This document summarizes stem cell research and development in Indonesia. It discusses the three main types of stem cells - embryonic, adult, and induced pluripotent stem cells. It also describes several studies that have been conducted in Indonesia using mesenchymal stem cells isolated from different tissues and human induced pluripotent stem cells generated from patients with diseases like alpha-thalassemia. The document emphasizes the importance of research collaborations and grants to advance stem cell research in Indonesia.

1. Stem Cell Research
and its Development in Indonesia
Ita M. Nainggolan
Webinar Kolaborasi BRIN dan ASPI, 16-02-2022

2. STEM CELL TYPES
1.Embrionic Stem Cells
2.Adult Stem Cells
3.Induced Pluripotent Stem Cells
CELL SOURCES
1.Human
2.Animal

3. ▪ Morphology of GDMSC at the fourth passage show a spindle-
like formation and fibroblast-like cells attached to the base of
the plate culture
▪ MSC markers: CD44, CD73 CD90 CD105 CD200 (positive)
Gingival-Derived Mesenchymal Stem Cell from Rabbit
(Oryctolagus cuniculus): Isolation, Culture, and
Characterization Eur J Dent 2021;15:332–339

4. • Gingival-Derived Mesenchymal Stem Cell from Rabbit
(Oryctolagus cuniculus): Isolation, Culture, and
Characterization Eur J Dent 2021;15:332–339
▪ HSC markers: CD34, CD45 (negative)
▪ Alizarin red examination confirmed osteogenic differentiation
(14 day culture, red-violet and brown deposit)

5. Glucosamine decreases the stemness of human ALDH+
breast cancer stem cells by inactivating STAT3
ONCOLOGY LETTERS 16: 4737-4744, 2018
• Human Breast CSCs (aldehyde dehydrogenase-positive/ALDH+)
• MCF7 (Human adherent epithelial adenocarcinoma cell line)

6. Combined Hydroxyapatite Scaffold and SHED Modulating Alveolar
Bone Regeneration via Regulating Receptor Activator of Nuclear
Factor-Κb and Osteoprotegerin System
Critical Reviews in Biomedical Engineering 40(5):363-408
• Analyze the Osteoprotegerin (OPG) and Receptor Activator of NF-Κb ligand
(RANKL) expression after the application of Hydroxyapatite scaffold and SHED.
• Hydroxyapatite scaffold and Stem cells from Human Exfoliated Deciduous Teeth
increase OPG and decrease RANKL expression that has a high potential to be
used as an effective alternative tissue engineering biomaterial for alveolar bone
defect regeneration.
Iran J Med Sci September 2019; Vol 44 No 5

7. The Influence of Wharton Jelly Mesenchymal Stem Cell toward
Matrix Metalloproteinase-13 and RELA Synoviocyte Gene
Expression on Osteoarthritis
• To identify the influence of Wharton Jelly Mesenchymal Stem Cell
(MSC-WJ) on MMP-13 and RELA expression gene in synoviocyte by
in vitro.
• RELA and MMP-13 involved in cartilage degradation
• The sample used derived from synovial tissue of OA patients who
underwent Total Knee Replacement (TKR) surgery.
• MSC-WJ in OA synoviocyte significantly reduced the expression
of MMP-13 and RELA gene.

8. Proteomic analysis of hypoxia and non-hypoxia
secretome mesenchymal stem-like cells
from human breastmilk
Saudi Journal of Biological Sciences 28 (2021) 4399–4407
• To identify the proteomic analysis of secretome mesenchymal
stem-like cells under hypoxia compared to non-hypoxia from
human breastmilk stem cells
• secretomes under hypoxia (A) and non-hypoxia (B) and
analyzed for LC-MS to identify the peptide structure
• The human breastmilk cells contain mesenchymal stem-like
cells and a high concentration of CD44, CD73, CD90, and
CD105 as surface markers at third passage culture.
• The hypoxic hBSC secretome produces a higher protein level
compare to non-hypoxia. The transforming growth factor -b
was found in the hypoxic hBSC secretome as a modulator of
VEGF-mediated angiogenesis.

9. Proteomic analysis of hypoxia and non-hypoxia
secretome mesenchymal stem-like cells
from human breastmilk
Saudi Journal of Biological Sciences 28 (2021) 4399–4407
• To identify the proteomic
analysis of secretome
mesenchymal stem-like cells
under hypoxia compared to
non-hypoxia from human
breastmilk stem cells
• secretomes under hypoxia (A)
and non-hypoxia (B) and
analyzed for LC-MS to identify
the peptide structure
H NH

10. A New Era of Medicine

12. • human embryonic stem cells (hESCs) and human induced
pluripotent stem cells (hiPSCs) hold great promise for clinical
applications.
✓self-renewal
✓differentiation capacities
• potential sources of large quantities of differentiated cells for
drug screening, toxicology studies, biomolecule production,
and cell therapy.
• hiPSCs usually made from patients with known mutated
diseases
Human pluripotent stem cells (hPSCs)

13. Comparison of the lifecycles of non-integrating SeV
vectors and integrating vectors

14. Non-viral delivery methods

16. Generation of human induced pluripotent stem cell line from
Alzheimer’s disease patient with PSEN2 N141I mutation using
integration-free non-viral method
Stem Cell Research 47 (2020) 101892
• Skin fibroblasts from an 81-year-old female Alzheimer’s
disease (German family) were obtained from the Coriell
Institute
• Familial Alzheimer’s Disease-linked Presenilin-2 (PSEN2) single
point mutation (N141I)
• Self-replicating RNA reprogramming vector, ReproRNA™-
OKSGM Kit (STEMCELL Technologies)

17. Characterization and validation
A. Mutation analysis; B. Genotype identity
C. Morphology and phenotype (Expression of pluripotency markers
OCT4, LIN28, NANOG, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, SOX2

18. Characterization and validation
D. RT-qPCR: Cells expressed POU5F1 (OCT4), NANOG, SOX2,
LIN28A, ZFP42 (REX1), CDH1 (E-CAD)

19. -Thalassemia as a model disease
ALPHA THALASSEMIA
inherited disease characterized by a reduction or an absence of
alpha globin chain due to alpha globin gene mutations.
b.
- O2 transport throughout the
body
- 2 -globin chains
- 2 -globin chains
- 4 heme molecules

20. -Thalassemia mutation
Cd 59 α2-globin gene (GGCgly →GACasp)
Non-deletional mutation commonly at termination codon and
missense mutation that resulted in unstable Hb dan unstable α
globin chain
20
➢ Change the amino acid Gly (neutral) →
Aspartate acid (negative charge)
➢ Hb Adana is one of common non-deletional
mutations found in Indonesia.
➢ Homozygote state of this mutation can
cause Hydrops Fetalis Syndrome.
(Nainggolan et al, 2010)

21. CytoTuneTM iPS 2.0 Sendai Reprogramming
❑ Sendai virus (SeV) is a respiratory virus of mouse and rat,
classified as mouse parainfluenza virus type I belonging to the
Paramyxoviridae family; Enveloped virus of 150–250 nm in
diameter; Single chain RNA (15,384 bases) in the minus sense.
❑ The reprogramming vectors include the four Yamanaka
factors, Oct, Sox2, Klf4, and c-Myc
❑ Multiple methods to generate iPSCs:
➢ Retroviral vectors (require integration into host
chromosome to express reprogramming genes)
➢ DNA based vectors (exist episomally, do not require
integration) host chromosomes at certain frequencies)
➢ CytoTune™ 2.0 reprogramming vectors do not integrate
into the host genome or alter the genetic information of
the host cell.

22. Advantages of CytoTune™-iPS 2.0 Sendai
Reprogramming Kit
• No genotoxicity
• Wide range of targets
• High transduction efficiency
• Short contact time
• High level of expression of the transgenes.
• Fast expression of the transgenes (6–10 hours after
transduction)
• Zero footprint
• No production of infectious particles by the transduced cells.
• Derived from a virus that is non-pathogenic to humans.

23. PBMC Isolation
https://researcher.sanguinebio.com/ficoll-is-not-fickle-reliable-isolation-of-pbmcs-from-whole-blood/

24. Different Types of Growth Surface

25. The major steps required for reprogramming PBMCs using the
CytoTune™-iPS 2.0 Sendai Reprogramming Kit to generate
iPSCs cultured MEF feeder

26. Early Colony of iPSC
Characteristic of Induced Pluripotent Stem Cells
Compact colonies (or monolayer)
Defined colony edges
High nucleus-to-cytoplasm ratio
Colony of iPSC

27. Validation of Human Induced Pluripotent Stem Cells
Data of PRBM Eijkman, unpublished
• Human alpha thalassemia patient iPSCs (p18,19)
• Culture on day 5 and 10 forming optimal iPSC colonies with typical
characteristic of iPSC
D5, 200x D10, 40x

28. Validation of Human Induced Pluripotent Stem Cells
Data of PRBM Eijkman, unpublished
• Exclusion of CD13
• hiPSC profiling shows positive for TRA-1-60 and SSEA-4 markers

29. Conclusion
▪ MSC, HSC and iPSC research has been carried out in Indonesia
▪ MSC research is currently still very much in demand,
especially as a disease model and drug screening
▪ Research grants, whether individual, group, internal or
external grants can help carry out expensive stem cell
research
▪ Research collaboration is very useful in the early stages of
new research, such as iPSC
▪ Collaboration with clinicians/hospitals is needed so that
research results can be continued, especially for
patients/community in general