Transplante de células tronco em Hepatologia

Transplante de células tronco
em Hepatologia
VI WORKSHOP INTE RNACIONAL DE ATUALIZAÇÃO E M
HE PATOLOGIA
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)



Mesenchymal stem cell (MSC)



main stem cell population within the BM
give rise to all mature blood lineages via erythroid,
myelomonocytic and lymphoid precursors
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
Improvement of liver
regeneration
process
Decrease in
hepatic fibrosis
What is the mechanism?
Which type of stem cell?
Improvement of
liver function and
survival rate
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
enriquecidas foram infundidas na artéria hepática
mononucleares
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)
Baseline
1 month
4 months
Min
Max
Mean
Median
Sta Dev
Relative mean
change from
baseline (%)
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
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
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
Albumin
(g/dl)
Baseline
1 month
4 months
INR
Baseline
1 month
4 months
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:



Laboratorial tests:


complete clinical and laboratorial evaluation
abdominal magnetic resonance imaging
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
Control
p value
N=15
Intervention
(BMC therapy)
N=15
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
Median
Age
Complications

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
Effect on liver function
1
2
3
4
5
Serum Albumin Levels
baseline 30 days 60 days 90 days 180 days 360 days
baseline 30 days 60 days 90 days 180 days 360 days
control
intervention
Albumin – Descriptive Analysis
Mean Relative change from baseline
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%
Time point
0
1
2
3
4
5
6
7
8
Serum Bilirubin Levels
baseline 30 days 60 days 90 days 180 days 360 days
baseline 30 days 60 days 90 days 180 days 360 days
control
intervention
6
8
CP
10
12
14
Child Pugh Score
baseline 30 days 60 days 90 days 180 days 360 days
baseline 30 days 60 days 90 days 180 days 360 days
control
intervention
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%
20
10
0
MELD
30
40
MELD
baseline 30 days 60 days 90 days 180 days 360 days
baseline 30 days 60 days 90 days 180 days 360 days
control
intervention
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
Days
0, 30, 60, 90
Time points
Days
0, 30, 60, 90, 180, 360
Slope
Slope
Control
BMC
pvalue
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
Parameter
Control
BMC
pvalue
Endpoints
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
Albumin analysis between g roups from baseline to 48 weeks
50.00
GROUP
P= 0.036
P= 0.045
P= 0.008
P= 0.018
Transplantation
Control
Albumin (g/ dL)
40.00
30.00
20.00
10.00
00.00
Baseline
1 week
2 weeks
3 weeks
4 weeks 12 weeks 24 weeks 36 weeks 48 weeks
TIME
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!