Brief Title
Allogenic Bone Marrow Derived Mesenchymal Stem Cell Therapy in Cases of Hemophilia
Official Title
Allogenic Mesenchymal Stem Cells in Hemophilia: a Pilot Study
Brief Summary
Hemophilia is caused by a single-gene defect resulting in familial bleeding disorder. Small
increase in gene products could transform a severe form of hemophilia into a mild one. Stem
cells from extrahepatic sources are being considered for clinical applications in liver cell
therapy as they possess high in vitro culture potential and could be used in transplant
procedures. We studied the differentiation of bone marrow hematopoietic stem cells (BM-HSCs)
from hemophilia patients' relatives into factor 8 (FVIII)-producing hepatocyte-like cells
aiming to expand patients' donor options for partial replacement of mutant liver cells by
healthy cells in hemophilia A patients which could manage the severity of the bleeding
disorder.
BM-HSCs from hemophilic families will be cultured in short-liquid hepatic induction medium.
Appearance of hepatic phenotype will be evaluated by alpha-fetoprotein expression using
immunocytochemistry. Functional evaluation of transdifferentiation will be done through
detection of albumin synthesis using microalbumin assay kit, factor VIII activity by
one-stage clotting assay and expression of FVIII messenger RNA( mRNA) by reverse
transcription ( RT-PCR).
Inducing the differentiation of BM-HSCs by in-vitro manipulation may become a valuable tool
to provide a cell source for hepatocyte transplant procedures for treatment of hemophilia
patients.
Detailed Description
Hemophilia A is an X-linked bleeding disorder caused by a deficiency or abnormality of factor
VIII. It is the most common inherited coagulation protein deficiency with an incidence of
approximately 1 in 10,000 males. More than 75% of hemophilic patients suffer the severe type
of the disease.
Hemophilia treatments are readily available in developed countries; however, it is estimated
that 70% of people with this disease worldwide are undiagnosed or undertreated. Moreover,
about 20% of hemophilia A patients develop inhibitors to treatment and consequently are
difficult to treat.
Exogenous factor 8 replacement for hemophilia patients presents a great financial and medical
challenge. The optimum therapeutic option for these patients is to provide endogenous
secretion of the factor. This was proposed through liver transplantation. Liver
transplantation in human and canine hemophilia A results in an increase in factor VIII levels
to normal and thus cures the bleeding diathesis. Given the problems of donor availability,
major operative procedure and the need for lifelong immunosuppression, cell-based therapy
using isolated hepatocytes has been proposed as a promising option to treat clotting
disorders. The therapeutic effectiveness of human hepatocytes transplanted under the kidney
capsules of mice has been demonstrated. Transplantation of wild-type rats with deficient
bilirubin conjugation after ischemia/reperfusion damage resulted in 30% decrease in serum
bilirubin, the appearance of bilirubin conjugates in bile and the expression of normal
glucuronyltransferase enzyme denoting that transplantation of a small number of hepatocytes
can result in partial correction of functional defects.
Although cellular transplantation of hepatocytes solves the operative risk, it has the
disadvantage of difficult propagation of hepatocytes in vitro.
An alternative to hepatocyte transplantation is the use of in-vitro transdifferentiated bone
marrow derived stem cells.
In the past few years, a novel option to regenerate damaged liver from bone marrow-derived
cells has been proposed by many investigators. Studies showed that bone marrow cells not only
differentiated into hepatic and liver sinusoidal endothelial cells but they also expressed
the intact gene of the FVIII A3 domain.
Mesenchymal stem cells have many advantages as candidates for cellular therapy. They can be
propagated in-vitro, do not evoke immune reaction as they express only human leukocyte
antigen (HLA)-G, and have been proven to adopt hepatocyte phenotype in vitro.
In hemophilia A patients, the mother is a carrier, the father is completely normal, and the
female siblings have a 50% chance of being normal or carrier. Thus, the possibility of
finding an HLA-matched donor with normal FVIII activity in the family is present. The use of
mesenchymal stem cells (BM-MSCs) have the following advantaged over BM-HSCs:
1. MSCs can differentiate into both hepatic and endothelial phenotypes
2. MSCs do not express HLA antigens except HLA-G which caused immunosuppression, thus
matching for MSC transplantation can be easier.
The hypothesis of the present study is to use allogenic bone marrow derived mesenchymal stem
cells induced to adopt hepatocyte phenotype in vitro as a cellular therapy product in
hemophilia patients.
Donors will be subjected to:
1. Bone marrow aspiration under local or short general anaesthesia, 40-60ml BM will be
collected on heparinized syringes.
2. Mononuclear cell fraction will be separated using SEPAX machine (Biosafe)
3. MSCs will be isolated using plastic adherence and subjected to hepatic induction using
sequential fibroblast growth factor and hepatocyte growth factor addition under GMP
conditions.
4. Verification of hepatic induction will be done using morphological, molecular and
proteomic screening.
5. Cells will then be harvested using 0.25% trypsin, washed and suspended in sterile saline
in a dose of 2 million cells per kg body weight in a final volume of 5ml and injected
into the hepatic parenchyma under sonographic monitoring.
Study Phase
Phase 1
Study Type
Interventional
Primary Outcome
Assessment of Safety / Efficacy
Secondary Outcome
Assessment of Coagulation Profile
Condition
Hemophilia
Intervention
Cellular therapy
Study Arms / Comparison Groups
Cellular therapy
Description: Cellular therapy : injection of mesenchymal stem cells subjected to hepatogenic induction
Publications
* Includes publications given by the data provider as well as publications identified by National Clinical Trials Identifier (NCT ID) in Medline.
Recruitment Information
Recruitment Status
Biological
Estimated Enrollment
20
Start Date
August 2014
Completion Date
February 2016
Primary Completion Date
August 2015
Eligibility Criteria
Inclusion Criteria:
- Laboratory diagnosis of factor 8 deficiency
- Dependent on exogenous factor 8 therapy
Exclusion Criteria:
- Liver disease
- History of allergy to factor therapy
- Abnormal spleen by sonography
Gender
All
Ages
6 Years - 40 Years
Accepts Healthy Volunteers
No
Contacts
hala Gabr, M.D., +202-23644460, [email protected]
Location Countries
Egypt
Location Countries
Egypt
Administrative Informations
NCT ID
NCT02108132
Organization ID
SC-HEM
Responsible Party
Principal Investigator
Study Sponsor
Cairo University
Collaborators
Affiliated Hospital to Academy of Military Medical Sciences
Study Sponsor
hala Gabr, M.D., Principal Investigator, Cairo University
Verification Date
April 2014