Stem Cell Therapy of Liver Disease

Transcription

1 Stem Cell Therapy of Liver Disease Stephen Strom, Ph.D. Torsten och Ragnar Söderberg Professor Cell Transplantation and Regenerative Medicine Department of Laboratory Medicine, Division of Pathology Karolinska Institutet

2 Potential Conflict of Interest: Stockholder Stemnion, LLC Yecuris NO Stemnion or Yecuris cells or products were used in these studies 2

3 Hepatocyte Transplantation Bridge patients to whole organ transplant. Repopulation of liver in FHF without whole organ transplantation. Correction of metabolic defects without whole organ transplantation

4 Donor Liver for cell isolation

5 Catheter placed into umbilical vein Hepatocyte Transplantation through the Umbilical Vein Falciform Ligament Liver Inferior Vena Cava Ductus Venosus Umbilical Vein Catheter Umbilical Vein 5

6 Hepatocyte Transplant Procedure

7 Cell Infusion

8 Hepatocyte Transplantation Hepatocyte transplants are effective.. However: Extreme shortage of useful livers and hepatocytes for transplants. Searching for stem cell source for hepatocytes

9 Stem Cell Source Human Amnion Epithelial cells as a possible source of hepatocyte-like cells for transplantation. 9

10 Human Amnion Epithelial Cells Placental Tissue Amnion Not cord blood or Warton s Jelly Not MSC, mesenchymal stem cells Chorion Express pluripotency genes (Oct4, Nanog, Sox2) Express surface markers associated with ES,iPS cells (SSEA3, SSEA4, TRA1-60, TRA1-81) Differentiate to cells from all 3 germ layers in vitro Non-tumorigenic Low immunogenicity Decidua Miki et al., Stem Cells,

11 Human Amnion Epithelial Cells Remarkable stem cells 11 Anti-Inflammatory / Immunomodulatory properties, like MSC express and secrete IL-6, TGF beta 1, PEG2, FasL, IDO suppress mitogen or antigen stimulated splenocyte or PBMC proliferation, suppress T-cell proliferation (Parolini, Manuelpilla, Banas, more ) Very Low Immunogenicity; not recognized, not rejected Express low levels of HLA Class 1A NO expression of HLA class II and co-stimulatory molecules, CD40, CD80 haec express HLA-G HLA-G is the Key molecule that induces true immunological tolerance, maternal tolerance to the developing fetus. HLA-G is tolerogenic to dendritic cells, Induces immunmosuppressive regulatory T cells HLA-G up-regulates killing-inhibitory receptors ILT-2, ILT- 4 on NK and T cells High HLAG expression is correlated with reduced graft loss with skin, heart, liver, kidney, and combined liver kidney Tx Confirmed tolerant OLT recipients years off of immunosuppressive agents (>50 fold) Control and Acute Rejection (5-9 U/ml) Stable liver function (55U/ml) TOL (325 u/ml)

12 Immunostain, anti-hlag, human amnion hae express and secrete HLA-G HLA-G detected in purified exosomes

13 Placenta, maternal side down.

14 Peal amnion membrane

15 Histology of Placenta Tissue (H&E) and Before/After Trypsinization Before Amnion Amniotic Epithelial cell Amniotic Mesenchymal cell Chorion After x400 Decidua x100 x400

16 Anti-Human Albumin, SCID Mouse LIver HAS 11 1:500 O/N 4 C D@M -FITC 1:500 2hr RT X 400

17 hae Cells Differentiate to Hepatocyte-like Cells, In Vivo In SCID mice receiving AE cell transplant, a number of genes are expressed at or near normal adult levels CYPs: CYP3A4, 3A7, 1A2, 2B6, 2C8, 2C9, 2D6, 7A1 Transporters: PGP (MDR1), MRP2, BSEP Genes involved in metabolic liver disease: OTC, G6P, A1AT, UGT1A1

18 Previous HTx studies with the MSUD mouse model resulted in a partial correction Skvorak, et al., 2011.so? HYPOTHESIS: COULD HUMAN AMNION EPITHELIAL CELLS SUBSTITUTE FOR HEPATOCYTES IN TRANSPLANT PROCEDURES? INFUSION OF PROFICIENT CELLS INTO THE LIVER COULD INCREASE BCKDH ACTIVITY AND IMPROVE MSUD PHENOTYPE. 18

19 MSUD: A deficiency in the BCAA Pathway Val Leu Ile Disease Symptoms neurological dysfunction brain damage BCAA / BCKA toxicity seizures neuron failure energy deficiency ammonia accumulation coma and death maple syrup smell Figure made using Pathway Builder 2.0

20 1 million cells/mouse transplanted directly to liver (birth) hae Tx 2 million cells/mouse transplanted directly to liver (3+ weeks) 1 week 3 weeks (Homanics et. al., BMC Med. Gen. 2006, 7:13) imsud mice 16-fold increase BCAA/ala VS wildtype 5-6% of normal BCKDH activity Survive to ~3-4 weeks of age Fed a Normal Protein Diet Throughout Study 5 weeks (35d) Long term study 14 weeks (100d) Liver analysis Enzyme activity AA and Neurotransmitters (Blood and Brain) Survival and Growth KJ Skvorak, et al. 2012

21 hae Transplants for MSUD

22 hae Transplants for MSUD

23 hae Tx significantly improved serum and brain amino acid levels Leucine >60% Isoleucine..... >60% Valine >60% BCAA/ala..... >50% Alloisoleucine... >80% Phenylalanine..Normalized Tyrosine Normalized GABA Normalized Glutamate.....Normalized Ornithine Normalized These improvements were also seen at the 35 days Citrulline >70% timepoint in brain and at both time points in serum.

24 Human phenylketonuria (PKU) -OMIM # Incidence: 1:10,000 births Phe Tyrosine Dopamine DOPAC HVA Autosomal recessive inborn error of metabolism resulting from phenylalanine hydroxylase (PAH) deficiency. 3-MT PAH catalyzes hydroxylation of Phe to Tyrosine (rate limiting step), the precursor to Dopamine. Hyperphenylalaninemia is toxic to CNS, IQ and memory loss, concentration problems, mood disorders, mental retardation, Seizures, hypopigmentation of eyes and hair, developmental delays, Maternal PKU syndrome. Dietary intervention is the most common treatment, although noncompliance is a problem, and cognitive effects are possible even with attentive care. 24

25 haec transplants of Pah enu2 mice Infusion of human haec 1 million cells/mouse transplanted directly to liver (birth) hae Tx 5 million cells/mouse transplanted directly to liver first 3 weeks of life Pah enu2 1 week 3 weeks Fed a Normal Protein Diet Throughout Study 5 weeks (35d) Long term study 14 weeks (100d) Liver analysis Enzyme activity AA and Neurotransmitters (Blood and Brain) Survival and Growth

26 Phenylalanine (umol/l Serum) Phenylalanine (nmol/gr Brain) 3-MT (nmol/gr Brain) Serum and brain Phe and Brain 3MT levels in mice +/- hae transplants WT (13) hae Tx (13) HTx (10) PKU (14) 0 WT (18) hae Tx (30) HTx (27) PKU (8) 0.0 WT (8) hae (19) Hep (18) PKU (8) Transplanted Animals (n) 60 % reduction in serum Phe, >80% reduction of brain Phe 26

27 Complement Factor H (CFH) Main plasma regulator of alternative pathway for complement activation. Loss of function results in renal pathologies due to C3 accumulation in endothelium Atypical haemolytic uraemic syndrome (ahus) C3 glomerulopathy. CFH produced mainly in the liver Cfh -/- mouse model

28 Complement Factor H (CFH) Pilot Study, Giuseppe Remuzzi, Mario Negri Institute, Bergamo, Italy. Transplant hae into young cfh -/- mice Analyze serum C3 levels Inversely correlated with C3 activation Deposition on tissues results in low serum levels. Analyze deposition of C3 on glomerular endothelial cells

29 Complement Factor H (CFH) hae transplants: Provides CFH 3x increase in C3 levels Reduced C3 deposition on glomerular endothelium A Group 5 B Group 6 C Group 5 C3 : green Lectin: red DAPI: blue

30 50% reduction of orotic acid in the urine of OTC-deficient (spf-ash) mice, following 2 injections of 0.7 million haec on days 2 and 5 of life 30

31 A 2 S 2 R 3 Score Ammonia Challenge Time (min) WT OTC OTC-hAEC 31

32 Conclusions Stem cell surface markers and gene expression profile of hae are similar but not identical to pluripotent stem cells (hes or ips). When transplanted into the liver of mice, human - AE express mature liver genes at levels observed in normal human liver. hae Transplants correct the lethal effects, amino acid and neurochemical imbalances and systemic toxicity observed in a mouse models of four metabolic liver diseases (MSUD, PKU, OTC and CFH deficiency), and acute liver failure.

33 Conclusions hae, like MSC, have immunomodulatory properties. haec may escape immune recognition, post transplant. MAY be possible to perform haec transplants without immunosuppression of the patient. This will be tested. We are moving hae isolation and banking to GMP level and seeking full regulatory approval for transplants to treat liver disease

34 Acknowledgements University of Pittsburgh Kristen Skvorak, Kenneth Dorko Roberto Gramignoli, Veysel Tahan, Fabio Marongiu, Gregg Homanics, K. Michael Gibson, Jerry Vockley, Marc Hansel.G. Phil Karolinska Institutet Roberto Gramignoli, Ph.D Massoud Vosough Kristina Kannisto Christina Hammarstedt Raghuraman Shrinivasan Mihaela Zabulica Funding NIH-DK,57956, and DK086135, Pfizer, PKU Alliance,