37-54 ⁵

HC03- (mmol/L) 24.9-30.3 ⁶ 23-28 ⁵

TC02 (mmol/L) 17-26 ⁷ 24-29 ⁵

BE(B) (mmol/L) 1.3-5.1 ⁶ -3-+3 ⁵

s02c (%) - 75 ⁵

1 I ,i\ Ot I IH1C1 IOI1

ALB (mg/dL) 1. ^{^} 2-2.2υ ^' 3.7-5.3 ⁵

AST (U/L) 30-85 ² 5-45 ⁵

ALT (U/L) 14-84 ² 0-40 ⁵

ALP (U/L) 124-477 ² 10-100 ⁵

TB (mg/dL) 0.1-0.3 ² 0.2-1.6 ⁵

DB (mg/dL) 0-0.15 ³ 0.00-0.45 ⁵

GGT(U/L) 55-113 ² 4-60 ⁵

Prothrombin time (sec.) 5.5 to 13.9 ⁴ 8.0-12.0 ⁵

INR (ratio) 0.85-1.15 ⁴ 0.85-1.15 ⁵

APTT (sec.) 16.3 to 57.1 ⁴ 23.9-35.5 ⁵

Lactate (mmol/L) 3.48-4.12 ¹ 4.5-19.8 ⁵

Ammonia (ug/dL) 10.6-88.8 ⁷ 0-69 ⁵

i ISi

WBC (10e3/uL) 12.8-20.8 ^' 4.5-11.0 ⁵

RBC (10e6/uL) 4.9-8.9 ² 4.2-6.2 ⁵

Hemoglobin (g/dL) 5.3-14.3 ² 12-18 ⁵

Hematocrit (%) 26.3-51.7 ² 37-54 ⁵

MCV (fL) 46.6-77.2 ² 80-96 ⁵

MCH (pg) 11.2-18.0 ² 27.5-33.2 ⁵

MCHC (g/dL) 19.6-28.7 ² 33.4-35.5 ⁵

Platelet (10e3/uL) 148-329 ² 150-350 ⁵

Neutrophil band (%) 0-4 ⁸ 0-5 ⁵

Neutrophil Seg. (%) 44.3-58.7 ² 45-75 ⁵

Lymphocyte (%) 26.5-45.3 ² 20-45 ⁵ Atypical Lymphocyte (%) - 0-5 ⁵

Monocyte (%) 0-13 ² 0-9 ⁵

Eosinophil (%) 0-8.0 ² 0-8 ⁵

Basophil (%) 0-3.0 ² 0-1 ⁵

Ki'iiiii funct ion

BUN (mg/dL) lu.S-Z l .u ' 7-20 ⁵

Creatinine (mg/dL) 0.6-1.5 ² 0.5-1.5 ⁵

1: J Swine Health Prod. 2013;21(3):148-151.

2: Taitung animal propagation station, Livestock research institute, Council of agriculture.

3: Lab Anim Res. 2012 Dec;28(4):245-53.

4: The FASEB Journal. 2006;20:A655-A656.

5: Department of pathology and laboratory medicine, Taipei veterans general hospital.

6: Can Vet J 1974;15(10):282-285.

7: Clinical Examination of Farm Animals Appendix 3 (2012)

8: Merck Veterinary Manual (http://www.merckvetmanual.com/)

[0041] In one embodiment, the method of the invention prolongs the survival of the subject from ALF and rescues the liver injury in the subject.

[0042] In one embodiment, the method of the invention prevents hepatocyte loss in the first three days and stays in diseases liver up to one month in the subject. [0043] In one embodiment, the method of the invention reduces one or more of the following serum levels: aspartate aminotransferase, alanine aminotransferase, albumin and alkaline-phosphatase after 3 days.

[0044] In one embodiment, the method of the invention replenishes platelet count in around 1 week.

[0045] In one embodiment, the liver tissue regeneration is promoted by prevention of extensive necrosis in the first 2 weeks and differentiation into functional hepatocytes in the liver after 2 weeks.

[0046] In one embodiment, hepatocyte growth factor (HGF) is produced and is responsible for first-week therapeutic benefits, while vascular endothelial growth factor is observed when hepatocyte differentiation occurs. [0047] In one embodiment, the method of the invention prolongs the survival and prevention of lethal sequelae from ALF in the acute stage and supports liver function by hepatocyte differentiation in the subacute stage.

[0048] In some embodiments, the mesenchymal stem cells are mesenchymal stem cell population (MSC), adipose tissue-derived stem cell (ADMSC) population, orbital fat-derived stem cell (OFSC) population or quadri -positive stromal cell (QPSC) population. In one embodiment, the QPSCs are those described in U.S. Application No. 14/615,737, which has at least 70% cell homogeneity and expresses cell markers of CD273, CD46, CD55 and CXCR4 but not CD45, wherein CD273 is strongly expressed at an intensity over 70%. In one embodiment, the ADSCs are those OFSCs described in US 20120288480, which express at least CD90, CD 105, CD29, CD44, CD49b, CD49e, CD58 and HLA-ABC but do not express CD133, CD31, CD106, CD146, CD45, CD14, CD117. Preferably, the stem cell is QPSC population.

[0049] In one embodiment, the single unit dose of the mesenchymal stem cell population used in the method of the invention ranges from about 0.5 x 10 ⁴ to about 5.0 x 10 ⁸ cells/kg body weight. In some embodiments, the unit dosage ranges from about 0.5 x 10 ⁴ to about 5.0 x 10 ⁷ , about 0.5 x 10 ⁴ to about 5.0 x 10 ⁶ , about 1.0 x 10 ⁵ to about 5.0 x 10 ⁸ , about 1.0 x 10 ⁵ to about 1.0 x 10 ⁸ , about 1.0 x 10 ⁴ to about 1.0 x 10 ⁷ , about 1.0 x 10 ⁴ to about 1.0 x 10 ⁶ , about 1.0 x 10 ⁴ to about 1.0 x 10 ⁵ , about 1.0 x 10 ⁵ to about 1.0 x 10 ⁸ , about 1.0 x 10 ⁵ to about 1.0 x 10 ⁷ , about 1.0 x 10 ⁵ to about 1.0 x 10 ⁶ , about 1.0 x 10 ⁶ to about 5.0 x 10 ⁸ , about 1.0 x 10 ⁶ to about 1.0 x 10 ⁷ , about 1.0 x 10 ⁷ to about 5.0 x 10 ⁸ , about 5.0 x 10 ⁵ to about 5.0 x 10 ⁶ , about 5.0 x 10 ⁵ to about 5.0 x 10 ⁷ or about 5.0 x 10 ⁵ to about 5.0 x 10 ⁸ cells/kg body weight cells/kg body weight. In a further embodiment, the single unit dose of the mesenchymal stem cell population ranges from about 0.5 to about 3.0 x 10 ⁶ cells/kg body weight, preferably, about 1.6 to about 2.4 x 10 ⁶ cells/kg body weight. [0050] The therapeutic agent refers to a drug, molecule, nucleic acid, protein, stem cell, composition or other substance that provides a therapeutic effect.

[0051] The invention indicates that the mesenchymal stem cell population decreases the mortality from ALF and prolong the survival time; in one embodiment, about 3 days in control group v.s. > more than 12 days in the mesenchymal stem cell population group. In the post op day 1, the mesenchymal stem cell population keeps the homeostasis of electrolyte, metabolism and hemodynamics from ALF-induced hypokalemia, hypoglycemia and hypoxemia. In the first 3 days, the invention inhibits the elevation of prothrombin time/international normalized ratio, serum creatinine and ammonia and maintain the levels within normal limit. With the mesenchymal stem cell population, serum levels of aspartate aminotransferase, alanine aminotransferase, albumin and alkaline-phosphatase are gradually reduced 3 days after the onset of ALF, and platelet count was replenished 1 week after ALF. Histopathological staining revealed that MSCs promote liver tissue regeneration by prevention of extensive necrosis in the first 2 weeks and differentiation into functional hepatocytes in the liver after 2 weeks.

[0052] The invention prolongs the survival and prevention of lethal sequelae from ALF in the acute stage by paracrine support mainly through HGFs and supported liver function by hepatocyte differentiation in the subacute stage. The time-dependent parameters in the invention provide a reference for clinical trial of MSC transplantation in treating ALF.

[0053] The present disclosure is further elaborated by way of the following examples and accompanying figures herein. However, these examples should not be construed to limit the scope of the present disclosure. Example

Materials and Methods

Animals

[0054] Sixteen healthy male mini-pigs weighing between 10 and 15 kg (22 and 35 lbs) were purchased from animal facility in Bali, New Taipei City. Animal housing followed the guidance of Division of Experimental Surgery at Taipei Veterans General Hospital (Taipei VGH), and all animal experiments were approved by the Institutional Animal Care & Utilization Committee of Taipei VGH (IACUC-2011-162). In this study, 6 animals were randomly enrolled into the control group while 10 were in the MSC group.

Mesenchymal Stem Cells

[0055] Human MSCs in this study are Stemchymal ^® , an ADMSC product manufactured by Steminent Biotherapeutics Inc. (SBI). MSCs were ex -vivo culture expanded and quality controlled followed SBI's standard operation procedures in a cell factory established in accordance with PIC/S Good Manufactory Practice guidelines. In brief, adipose tissue was harvested from healthy donors and immediately transported to SBI's processing facility at low temperature (0~5°C). MSCs were isolated, purified and maintained in SBI's proprietary culture medium during culture expansion. MSCs with passage at 12 were then packaged in a cryopreservation bag, and the product (Stemchymal ^® ) was sent for quality certification and cryopreservation. The quality control for Stemchymal ^® is composed of in- process control and product-release tests, which includes but is not limited to viability, sterility, mycoplasma tests, endotoxin assessment, MSC phenotyping (positive for CD 73, CD 90, and CD 105, negative for CD 34, CD 45, CDl lb, CD 19 and HLA-DR), and tri- lineage differentiation capacity (osteogenic, chondrogenic and adipogenic differentiation). Experimental Protocol and Cell Transplantation

[0056] Miniature pigs were fasted overnight before surgery. Under general anesthesia

(GA) with 1.5% isofluorane through inhalation, ventilation was provided through oral- tracheal intubation with oxygen supplied at a constant rate of 16L/min. After laparotomy, a 7- Fr catheter was surgically placed into the inferior vena cava in order to obtain blood samples. Surgical procedures to induce acute liver failure were as follows: (1) Permanent ligation of the left hepatic artery, followed by temporary occlusion of the right hepatic artery and right portal vein for 1.5 hours. (2) Re-perfusion of the right hepatic artery and right hepatic vein 1.5 hours after occlusion. (3) Ligation of the left hepatic vein. (4) 2.4 x 10 ⁷ cells (1.6-2.4 x 10 ⁶ MSCs/kg body weight) suspended in 100 mL of normal saline (MSC group) or 100 mL normal saline (control group) was infused intravenously via the splenic vein with 20-minute duration. (5) Closure the wound and recover animals from anesthesia.

Blood Examinations

[0057] During the surgical procedure, a single-lumen central venous catheter (Arrow

International, Reading, PA) was placed through the infra-renal vena cava and fixed by 5-0 prolene purse-string suture for blood sampling 1 mL whole blood for blood gas analysis collected from the catheter and data was tested by Nova PHOX blood gas analyzer (Diamond Diagnostics, MA, USA). Samples for CBC and biochemistry tests were obtained during and after the operation from the same catheter. For CBC test, 2-3 mL whole blood in a EDTA- containing blood tainer (BD, Franklin Lakes, NJ USA) and data were analyzed by automated cell count analyzer Sysmex XE-2100™ (Sysmex Cooperation, Kobe, Japan). For biochemistry tests, 3-5 mL whole blood in a serum separation tube containing clot activator and gel (BD, Franklin Lakes, NJ USA), and serum were analyzed by automated clinical chemistry analyzer (AD VIA 1800, Siemens, Ireland).

Histopathology

[0058] Liver tissues were obtained through biopsy before surgery, 30 minutes after the re-perfusion during the surgery, bi-weekly liver biopsy in the follow-up period via laparotomy under GA, and through autopsy. Tissues were harvested, fixed in formalin and embedded in paraffin blocks. Paraffin-embedded tissues were sectioned at a thickness of 3-5 □ m and stained with hematoxylin and eosin (Sigma- Aldrich, St. Louis, MO, USA). The histopathology was evaluated under a light microscope and tissue sections were captured with the 200x magnification in each sample.

Immu nostaining

[0059] The section tissue slides were treated with xylene for paraffin removal. The slides were rehydrated and then boiled for 10 minutes in antigen retrieval buffer (10 mM sodium citrate with 0.1 % tween 20, pH 6). Endogenous peroxidases and phosphatases were blocked by Dako Dual Endogenous Enzyme Block reagent (Dako, Santa Clara, California) for 10 minutes. The slides were double-stained with sheep antibody against porcine/human albumin (ab8900, Abeam, Cambridge, UK) and mouse antibody against human nucleus (MAB1281, EMD Millipore, Darmstadt, Germany) for overnight. Following staining by rabbit anti-sheep IgG H&L conjugated with alkaline phosphatase (ab97127, Abeam, Cambridge, UK) and goat anti-mouse IgG H&L conjugated with peroxidase (ab6789, Abeam, Cambridge, UK) were used for enzymatic detection. All samples were assessed under microscope (ECLIPSE Ti-U, Nikon, Tokyo, Japan). Images were acquired using NIS- Elements version 4.3 (Nikon, Japan).

[0060] Stemchymal® (ADMSC) at passage 8 and 12 were thawed, washed in PBS and centrifuged at 330 x g at room temperature for 5 minutes (Eppendorf 581 OR). Cell pellet (^ lxlOE6 cells) was lysed and preserved in 3ml of Trizol reagent for microarray analysis

(Agilent Human Gene Expression v3 8x60K Microarray). The entrusted party was Welgene Biotech. Co. Ltd. A total of over 58,000 gene probes was tested. The raw intensity values corrected by background intensity were normalized by quantile between samples. The normalized intensity with a Signal-to-Noise Ratio (SNR) > 1 was seen as detectable. Quantitative Polymerase Chain Reaction

[0061] RNA was extracted and quantified from the liver tissues. Then, the RNA was reverse-transcribed to cDNA by MMLV reverse transcriptase (EPICENTRE Biotechnologies, Madison, WI, USA). The primer sequences are described in Table 1. Using the mRNA expression of pig GAPDH for normalization, the mRNA expression of human HGF, EGF, VEGFs, IGFs, FGF-7, and TGFs were further analyzed by quantitative polymerase chain reaction (TaqMan Fast Advanced Master Mix, Thermo Fisher Scientific, Waltham, MA, USA).

Table. 1 Primer list

Statistics

[0062] All values were expressed as means ± standard deviation (SD). A P < 0.05 was considered statistically significant between the control and MSC group at the same time point (*), between each time point and before op in the control group (#) and between each time point and before op in the MSC group ($).

Example 1 Establishment of ALF Mini-Pig Model

[0063] The baseline data of body weight, blood gas, liver function and renal function of mini-pigs between the control and MSC groups were of no statistical significance. Animals in this study showed a normal CBC profiling before surgery (Table 2). Venous blood sampling was collected from inferior vena cava, and mini-pigs received surgically induced ALF as the description in the material and methods. A dark red appearance in a liver represented an ischemic change, while a bloody color in a liver indicated reperfusion injury. All animals were intensively monitored by blood gas tests in the first 24 hours after surgery, and survival was recorded during the study. We performed the regular blood examinations and liver biopsy during the follow-up period, and en bloc liver was removed when an animal passed away from ALF or completed a 28-day observation.

Table 2. Baseline data

Control Group (n=6) MSC group (n=10) p value

Body Weight (kg) 12.52±1.755 13.25±1.399 0.371

lilood gas

pH 7.3745±0.25647 7.4091±0.11806 0.5186 pC02 (mmHg) 64.25±62.953 45.41±16.107 0.375 p02 (mmHg) 63.25±8.501 60.59±11.695 0.637

Na+ (mmol/L) 131.03±3.736 132.78±2.728 0.297

K+ (mmol/L) 3.837±0.1700 3.966±0.7123 0.672

Ca++ (mmol/L) 1.075±0.1127 1.131±0.1075 0.3385

Glucose (mg/dL) 307.5±241.70 197.3±69.18 0.19 Hct (%) 27.8±3.87 29.5±2.17 0.28

HC03- (mmol/L) 27.57±3.680 27.48±3.146 0.961 TC02 (mmol/L) 29.53±5.385 28.89±3.573 0.777 BE(B) (mmol/L) 1.93±2.885 3.08±1.718 0.331 S02c (%) 88.70±7.663 88.91±7.145 0.959 I .IM function

ALB (g/dL) 2.68±0.271 2.87±0.116 0.074 AST (U/L) 51±35.1 34.5±5.76 0.16 ALT (U/L) 43.7± 11.40 48.4±13.35 0.48 Alk-P (U/L) 128.8±63.69 157.1±49.77 0.34 TB (mg/dL) 0.04±0.055 0.04±0.055 1 DB (mg/dL) 0.03±0.052 0.01±0.038 0.459 GGT(U/L) 29.0±3.35 34.5±7.70 0.12

Prothrombin time (sec.) 12.15±1.013 12.06±0.401 0.803 INR (ratio) 1.152±0.0968 1.144±0.0369 0.8226 APTT (sec) 18.18±1.601 18.30±2.712 0.926 Lactate (mmol/L) 3.58±1.459 3.86±0.810 0.63 Ammonia (ug/dL) 222.7±150.18 158.6±84.23 0.29

Control Group (n=6) MSC group (n=10) p value

WBC (10e3/uL) 17.02±4.104 14.46±5.305 RBC (10e6/uL) 5.468±0.3117 5.78±0.300 Hemoglobin (g/dL) 9.58±1.038 10.17±0.756 Hematocrit (%) 33.48±4.581 34.05±2.192 MCV (£L) 61.07±5.7833 59.00±4.045 MCH (pg) 17.50±1.213 17.60±1.083

MCHC (g/dL) 28.73±1.078 29.85±0.963

Platelet (10e3/uL) 345.2±92.53 336.8±79.797

Neutrophil band (%) 3.2±2.77 2.6±3.13

Neutrophil Seg. (%) 31.2±10.52 42.3±17.20

Lymphocyte (%) 56.2±1 1.84 43.2±15.12

Atypical Lymphocyte (%) 4.2±3.63 3.5±1.69

Monocyte (%) 4.7±0.82 6.1±1.29

Eosinophil (%) 1.5±0.55 0.9±0.88

Basophil (%) N.D. 0.2±0.42

Ucnnl funcl ion

BUN (mg/dL) 14.72±9.913 17.66±7.387

Creatinine (mg/dL) 0.78±0.126 0.77±0.155

Example 2 Survival Improvement by MSC treatment

[0081] Comparison on survival rate between the control and MSC group, MSCs potentially prolonged the mini-pig survival from ALF (Fig. 1A). In the control group, the survival rate was 33.3% at 2 days (2/6), 16.7% at 4 days (1/6), and 1 animal survived for 1 1 days. In the MSC group, the survival rate was 90% at 2 days (9/10), 60% at 4 days (6/10), and 4 of the ten animals survived for more than 14 days, and 1 animal was still alive at the end of observation (Fig. 1A). The mean survival time for untreated mini-pigs in the control group was 3.83 days, and the average survival time for MSC treated mini-pigs was > 12.5 days. The lethal ALF made a necrotic appearance on both the left and right lobe liver, and the dark black over the left lobe was a consequence of un-circulation (Fig. IB). In the living recipient, the left lobe of liver showed an atrophic change, and the right lobe liver appeared a pink color after MSC transplantation for 2 weeks (Fig. 1C).

Example 3 MSCs Differentiation into Hepatocyte in the Liver 2 Weeks after Transplantation

[0082] To evaluate liver condition after MSC transplantation, bi-weekly liver biopsy in the living recipients was performed. Normal liver section showed a typical liver lobule with sinusoid surrounding the hexagonal hepatocytes and forming a central vein, and portal triad could be found in the interlobular connective tissue (Fig. 2A). In a liver with ALF, extensive hepatocyte loss and necrosis were noted, and lobular components could not be identified during autopsy (Fig. 2B). MSCs were transplanted into a congested liver when the animal suffered from ALF (Fig. 2C). Two weeks after MSC treatment, no extensive hepatocyte necrosis appeared but relative enlarged sinusoid could be found in the liver tissue (Fig. 2D). Four weeks after transplantation, the recipient's liver tissue presented a normal histology (Fig. 2E), indicating ongoing hepatocyte regeneration in the damaged liver.

[0083] We further investigated the fate of transplanted cells by double staining of human/porcine albumin and human nucleus in the liver. Human/porcine albumin normally expressed in the healthy liver (Fig. 2F), and loss of albumin in the hepatocyte could be easily found from an animal dead from the ALF (Fig. 2G). When MSCs were initially transplanted into an animal with ALF, most cells clustered near the central vein area (Fig. 2H). Few human nuclear staining positive cells were observed in the liver parenchyma 2 weeks after MSC transplantation (Fig. 21), and albumin-expressing human cells in liver parenchyma could be found in liver sections from a survived animal from 2-4 weeks after MSC transplantation (Fig. 21 and J). Example 4 MSCs Rescue Thrombocytopenia 1 Week after Transplantation

[0084] In clinic, 60% of patients with liver failure accompany systemic inflammatory syndrome with or without infection, and thrombocytopenia is associated with multi-organ failure in a patient with liver disease. CBC data showed that neither ALF nor MSCs affected the white count level of mini-pigs in this study (Fig. 3A). Increased red cell count (Fig. 3B), hemoglobin (Fig. 3C) as well as decreased platelet count (Fig. 3D) were noted in control group before death. MSCs did not affect he levels of red cell count (Fig. 3B) and hemoglobin (Fig. 3C) but specifically replenished the platelet count 1 week after the onset of ALF (Fig. 3D).

Example 5 MSCs Prevention Extensive Hepatocyte Loss in Individuals Survived from the First 3 Days

[0085] Data from figure 3 demonstrated that MSCs possessed in vivo hepatocyte differentiation in porcine. However, it took 2 to 4 weeks for MSCs to mature the hepatocyte differentiation, and that was too late to save life from ALF in the first 3 days (Fig. 1 A). AST, ALT and ALP are cellular enzymes in the hepatocytes or cells in bile tract and levels of those intracellular enzymes in the serum represent the amount of hepatocyte loss. As shown in figure 5, surgery induced a fulminant hepatocyte loss evidenced by sharply elevating the serum AST, ALT and ALP on post operation day 1. MSCs did not alter the serum levels of AST (Fig. 4A), ALT (Fig. 4B) in the first 3 days, and slightly reduced serum ALP on Day 1 and 2 after surgery (Fig. 4C). However, transplanted cells prevented persistence hepatocyte loss, and gradually reduced serum AST, ALT and ALP levels after 3 days in those survived animals (Fig. 4). Example 6 Liver Function Restoration and Lethal Complication Prevention from Day 1 of MSC Transplantation

[0086] Change in PT and INR indicate acute, severe liver dysfunction resulting in insufficient coagulation. In this study, prolonged PT (Fig. 5A) and increased INR (Fig. 5B) were noted since the first day of ALF, and extensive bleeding tendency occurred on the third day. With MSC treatment, neither abnormal on PT nor INR were noted during the follow-up period (Fig. 5 A and B). Renal failure and hepatic encephalopathy are major sequelae of ALF leading to mortality. Serum creatinine (Fig. 5C) and ammonia (Fig. 5D) levels were quickly response to ALF since the first day onset. However, MSCs abrogated the ALF-induced renal dysfunction and elevation of ammonia from Day 1 after transplantation (Fig. 5C and D). Example 7 MSCs Maintenance Blood Gas Homeostasis from Day 1 of Transplantation

[0087] During liver injury, hemodynamic changes, electrolyte as well as metabolic imbalance are frequently presented. Data from time-dependent blood gas monitoring illustrated that MSCs kept a constant and stable electrolyte levels and prevented ALF induced a transient hypernatremia immediately after the surgery (Fig. 6A), hypokalemia 2-16 hours after the surgery (Fig. 6B) and hypocalcemia during the surgery (Fig. 6C). Surgery-related increase in blood sugar level was observed in both control and MSC groups, but ALF- induced hypoglycemia after the surgery did not occur in MSC group (Fig. 6D). Animals in both control and MSC groups suffered from hypoxemia starting from 8 hours after the onset of ALF, but MSCs kept a higher p0 ₂ in the venous circulation than the control (Fig. 6E). Oxygen saturation significantly dropped at 16 and 24 hours after the surgery, and MSCs maintained the oxygen saturation around 85-90% in the critical period (Fig. 6F). On the first day of surgery, pC02 (Fig. 6G), total C0 ₂ (Fig. 6H), HC0 ₃ ^" (Fig. 61) levels and pH value (Fig. 6J) in the venous circulation were relatively insensitive to ALF. In addition, the increased BE was only observed in the control group after surgery (Fig. 6K).

Example 8 Hepatocyte Growth Factor from MSCs Paracrine Support Liver Homeostasis from Day 1 of Transplantation

[0088] Hepatocyte growth factor (HGF), epithelial growth factor (EGF), vascular endothelial growth factors (VEGFs), insulin growth factors (IGFs), fibroblast growth factor (FGF) -7, and tissue growth factors (TGFs) have been reported to involve liver regeneration. Using microarray analysis with multiple primers to detect the expressions of the above cytokines, we demonstrated that MSCs possessed the constant ability from passage 8 to passage 12 to support liver regeneration by expressing HGF (Fig. 7 A), EGF (Fig. 7B), VEGF-A (Fig. 7C), VEGF-B (Fig. 7D), IGF-1 (Fig. 7E), IGF-2 (Fig. 7F), FGF-7 (Fig. 7G), TGF-a (Fig. 7H), TGF-βΙ (Fig. 71), TFG-p2 (Fig. 7 J), and TGF-p3 (Fig. 7K) in vitro. Moreover, q-PCR on specimens from liver in each time point revealed that only human HGF was detectable in a liver with acute liver failure during Day 1 to 2 weeks after MSC transplantation (Fig. 8A), and flare-up of human VEGF expression in a regenerative liver after 3 weeks of MSC transplantation could be observed (Fig. 8B).