APLIKASI TERAPI STEM CELL

AG. Soemantri
BAGIAN ILMU KESEHATAN ANAK FAKULTAS KEDOKTERAN UNDIP / RSUP DR KARIADI SEMARANG

PENDAHULUAN (1)
UU RI No. 36 tahun 2009 tentang kesehatan, yang menyangkut masalah transplantasi Pasal 64 ayat 1, ayat 2, ayat 3 Pasal 65 ayat 1, ayat 2, ayat 3 Pasal 66 Pasal 70 ayat 1, ayat 2, ayat 3

PENDAHULUAN (2)
Frischzellenthherapeutica (i.e. cell xenotransplants) are outside of regulatory controls of the State because they are neither drugs nor (therapeutica, (arzneimittel in German), but are another form of medication, individually prepared by a physician, for his specific patient, by agreement with his patient, confirmed by the signed informed consent prescribed by law (February, 16, 2000) 2. They are prepared for one time use only
1.

PENDAHULUAN (3)
3. They have no shelf-life 4. They are not distributed through the

usual channels, i.e. through pharmacies 5. They have to be implanted on a date set prior to the start of their preparation 6. Their implantation has to be carried out by the same physician who wrote a prescription for the stem cell xenotransplants for the named patient.

PENDAHULUAN (4)
BUNDESVERFASSUNGSGERICHT Der Bund ist nach Art. 74 Abs. 1 Nr. 19 GG nicht befugt, die Herstellung solcher Arzneimittel zu regeln, die der Arzt zur Anendung bei eigenen Patienten herstellt (February, 16, 2000) The International Xenotransplantation Association Consensus Statement on Conditions for Undertaking Clinical Trials of Porcine Islet Products in Type 1 Diabetes (July, 27, 2009)

DEFINISI STEM CELL (SEL PUNCA)

Stem Cell adalah sel yang mampu memperbanyak diri dalam waktu yang lama dan dapat berkembang menjadi berbagai sel khusus / spesifik yang membentuk jaringan. Potency kemampuan berdeferensiasi kearah sel tertentu Self-renewal kemampuan memperbanyak melalui siklus dari pembelahan sel sambil mempertahankan kondisi undifferentiated

KRITERIA STEM CELL ( SEL PUNCA )

Belum mempunyai fungsi 2. Kemampuan untuk berproliferasi menjadi sel yang identik dengan sel asalnya 3. Berubah menjadi sel tipe lain dengan fungsi yang spesifik
1.

1.

GENERAL ISSUES - Stem Cell Biology - Differentiated parental DNA Chain causes stem cell pattern of cell-type switching in Schizosaccharomyces pombe - On equivalence groups and the Notch/LIN-12 - Cell Cycle Control, Cheekpoints, and Stem Cell Biology - Senescence of Dividing Somatic Cells - Repopulating Patterns of Primitive Hemato poietic Stem Cell

2.

EARLY DEVELOPMENT - The Drosophila Ovary : An in vivo stem cell system - Male Germ-line stem cell - Primordial Germ cells as stem cell - Embryonic stem cells - Embryonal Carcinoma cells as embryonic stem cells - Trophoblast stem cells

3.

MESODERM - Hematopoietic stem cell : molecular diversification and developmental irrelationships - Hematopoietic stem cell : Lymphopoiesis and the problem of commitment versus plasticity - The hemangioblast - Mesenchymal stem cells of human adult bone marrow - Fate mapping of stem cell

4. ECTODERM - Stem cell and neurogenesis - Epidermal stem cell

5. ENDODERM - Liver stem cell - Pancreatic stem cell - Stem cells in the Epithelium of the small intestine and colon

BASIC CONCEPT OF CELLULAR AND MOLECULAR BIOLOGY

DOGMA CENTRAL

DNA

RNA

Protein

Mathematical Biosciences Institute (Ohio State Univ), 2 October 2003

3 MOLECULES OF LIFE
All life depends on three critical molecules: DNAs Hold information on how cell works RNAs Act to transfer short pieces of information to different parts of cell Provide templates to synthesize into protein Proteins Form enzymes that send signals to other cells and regulate gene activity Form bodys major components (e.g. hair, skin, etc.)

ANOTHER VIEW OF CENTRAL DOGMA

A gene is expressed in 3 steps: 1) Transcription: RNA synthesis 2) Splicing: removal of intron sequence from RNA

3) Translation: Protein synthesis

TRANSCRIPTION
Transcription is initiated by a complex of transcription factors binding to the promoter site located upstream of the gene An enzyme, RNA polymerase II, travels along the gradually unzipped DNA template and polymerizes nucleotides into an RNA. The sequence of nucleotides on DNA template determines the sequence on RNA by following the rule of base-pair complementarity, i.e., A U, T- A, C G, G C. Transcription continues until entire gene is copied to RNA.

Animation

Source of diagram: http://ghs.gresham.k12.or.us/science/ps/sci/ibbio/chem/nucleic

SPLICING

pre mRNA Exon 1 Intron 1 Exon 2 Intron 2 Exon 3

mature mRNA

TRANSLATION (1)
By translation, the nucleotide sequence on mRNA

determines the amino acid sequence by genetic code.

Genetic code: three base pairs of RNA (called a codon) determine one amino acid based on a fixed table. Translation always starts at AUG (start codon), and ends with any of UAA, UAG, or UGA (stop codon)

TRANSLATION (2)
Transfer RNAs (tRNAs): small RNA molecules. Most of the tRNAs function as carriers of amino acids and participate in protein synthesis. For example, the tRNA with the anticodon CGG corresponds with the codon GCC and attaches alanine amino acid onto the peptide chain. Ribosome: a complex of protein and rRNA
Animation
Source of diagram: http://www.wiley.com/legacy/college/boyer/0470003790/structure/tRNA/

MODERN TECHNOLOGY IN MEDICINE

Modern technology: Diagnostic Early Screening Prognostic Follow up Human genome: Gene ther. in 2010 Individualized Ther. Stem cell Antisense Biomedical Engineering New approaches: Genome Analysis: Expression gene analysis Micro Array Protein analysis New hopes: Cancer Vascular/Heart dis. Diabetes Alzheimers Parkinson

HOW MUCH DNA DO WE HAVE?

humans have 2 x 23 chromosomes EACH cells contains 6 billion bases DNA that is 1 meter of DNA a human being has >100.000.000.000.000 cells that is 100 billion km of DNA

CELL CELL COMMUNICATION

Receptors Signal transductions Second messengers molecules Nuclear transcription factors Gene activations Protein Respons

CELLS CAN WITHDRAW FROM THE CELL CYCLE AND DISMANTLE THE REGULATORY MACHINERY
G0 is a quiescent state Cdks and cyclins disappear Some cells enter G0 temporarily and divide infrequenty (I.e. hepatocytes) Other differentiated cell types (neurons) spend their life in G0

Cell division increases the number of somatic (body) cells, and consists of:
Mitosis (division of nucleus) Cytokinesis (division of cytoplasm)

Cells

Apoptosis (cell death) decreases the number of cells. Both cell division and apoptosis occur during normal development and growth.

Start checkpoint Controls G1/S transition Commits cell to another round of cell division G2/M checkpoint Checks that DNA is fully replicated M checkpoint Is DNA division (chromosome segregation) progressing correctly?

CHROMOSOMES
genomic DNA is very long humans, 6 x 109 bp ~1.8 meters total length total length reduced via associates with proteins individual DNA molecules condensed into chromosomes humans have 22 pairs + X/X or X/Y (ie, diploid)

upstream

exon intron exon

downstream

Initiation codon

termination codon

3 5

CENTRAL DOGMA OF MOLECULAR BIOLOGY

DNA Sequence (splited by genes)

RNA

Amino Acid sequence

protein

phenotype

Adapted from http://www.bioinfbook.org/

CENTRAL DOGMA
Gene
Transcription

Genome

RNA

Transcriptome

Translation

Protein

Proteome

REGULATORS : PROLIFERATION, DIFFERENTIATION, APOPTOSIS, REPAIR

Genome : Genes :
Cell cycle Differentiati o Apoptosis Repair Metabolism etc Transcriptome

Proteome Protein:
cyclin, CDK,CDKI GF : GM-CSF, FGF Bcl-2, p53, caspase Gadd, enzym repair dll

RNA RNA RNA RNA

RNA
RNA

HOW TO UNDERSTOOD ??
GENE: Cytogenetics FISH DNA analysis Southern blot +/PCR specific MLPA Sequencing : SSCP

Gene activ: RNA Northern blot RT- PCR RNA-se protection Assay Micro array R-ISH Gene active : Protein

Western Blot
SDS-PAGE ELISA FISH

APLIKASI STEM CELL (SEL PUNCA)

Tidak semua dibicarakan satu persatu tentang aplikasi Yang disinggung yang pernah dijalankan oleh RSUP Dr Kariadi / FK UNDIP

BERDASAR ASAL SEL YANG DIPAKAI

Autotransplantasi Dari satu bagian tubuh ke bagian tubuh yang lain pada individu yang sama Allotransplantasi Antar individu pada spesies yang sama Xenotransplantasi Antar spesies yang berbeda

MESODERM
Sel yang berasal dari Mesoderm ini yaitu Mesenchymal Stem Cells of Human Adult Bone Marrow, yang digunakan untuk cangkok sumsum tulang - Metoda Allolog - Metoda Autolog

Cangkok Sumsum Tulang telah dilaksanakan di RSUP Dr. Kariadi tahun 1987, 2 kasus dengan autolog untuk penderita dengan diagnosis AML M(2) Salah satu sudah lulus Sarjana Kedokteran dan Ekonomi Satu kasus dengan metoda allolo untuk penderita thallasemia homo zygote

ENDODERM
Cangkok hati dilaksanakan di RSUP Dr Kariadi pada tanggal 1 Oktober 2006 dengan atresia billier, anak U laki-laki umur 15 bulan dengan donor dari ibu kandungnya. Cangkok hati II dilaksanakan di RSUP Dr Kariadi pada Juni 2009 dengan Allagile Syndrome, anak A laki-laki umur 7 tahun dengan donor sukarela.

XENOTRANSPLANTASI AMANKAH ?? Legalitas (+)

Keputusan MA German No 1 BvR420/97, European Community Council directive 2001/83/EC, UU seluruh negara Uni eropa

Jaminan keamanan (+)

Hewan yang dipakai (kelinci) memenuhi persyaratan yang dikeluarkan oleh : Guidelines on Infectious Disease Issues in Xenotransplantasi Merupakan hewan dari galur tertutup yang diisolasi dan diawasi dari kemungkinan tertular penyakit (diawasi 7 turunan )

EVIDENBASED HASIL PENELITIAN TERAPI CANGKOK STEM CELL

3000 penderita SD yang diterapi 25% bisa sekolah secara normal Outcome terapi : terjadi peningkatan TB, LK, IQ, indeks volume otak, perkembangan mental, perkembangan motorik

Evaluasi mental dan psikologis 83 penderita SD yang diterapi : terjadi penurunan hiperaktivitas, peningkatan konsentrasi dan peningkatan kemampuan bahasa ekspresif
Evaluasi imunologis : terjadi peningkatan IgA dan penurunan IgG Menurunnya frekuensi dan beratnya infeksi bakteri

EVIDENCE BASED
Pada Cangkok Stem Cell (CST) usia 3 tahun
kenaikan MDI > 50 point terjadi pada : 17 % bila tidak di CST 58 % pada CST I 71 % pada CST II

Pada usia 4-9 tahun

kenaikan 25-49 point

Figure 2. Phosphorylation targets of DYRK1A. The Hsa21-encoded kinase DYRK1A has been shown to phosphorylate a multitude of targets, which have been implicated in a number of biological processes and DSassociated phenotypes, including endocytosis and AD.

RINGKASAN
Telah diutarakan konsep dasar biomolekuler, siklus sel Definisi dan jenis-jenis, kriteria stem cell (sel punca) Dasar hukum Dasar keberhasilan asosiasi stem cell Aplikasi yang pernah dilakukan di RSUP Dr Kariadi/FK UNDIP : cangkok sumsum tulang, cangkok hati, cangkok stem cell