Stem Cell on animal & it's application

1. Drh. Yuda Heru Fibrianto, MP., PhD.
Bagian Fisiologi Fakultas Kedokteran Hewan Universitas Gadjah
Mada Yogyakarta 19012013

2. The Stem Cell
Concept
A 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.

3. Cell Therapy
 A 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.

4. Human and Animal
Stem Cell
Embryonic Infant
Fetal Adult
Umbilical Wharton’s
Blastocyst Gonadal ridge Abortus Jelly Germ line
cord blood Somatic
(5-7 days) (6 weeks) (Fetal tissues)
Spermatogonia Oogonia
Embrionic Embrionic Fetal stem Umbilical cord Umbilical cord
stem cells germ cells cells blood stem cells matrix stem cells
Hemopoietic Mesenchymal Pancreas?
Liver Epidermal Neuronal Eye Gut
Bone Peripheral (skin, hair)
marrow blood Bone marrow
stroma

6. Therap sel dan produk sel pada
hewan
 Lebih berkembang
 Sebagai hewan coba
 Hewan model
 Etika dapat diterima

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

8. Effect of advanced kidney cell-derived protein
extract (AKCPE) on treatment of cronic renal
failure in dog and cat (fibrianto dkk., 2012)
SDM Hb PCV MCH MCV MCHC BUN Creat
(106/μL) (g/dL) (%) (pg) (fl) (%) (mg/dL) (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

9. Dog I Dog II Dog III Dog IV Dog V Cat
BUN 0 69 46 62.3 174 160.3 50
11 59.4 11.5 48.7 - 111.8 46
18 - 46 23 - - 23
Creatinin 0 2 2 4.7 4.97 5.7 1
11 2.07 1 4.2 - 3.1 1
18 - 1 4 - - 1
RBC 0 2.13 6.65 3.43 9.19 4.53 1.18
11 9.11 3.13 3.76 - 4.36 1.79
18 - 4.83 3.34 - - 2.71
Hb 0 3.5 6.1 8.6 21.1 9.4 1.7
11 17.6 7.5 9.2 8.7 2.8
18 - 7.8 4.4 - 3.5

11. 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)

12. a
c
b d
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)

13. 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

14. Stem cell based therapeutical
approach of male infertility by
teratocarcinoma derived germ cells
Nayernia et al., 2004. Human Molecular
Genetics 13, (14):1451–1460
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).

15.  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. ).

16. 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)

17. BM-derived cells used in various animal models

19.  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

20. 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 trial
 Neurons 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.

21. 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.

22. Neurol Med Chir (Tokyo). 2010;50(2):98-105
 Seizure 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.

25. 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

27. Umbilical Cord Blood-Derived Multipotent Stem
Cells for Buerger's Disease and Ischemic Limb
Disease Animal Model. Sung-Whan Kim , Hoon Han , Gue-Tae Chae ,
1 2 1
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
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.
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).

28. 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).

30. Tekhnik kedokteran tinggi