Stem sel pada hewan dan aplikasinya

Drh. Yuda Heru Fibrianto, MP., PhD.Bagian Fisiologi Fakultas Kedokteran Hewan

Universitas Gadjah Mada Yogyakarta 19012013

The Stem Cell ConceptA stem cell is an undifferentiated, dividing cell that gives rise to a daughter cell like itself and a daughter cell that becomes a specialized cell type.

Cell TherapyA treatment intended to regenerate or rejuvenate

the body by injecting it with healthy live or freeze-dried cells derived from organs or embryos. Sometimes called fresh or live cell therapy.

Performed to treat specific diseases and disorders: arthritis, lupus, cancer, HIV infection, cardiovascular and neurological disorders, and Parkinson's disease.

Also used to stimulate the immune system, revitalize bodily organs, and slow the effects of aging, including memory loss and sexual dysfunction.

Human and Animal Stem

Cell

Embry-onic

FetalIn-fant Adul

t

Blastocyst

(5-7 days)

Gonadal ridge

(6 weeks)

Embrionic stem cells

Embrionic germ cells

Fetal stem cells

Abortus

(Fetal tis-sues)

Umbilical cord blood

Wharton’s Jelly

Umbilical cord blood stem

cells

Umbilical cord matrix stem

cells

Spermatogo-nia

Oogonia

Germ line So-matic

HemopoieticGut Eye

MesenchymalLiver Epidermal

(skin, hair)Neu-ronal

Pancreas?

Bone mar-row

Peripheral blood Bone marrow

stroma

Therap sel dan produk sel pada hewanLebih berkembang Sebagai hewan cobaHewan model Etika dapat diterima

Effect of advanced kidney cell-derived protein extract (AKCPE) on treatment of cronic renal failure in dog and cat (fibrianto dkk., 2012)

Effect of advanced kidney cell-derived protein extract (AKCPE) on treatment of cronic renal failure in dog and cat (fibrianto dkk., 2012)

SDM(106/μL)

Hb (g/dL)

PCV (%)

MCH (pg)

MCV (fl)

MCHC (%)

BUN (mg/dL)

Creat (mg/dL)

Ke-0 5,19 9,74 31,46

18,92

63,59 29,21 102,32 3,87

Ke-11 5,09 10,75 36,26

22,07

77,93 29,33 57,85 2,59

Ke-18 4,09 6,1 27,5 14,66

66,13 22,4 34,5 2,5

Dog I Dog II Dog III Dog IV Dog V Cat

BUN 01118

6959.4-

4611.546

62.348.723

174--

160.3111.8-

504623

Creatinin

01118

22.07-

211

4.74.24

4.97--

5.73.1-

111

RBC 01118

2.139.11-

6.653.134.83

3.433.763.34

9.19--

4.534.36-

1.181.792.71

Hb 01118

3.517.6-

6.17.57.8

8.69.24.4

21.1 9.48.7-

1.72.83.5

Tissue engineering and therapeutic cloning in an effort to produce genetically identical renal tissue in an animal model (Bos taurus) Bovine skin fibroblasts from adult Holstein

steers were obtained by ear notch and single donor cells were isolated and microinjected into the perivitelline space of donor enucleated oocytes (nuclear transfer).

Blastocysts were transferred to the uterus of progestin-synchronized recipients permit further in vivo growth.

After 12 weeks cloned renal cells were harvested, expanded in vitro, then seeded onto biodegradable scaffolds.

The constructs (consisting of cells + scaffolds) were then implanted into the subcutaneous space of the same steer from which the cells were cloned to allow for tissue growth (Hipp and Atala 2004)

a. Combining therapeutic cloning and tissue engineering to produce kidney tissue, an illustration of the tissue-engineered renal unit

b. Renal unit seeded with cloned cells, three months after implantation, showing the accumulation of urinelike fluid

c. Clear unidirectional continuity between the mature glomeruli, their tubules, and the polycarbonate membrane.

d. Elispot analyses of the frequencies of T-cells that secrete IFN-gamma after primary and secondary stimulation with allogeneic renal cells, cloned renal cells, or nuclear donor fibroblasts. (Hipp and Atala 2004)

a

d

c

b

Succesful transplantation of bovine testicular cells to heterologous recipients

Histology of donor testes. In calves with SC between 18-20 cm, 45% of tubules contained a single layer of epithelium composed of Spermatogonia (arrows) and Sertoli cells (arrowhead) (A), while the remaining tubuleswerecomposedof2–4 layers with spermatocytes (arrows) (B). In calves with SC between 21 and 22 cm, nearly 53% of tubules contained 4–6 layers epithelium and spermatogenesis had progressed to Production of spermatids (arrow) (C). Bar Z 50 mm. Herrid et al. Reproduction 2006; 132:617-624

Stem cell based therapeutical approach of male infertility by

teratocarcinoma derived germ cells

Analysis of differentiation of SSC1 cells in vivo. (A) A fluorescent microscopic picture of a section of a 3 week transplanted testis showed proliferation of GFP positive cells (2.1% of tubuli). (B) Eight weeks after transplantation, GFP positive cells migrated to the basement membrane and colonized the tubules (3.8 colonies per 107 cells). These cells were able to initiate spermatogenesis and to differentiate into spermatids after 3 months (D) and into mature sperm after 7 months (F) of transplantation (3.0 colonies per 107 cells). A higher magnification of a spermatid and a sperm cell are shown at the right corner of the pictures. The other non-transplanted testis served as an internal control (C and E), no regeneration of spermatogenesis was observed. Hemalaun-eosin staining (G) and the corresponding fluorescence picture (H) showed the GFP positive cells in the periphery of seminiferous tubules (arrow) and their differentiation into sperm (arrow). A higher magnification of a sperm cell is shown at the right corner of the picture. (I) RT–PCR analysis of transplanted testis (TR) 3 months after transplantation, compared with a germ cell depleted but non-transplanted testis (NTR). Expression of genes specific for premeiotic (EGFP, Stra8), meiotic (SCP3, Pgk2) and postmeiotic (Tp2) stages shows that SSC1 cells were differentiated into postmeiotic cells. For control, RNA from SSC1 cells and testicular RNA (T) were used. GAPDH served as an internal standard. (J) DNA image cytometry analysis. The DNA contents were quantified by assigning an optical density to each pixel in the image and summing the optical density values for each nucleus. One hundred cells in luminal layer of the same sections in (G) and (H) were evaluated for DNA ploidy analysis. The presence of a haploid cell population (arrow) was confirmed. (K) As reference cells, mouse epidermis and lymphocytes were measured. (L and M) Characterization of differentiated cells. Testis sections were subjected to indirect immunofluorescence with antibodies to outer acrosomal membrane protein (L) and transition protein 2 (M) as primary antibodies and Cy3-conjugated (red) secondary IgG antibodies. Positive cells are shown (arrows).

Nayernia et al., 2004. Human Molecular Genetics 13, (14):1451–1460

Injeksi jantung tikus dengan sel jantung dari hESC + PSC teridentifikasi sel jantung manusia disokong oleh pembuluh darah tikus dan memperbaiki kemampuan untuk memompa darah (Murry. Heart Cells Derived from Human Embryonic Stem Cells Help Restore Rat Heart Function. Nature Biotechnology 2007; 25 (9):1015-1024. ).

Ability of hESDCMs to survive, function and integrate in the in vivo heart as “biological pacemaker”

Generation of a reproducible spontaneous cardiomyocyte differentiating system (Kehat et al., 2001) EBs (7-10 days in suspension) plated on top of gelatin-coated

culture dishes and observe microscopically for the appearance of spontaneous contraction

4-22days after plating, in 8.1% EBs rhythmically contracting appear

Cell transplated to the posterolateral region of the left ventricle in swine model of slow heart rate after grafting, a new ectopic ventricular rhythm was detected

in 11 of 13 animal studies, in 6 was characterized by sustained and long term activity

Electrophysiological mapping: ectopic ventricular rhythm originated from the area of cell transplantation and pathologic studies validated the presence and integration of the grafted (Caspi and Gepstein, 2006)

BM-derived cells used in various animal models

The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver. Stock et al. Nat Protoc. 2010 Apr;5(4):617-27

iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes (Wernig et al., 2008)

Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients Suneet agarwal et al. Nature 464, 292-296 (11 March 2010)

Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Lian et al. Circulation. 2010 Mar 9;121(9):1113-23

hES cell-derived oligodendrocytes and their ability to remyelinate and restore function of the spinal cord in mice after injury by Keirstead et al., 2005 approved by FDA for phase I clinical trialNeurons derived from reprogrammed fibroblast

functionally integrate into the fetal brain and improve sympton of rats with Parkinson’s disease.

iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic By Wernig et al., 2008. (PNAS )

PLoS One. 2011 Mar 4;6(3):e17560

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model. Tønnesen J, Parish CL, Sørensen AT, Andersson A, Lundberg C, Deisseroth K, Arenas E, Lindvall O, Kokaia M.

Neurosurgery. 2011 Jan;68(1):213-22; discussion 222.Predifferentiated brain-derived adult human progenitor cells migrate toward ischemia after transplantation to the adult rat brain. Olstorn H, Varghese M, Murrell W, Moe MC, Langmoen IA.

The adult human brain contains neural stem/progenitor cells (AHNPCs) that can survive transplantation into the adult rat brain, migrate toward a lesion, and display limited neuronal differentiation in vivo

Epilepsia. 2010 Jul;51 Suppl 3:71-5.Effect of neuronal precursor cells derived

from medial ganglionic eminence in an acute epileptic seizure model. Calcagnotto ME, Ruiz LP, Blanco MM, Santos-Junior JG, Valente MF, Patti C, Frussa-Filho R,

Santiago MF, Zipancic I, Alvarez-Dolado M, Mello LE, Longo BM.Stem Cells. 2010 Jul;28(7):1153-64.Medial ganglionic eminence-derived neural stem cell grafts ease spontaneous seizures and restore GDNF expression in a rat model of chronic temporal lobe epilepsy. Waldau B, Hattiangady B, Kuruba R, Shetty AK.

Neurol Med Chir (Tokyo). 2010;50(2):98-105Seizure suppression in amygdala-kindled

mice by transplantation of neural stem/progenitor cells derived from mouse embryonic stem cells.

Shindo A, Nakamura T, Matsumoto Y, Kawai N, Okano H, Nagao S, Itano T, Tamiya T.ScienceDaily (Aug. 30, 2008) — Oregon Health & Science

University scientists have successfully produced functional auditory hair cells in the cochlea of the mouse inner ear. The breakthrough suggests that a new therapy may be developed in the future to successfully treat hearing loss. The results of this research was recently published by the journal Nature.    

ScienceDaily (Aug. 3, 2009) — University of Florida researchers were able to program bone marrow stem cells to repair damaged retinas in mice, suggesting a potential treatment for one of the most common causes of vision loss in older people.

Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice

Human iPSCs were induced to MSC differentiation with a clinically compliant protocol. Three monoclonal, karyotypically stable, and functional MSC-like cultures were successfully isolated using a combination of CD24(-) and CD105(+) sorting. They did not express pluripotent-associated markers but displayed MSC surface antigens and differentiated into adipocytes, osteocytes, and chondrocytes.

Transplanting iPSC-MSCs into mice significantly attenuated severe hind-limb ischemia and promoted vascular and muscle regeneration. The benefits of iPSC-MSCs on limb ischemia were superior to those of adult bone marrow MSCs. The greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment after transplantation to induce vascular and muscle regeneration via direct de novo differentiation and paracrine mechanisms.

Functional MSCs can be clonally generated, beginning at a single-cell level, from human iPSCs. Patient-specific iPSC-MSCs can be prepared as an "off-the-shelf" format for the treatment of tissue ischemia.

By Lian Q, Zhang Y, Zhang J, Zhang HK, Wu X, Zhang Y, Lam FF, Kang S, Xia JC, Lai WH, Au KW, Chow YY, Siu CW, Lee CN, Tse HF. Circulation. 2010 Mar 9;121(9):1113-23

Umbilical Cord Blood-Derived Multipotent Stem Cells for Buerger's Disease and Ischemic Limb Disease Animal Model. Sung-Whan Kim1, Hoon Han2, Gue-Tae Chae1, Sung-Hoon Lee3, Sun Bo3, Jung-Hee Yoon4, Yong-Soon Lee3, Kwang-Soo Lee5, Hwon-Kyum Park M.D., Ph.D.5,*, Kyung-Sun Kang Ph.D.3,*STEM CELLS Volume 24, Issue 6, pages 1620–1626, June 2006

1. Necrotic lesion on right thumb of Patient 2. Patient showed necrotic lesion of right thumb before treatment (left). Umbilical cord blood-derived mesenchymal stem cell treatment can cure the necrotic lesion on day 120 after injecting (right).

Human umbilical cord blood-derived multipotent stem cells can salvage limbs in ischemic hind limb mouse model. Representative photographs of control medium (left) showed autoamputation within a week after ligation. Umbilical cord blood-derived mesenchymal stem cell treated ischemic limb showed limb necrosis (middle) and limb salvage (right) on day 28 after ligation.

Angiographic analysis of patient with Buerger's disease after transplantation with umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). Collateral branches and vascularities increased strikingly at the ankle and foot before (upper left), 30 days after (right), and 120 days after (lower left) UCB-MSC implantation

Angiography of hind limb mouse model on day 28 after femoral artery ligation. Umbilical cord blood-derived MSC-treated hind limb shows the artery (red arrow).

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