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!