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Transplante de células tronco
em Hepatologia
VI WORKSHOP INTERNACIONAL DE ATUALIZAÇÃO EM
HEPATOLOGIA
Centro de Convenções David Carneiro - Pestana Hotel
3 e 4 de agosto de 2012 - Curitiba - PR – Brasil 15 min
Andre Castro Lyra
Prof. Adjunto e Livre Docente do Depto de Medicina da
UFBA / Serviço de Gastro-Hepatologia c-HUPES
Coordenador do Serviço de Gastro-Hepatologia do Hospital São Rafael
Stem Cells – What are they?
Cells that have the ability to self-replicate and
give rise to other cell types.
What are the types of stem cells?
Embryonic Non- Embryonic
Bone Barrow Stem Cells (BMSC)
Haematopoietic stem cells (HSCs)
main stem cell population within the BM
give rise to all mature blood lineages via erythroid,
myelomonocytic and lymphoid precursors
Mesenchymal stem cell (MSC)
forms stromal tissue
can give rise to cells of mesodermal origin
Endothelial precursors
Alternative methods are necessary to
increase survival of patients on the liver
transplant waiting list or even to treat
advanced liver disease without
transplantation
Studies in animal models – BMC
transplantation
Decrease in
hepatic fibrosis
Improvement of
liver function and
survival rate
What is the mechanism?
Which type of stem cell?
Improvement of liver
regeneration
process
Stem cells and liver diseases
Animal models
Studies in humans
Safety and feasibility
Feasibility and safety of Bone Marrow Cell therapy in patients with liver disease
Yannaki E et al. Lasting amelioration in the clinical course of decompensated alcoholic cirrhosis with boost infusions of mobilized peripheral blood stem cells. Experimental Hematology 2006, 34:1583-1587
Gaia S et al. Feasibility and safety of G-CSF administration to induce bone marrow-derived cells mobilization in patients with end stage liver disease. J. Hepatology 2006, 45(1):13-9
Gordon My et al. Characterisation and clinical application of human CD34+ stem/progenitor cell populations mobilised into the blood by G-CSF. Stem Cells, 2006, 24(7):1822-30
Terai S et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006, 24(10):2292-8
Lyra AC et al. Feasibility and safety of transplanted autologous bone marrow mononuclear cells in patients with advanced chronic liver disease. World J. Gastroenterology 2007, 21;13(7):1067-1073
Khan AA et al. Safety and efficacy of autologous bone marrow stem cell
transplantation through hepatic artery for the treatment of chronic liver failure:
a preliminary study. Transplant Proc. 2008, 40(4):1140-4.
Characterization and clinical application of human CD34+ stem/progenitor
cell populations mobilized into the blood by G-CSF Gordon MY et al, Stem Cells, 2006
Aims: safety and tolerability of injecting autologous CD34+ into
patients with chronic liver failure
Methods: 5 patients; age 20-65 years with life expectancy < 3
months; unsuitable for liver transplantation;
Treated with 520 μg G-CSF / sc / daily - 5 days. Leukapheresis on day
5 immunoselection of CD34+ and subfractionated into adherent
and non adherent cells
Reinfusion of CD34+ via hepatic artery (2) or portal vein (3)
Patients followed with liver function tests up to 60 days after infusion
Results - Gordon MY et al, 2006
Long-term clinical results of autologous infusion of mobilized
adult bone marrow derived CD34 + cells in patients with chronic
liver disease N. Levicar`et al. 2008; Cell Prolif. 2008, 41 (Suppl. 1), 115–125
Imperial College London
Four patients showed an initial improvement in serum
bilirubin level, which was maintained for up to 6
months.
There was marginal increase in serum bilirubin in three
of the patients at 12 months,
In the fourth patient’s serum bilirubin increased only at
18 months post-infusion.
. Levicar`et al. 2008; Cell Prolif. 2008, 41 (Suppl. 1), 115–125
Levicar`et al. 2008; Cell Prolif. 2008, 41 (Suppl. 1), 115–125
Levicar`et al. 2008; Cell Prolif. 2008, 41 (Suppl. 1), 115–125
Improved Liver Function in Patients with Liver Cirrhosis
After Autologous Bone Marrow Cell Infusion Therapy Terai S et al, Stem Cells, 2006
9 patients with cirrhosis (HBV 3; HCV 5)
Follow up: 6 months
400ml of bone marrow were aspirated from the iliac crest
General anesthesia
Reinfusion of BMC (5,20 0,63 x 109) peripheral vein
No serious adverse event
Ascites improvement in 5 patients
Results - Terai S et al Stem Cells, 2006
AFP/PCNA increased at 4w. on liver histology p<0.05
Long-Term Follow up For the Patient of Autologous
Bone Marrow cell Infusion (BMI) Therapy For Liver
Cirrhosis Terai S et al, Hepatology , 246A, 2007
Total of 16 patients
Follow up – 15 months - 9 patients
Albumin was improved in all patients
Feasibility and safety of transplanted autologous bone marrow
mononuclear cells in patients with advanced chronic liver disease World Journal of Gastroenterology, 2007
André C. Lyra1,2, Milena B. P. Soares1,3, Luiz F. Silva1, Marcos Fortes1,
André Goyanna1, Augusto C. A. Mota3, Sheilla A. Oliveira3, Eduardo
L. Braga1,2, Wilson Carvalho1, Bernd Genser4, Ricardo R. dos
Santos1,3, Luiz G. C. Lyra1,2
1 Hospital São Rafael, Salvador, Bahia, 3 Centro de Pesquisas Gonçalo Moniz, Brazil;
2 Gastro-Hepatology Unit, Federal University of Bahia, Salvador, Bahia, Brazil;
Fundação Oswaldo Cruz, Salvador, Bahia, Brasil;
4 BGStats Consulting
População de Estudo
10 pacientes com cirrose hepática na lista de transplante hepático
(8 homens, 2 mulheres)
Diferentes etiologias – Child-Pugh B ou C
50 ml da medula óssea foram aspirados da crista ilíaca
Fração mononuclear foi preparada por centrifugação utilizando um
gradiente de “ficoll-hypaque”
Um mínimo de 100 milhões de células mononucleares
enriquecidas foram infundidas na artéria hepática
Resultados
Não houve complicações ou efeitos colaterais
específicos relacionados á infusão celular
Table 2. Distribution of serum bilirubin, albumin and INR levels of 10 patients with chronic liver failure at baseline, 1 and 4 months after transplantation of autologous BMC.
Bilirubin
(mg/dl) Min Max Mean Median Sta Dev
Relative mean
change from
baseline (%)
Baseline
1 month
4 months
1.20
0.50
0.72
4.83
3.56
4.16
2.78
2.19
2.10
2.45
2.28
1.87
1.16
0.91
1.04
-21
-24
Albumin
(g/dl)
Baseline
1 month
4 months
2.50
2.90
3.10
4.40
4.50
4.80
3.47
3.44
3.73
3.50
3.25
3.60
0.51
0.52
0.51
-1
+7
INR
Baseline
1 month
4 months
1.08
1.10
1.16
1.89
1.94
1.75
1.46
1.44
1.42
1.48
1.43
1.43
0.23
0.23
0.18
-1
-3
Autologous Infusion of Expanded Mobilized Adult Bone
Marrow-Derived CD34+ Cells Into Patients With
Alcoholic Liver Cirrhosis Madhava Pai, et al .
Am J Gastroenterol 2008;103:1952–1958
Patient Selection - inclusion criteria
Patients with biopsy-proven ALC - abstained from alcohol for
at least 6 months (n=9)
chronic liver failure
unsuitable for liver transplantation
life expectancy of at least 3 months
Madhava Pai, et al . Am J Gastroenterol 2008;103:1952–1958
Methods
G-CSF, 520 μg per day for 5 days
Ultrasound → spleen size during G-CSF administration
On day 5 → leukapheresis.
CD34+ cells were immunoselected
CD34+ cells were placed in culture for amplification for 7 days
Hepatic artery infusion
Madhava Pai, et al . Am J Gastroenterol 2008;103:1952–1958
Results
Mean Serum Bilirubin
N=9
Madhava Pai, et al . Am J Gastroenterol 2008;103:1952–1958
Results
Mean Child-Pugh Score
N=9
Madhava Pai, et al . Am J Gastroenterol 2008;103:1952–1958
Results
Mean Serum Albumin
Madhava Pai, et al . Am J Gastroenterol 2008;103:1952–1958
European Journal of Gastroenterology and Hepatology
2009
Métodos
N= 8 pacientes
Cirrose hepática e insuficiência hepática crônica de qualquer
etiologia;
MELD >10
Aspiração da medula óssea – 20 ml
Separação e cultura das células mesenquimais
3 ou 4 passagens
Diferenciação em hepatócitos
Infusão na veia porta / periférica
Resultados
Summary of Results – Human Studies of Safety
and Feasibility
BMC therapy is safe and feasible
Increase of albumin levels
Decrease of bilirubin levels
Improvement of Child-Pugh score
Stem cells and liver diseases
Animal models
Studies in humans
Safety and feasibility
Controlled studies
European Journal of Gastroenterology and Hepatology
2009 ahead of print
Aims
To evaluate the efficacy of BMC transplantation on liver function of patients with advanced chronic liver disease
Primary Endpoints:
Liver function scores (Child-Pugh / MELD)
Secondary Endpoints:
Total bilirubin
Albumin
INR
Methods
Pilot sample of 30 patients
Treatment allocation by simple randomization
Intervention (BMC Therapy) group: 15 patients
Control group: 15 patients
Evaluation at 6 prospectively defined time points:
Time 0: Baseline (date of randomization)
Time 1: 30 days
Time 2: 60 days
Time 3: 90 days
Time 4: 180 days
Time 5: 360 days
Baseline assessment and patients follow up
Baseline assessment: complete clinical and laboratorial evaluation
abdominal magnetic resonance imaging
Laboratorial tests: complete blood count, liver profile tests, serum blood
glucose, urea, creatinine, alpha-fetoprotein.
Follow-up: clinical and laboratorial evaluation at day 1, 30, 60, 90, 180,
360 after BMC transplantation.
US 3, 6 and 12 months
Bone marrow cells therapy
50 ml of bone marrow were aspirated from the iliac crest.
Bone marrow mononuclear cells (BMC) were purified by centrifugation of total bone marrow in a ficoll-hypaque gradient
A minimum of 88 millions of BMC were infused into the hepatic artery
RESULTS
BASELINE ASSESSMENT
Comparison between groups:
Intervention (BMC) vs control
Baseline assessment
Median
Control
N=15
Intervention
(BMC therapy)
N=15
p value
Age 51 (32-68) 55 (41-74) 0.075
Bilirubin
mg/dL
2.0
(0.9-6.4)
1.9
(0.7-3.3)
0.312
Albumin
g/dL
3.2
(1.8-4.8)
2.9
(2.3-3.8)
0.114
Child-Pugh 8
(6.0-11.0)
9
(6.0-13.0)
0.684
Meld 13
(8-21)
13
(8-20)
0.447
INR 1.43
(1.03-1.95)
1.42
(1.12-2.19)
0.678
Mild complications – pain, echymosis
No major complications or specific side effects related
to the infusion procedure were reported
All patients were discharged 48h after BMC infusion
Complications
Effect on liver function
Serum Albumin Levels
12
34
5
Alb
um
in (
mg/d
l)
control intervention
baseline 30 days 60 days 90 days 180 days 360 days baseline 30 days 60 days 90 days 180 days 360 days
Albumin – Descriptive Analysis
Mean Relative change from baseline
Time point Control
Intervention
(BMC Therapy) Difference
30 days -2% 14% 16%
60 days -6% 14% 20%
90 days 2% 16% 14%
180 days -2% 11% 13%
360 days -2% 12% 14%
Serum Bilirubin Levels
01
23
45
67
8
Bili
rubin
(m
g/d
l)
control intervention
baseline 30 days 60 days 90 days 180 days 360 days baseline 30 days 60 days 90 days 180 days 360 days
Child Pugh Score
68
10
12
14
CP
control intervention
baseline 30 days 60 days 90 days 180 days 360 days baseline 30 days 60 days 90 days 180 days 360 days
Child Pugh – Descriptive Analysis
Mean Relative change from baseline
Time point Control Intervention Difference
30 days 2% -11% -13%
60 days 5% -7% -12%
90 days 5% -8% -13%
180 days 6% -6% -12%
360 days 5% -2% -7%
MELD
010
20
30
40
ME
LD
control intervention
baseline 30 days 60 days 90 days 180 days 360 days baseline 30 days 60 days 90 days 180 days 360 days
MELD – Descriptive Analysis
Mean Relative change from baseline
Time point Control Intervention Difference
30 days 7% 0% -7%
60 days 12% -2% -14%
90 days 13% 2% -11%
180 days 6% -3% -9%
360 days 18% 6% -12%
INR – Descriptive Analysis
Relative change from baseline
Time point Control Intervention Difference
30 days 1% -1% -2%
60 days 9% -1% -10%
90 days 7% 1% -6%
180 days 1% -4% -5%
360 days 9% 1% -8%
Summary of
Random effects model
Results
Random effects model
Time points
Days
0, 30, 60, 90
Days
0, 30, 60, 90, 180, 360
Parameter Control BMC
p-
value Control BMC
p-
value
Endpoints
Albumin 0,03 0,14 0,035 0,01 0,05 0,151
Bilirubin 1,45 -0,02 0,099 0,75 0,12 0,134
INR 0,04 0,01 0,215 0,02 0,01 0,265
Child-Pugh 0,12 -0,19 0,039 0,08 0,01 0,854
MELD 0,63 0,04 0,085 0,34 0,08 0,189
Slope Slope
Material and Methods
N = 140 subjects - randomized
Group 1: 90 patients
G-CSF (Neupogen, Roche) for 5 days
Bone marrow aspiration – day 6
Autologous CD34+ and CD133+ stem cell infusion in the portal vein
Group 2: 50 patients who served as a control
received regular liver treatment
daily SC injection of distilled water
Age 20 to 60 years
Chronic liver insufficiency
Hosny Salama et al 2010
Serum Albumin Levels
Hosny Salama et al 2010
Serum Bilirubin Levels
Hosny Salama et al 2010
Prothrombin concentration
Hosny Salama et al 2010
Material and Methods
N = 153 subjects – non-randomized
Group A: 53 patients
Bone marrow aspiration - 100-120 mL
Autologous MMSC Culture
Infusion through the hepatic artery
Group B: 105 matched controls patients who served as a control
matched for age, sex, and some biochemical indexes
Age 15 to 75 years
Chronic Hepatitis B
Most were cirrhotic
Peng et al 2011
Peng et al 2011
GROUP
Transplantation
Control
Alb
um
in (
g/dL
)
Baseline 1 week 2 weeks 3 weeks 4 weeks
TIME
40.00
30.00
20.00
10.00
00.00
12 weeks 24 weeks 36 weeks 48 weeks
50.00
P= 0.045 P= 0.036 P= 0.008 P= 0.018
Albumin analysis between groups from baseline to 48 weeks
Peng et al 2011
Peng et al 2011
Estudos clínicos com células troncos nas doenças hepáticas: Conclusões
O transplante autólogo de células mononucleares da medula
óssea e de células mesenquimais parece ser uma opção
terapêutica promissora para pacientes com doença
parenquimatosa crônica do fígado avançada.
Novos estudos são necessários em pacientes com doença
hepática para definir:
O real papel da terapia celular
Qual é o melhor tipo de célula a ser utilizada
Se a adição de G-CSF proporciona benefícios
Se infusões repetidas poderiam melhorar os efeitos obtidos
Obrigado!