Clinical-gradehumanlivermesenchymalstemcells
forthetreatmentofNASH-Fibrosisthroughimmunomodulation
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KrisGellynck ,GuillaumeRommelaere ,MustaphaNajimi ,JohnTchelingerian ,CatherineLombard ,JoelleThonnard ,GiuseppeMazza ,EtienneSokal
1PrometheraBiosciences,rueGranbonpré11,1435Mont-Saint-Guibert,Belgium;2InstitutdeRechercheExpérimentaleetClinique,UniversitéCatholiquedeLouvain,Belgium
Results
Introduction
NASH/Fibrosis
Nonalcoholicsteatohepatitis(NASH),asevereformofnonalcoholicliverdiseases
(NAFLD),isoneoftheprominentliverdiseasesworldwide. Itisestimatedthat80%
ofobesepeopleandahighproportionofdiabeticpeople(40-70%)haveNAFLD5.
ResearchhasshownthattheprevalenceofNAFLDvariesbetween20-44%inthe
generalpopulation.Ahighprevalence(36–44%)ofNAFLDwasfoundinobese
childrenjustifyingthetermof“Thenextbigglobalepidemic”.
HepaStem is a cell suspension constituted of Heterologous Human Adult Liver-derived
Progenitor Cells isolated from normal adult human liver tissue. The cells are isolated,
expanded, cryopreserved and reconstituted according to a proprietary process. A phase
I/II clinical trial already showed the cell therapy was safe and well tolerated in 20
paediatric patients. It is intended to use HepaStem’s metabolic and mesenchymal
properties to treat genetic and chronic liver diseases.
CellinvivoeffectonfibroticcollagendepositioninaNASHmicemodel
A.
CellIdentity&Characterization
C.
B.
D.
Fig 1: A.: Volcanic plot comparing gene expression by Liver cell suspension vs HepaStem based on
micro-array analysis, showing the 12926 significantly different array features at adjusted p-value <
0.0001 and fold change >= 2. B., C. and D.: Quality control of MSC identity markers (CD90, CD73
(B.)) and impurity markers (CD45, CD31 (C.), CD133 and CK19 (D.)).
Cellphenotypeandsecretionresponsetoinflammation
Fig 2:
A.: Flow Cytometry Quantibrite analysis of the
immuno-phenotype shows a low immunogenic
phenotype. It showed a medium expression of
HLA Class I and HLA-G, and low expression of
HLA-Class II, even in Inflammatory conditions,
which increased the expressions of these
markers. (MFI standard: low: 15,4; medium low:
162; medium high: 700 and high 1902)
Material&Methods
Fig 5: Left: the liver-sections of the
NASH mice inj ected with Vehicle or a
single or triple inj ection of HepaStem
with and without immunosuppression,
stained with Sirius Red and quantitated
calculate the collagen deposition.
Immunosuppression on its own did not
alter the collagen deposition in the
animals, but a single and a triple
injection of 12,5x106 cells were both
significantly efficient to reduce the
collagen compared to vehicle only;
When
immunosuppression
was
administered, the triple injections lead
to a sufficient reduction with a
significant effect. As cells were injected
during the steato-hepatitis phase, the
reduction shows a inhibition of fibrosis
formation in the pericentral regions.
A.
B.
The proposed mechanism of action of HepaStem in NASH is a systemic inhibition of inflammation and
inhibition of stellate cell activation. The effect works through the secretion of several cytokines (HGF, IDO,
PGE2) in response to the liver inflammation. HGF has previously been shown to be involved in the inhibition of
hepatic stellate cell proliferation and collagen deposition by adult liver progenitor cells.
B.: HGF is secreted by HepaStem in vitro culture
under normal conditions. The addition of
inflammatory
cytokines
increases
these
secretions.
CellIdentity&Characterization
CellinvitroImmunomodulatoryFunctionalityonT-LymphocytesinanMLRandonDendriticcellgeneration
A full HepaStem gene expression profile analysis was done to evaluate the cell’s identity
and compared to Hepatocytes. In production, each Batch is quality controlled on it’s mesenchymal
identity (flow cytometric analysis of CD73 and CD90) and its impurity (CD45/CD31/CD133/CK19). The
immunogenic profile, +/- inflammatory conditions was analysed using flow cytometry (HLA-ABC, HLA-DR
and HLA-G). The HGF secretion profile of immunomodulatory cytokines by HepaStem was also evaluated
with and without the addition of inflammatory cytokines (IL1β, TNFα, IFNɣ and IFNα).
Fig 3: A.: HepaStem inhibitory effect on T-lymphocyte proliferation in an MLR reaction by 2 HepaStem batches. B.: Flow cytometric data of CD1a,
CD14 and CD83 (middle) and TNFα secretion (C.) after dendritic cell generation and maturation through LPS/INFγ response of monocytes with and
without co-culture with HepaStem clinical batches. Showing the inhibition of Dendritic cell generation and their functionality.
A.
B.
Discussion
C.
Fig 6: The multiple mode of action proposed to
treat chronic liver diseases with HepaStem
cells, to modulate inflammation through
migration to the liver and systemically by the
secretion of anti-inflammatory cytokines. GMP
cell expansion and manufacturing, before the
cryopreservation of a clinical grade easy-to_ ___reconstitute at the bed-side cell product.
CellinvitroImmunomodulatoryFunctionality
The anti-inflammatory effect of HepaStem was investigated towards T-lymphocytes and
Dendritic cells. In co-culture systems with CellTraceTM dye (CFSE) labeled T-lymphocytes were stimulated
by iDCs in a mixed leukocyte reaction using different HepaStem/T-cell ratios. The percentage of
proliferation of the responding T cells was assessed by flow cytometry by the analysis of the CSFE profile.
Similarly, monocytes were stimulated for dendritic cell (DC) generation and then matured through
LPS/INFγ stimulation, with and without HepaStem co-culture. The dendritic markers (CD14 (up for
monocytes, down for DCs) and CD1a and CD83 (down for monocytes, up for DCs) were analyzed using
flow cytometry together with the dendritic TNFα secretion measured using ELISA. Tests were done using
2 different clinical HepaStem batches.
NASHpreclinicalanimalmodels
In a preclinical high-fat STAM NASH mice model, NASH was induced in 28 male mice by a single
subcutaneous injection of 200 μg streptozotocin solution 2 days after birth and feeding with high fat diet
after 4 weeks of age. The treatment (1 injection at week 6, or a repeated injection at week 6, 7 and 8,
before necropsy at week 9) falls in the steatohepatitis phase of the NASH progression of this NASH mice
model. This means steatosis is already well established and fibrosis starts forming. Due to the xenotransplant situation in the preclinical study, immunosuppression might be required; but as the
immunosuppression could alter the NASH/fibrosis formation, a study arm with and another without the
use of immunosuppression (cyclosporine) were used. Safety evaluation was based on clinical
observation. Efficacy evaluation was based on histology , pathological examination and automated
quantification.
CopyrightPrometheraBiosciencesSA
16470.Promethera-Impression-poster-AASLD-01.indd 1
CellinvivoeffectoninflammationandNASscoreinaNASHmicemodel
Fig 4: The H&E staining of liversections of the NASH mice injected
with Vehicle or a single or triple
injection
of HepaStem
with
and
without immunosuppression. A
pathological NAS score was given by
analyzing the steatosis, inflammation and
ballooning in H&E, Oil Red O and
PAS
stained
sections.
Immunosuppression on its own did not
alter the steatosis, inflammation nor
ballooning, but a single injection of
HepaStem
combined
with
an
immunosuppression (cyclosporine) was
sufficient to show a significant reduction
in the inflammation score, which led to a
significant reduction (4,0 to 2,2)
compared
to
immunosuppression
without HepaStem injection. A triple
injection leads to a similar decrease in
several animals.
Contact:[email protected]
As a living medicinal product, HepaStem is proposed to treat NASH by decreasing the exaggerated systemic
and liver inflammation through the restoration of an immune balance, in a responsive way using multiple
mode of actions. A single HepaStem injection significantly decreased the NAS score, which was mainly
attributed to reduction in inflammation and the fibrosis will be reduced as a consequence, furthermore
HepaStem have been shown to secrete HGF, which inhibits the proliferation and collagen deposition of stellate
cells. In addition to the systemic action, MSCs could have a major tropism for the liver where it could have
local immunomodulatory effects and to a second extent an ability to secrete growth factors aiding to restore
liver functionality. Through liver tissue regeneration, fibrosis can be degraded and restructured which will lead
to a diminishing of fibrosis in case there is no new fibrosis formation as the inflammation and fibrosis
formation are inhibited by HepaStem.
Conclusion
The preclinical results suggest that clinical grade liver progenitor cells have anti-inflammatory anti-fibrosis and
anti-NASH effects, both in vitro and in an in-vivo NASH model. As these cells are GMP manufactured, and
cryopreserved as easy to use, off-the-shelf, easy to bring to the bed-side product, which have shown
safety/tolerability in a Phase I/II study treating metabolic disorders; this cell therapy is now ready for a first
clinical trial in NASH/fibrosis patients.
www.promethera.com
7/11/16 11:19