1. Human Inferior Turbinate : An
Alternative Tissue Source of
Multipotent Mesenchymal Stromal
Cells
Presenter –Madan Gupta
Guide –Prof S C Sharma

2. INTRODUCTION
 MSCs (mesenchymal stromal cells) - plastic
adherent fibroblast-like cells with extensive
proliferative capacity and differentiation
potential.
 Types of stem cells:
Embryonic
 non-embryonic.
Induced pluripotent stem cell (iPSC)

3. International Society of Cellular
Therapy
 Three criteria:
1)MSCs must be adherent to plastic under standard
tissue culture conditions.
(2)MSCs must express certain cell surface markers such
as CD73, CD90, and CD105, and lack expression of other
markers including CD45, CD34, CD14, or CD11b, CD79alpha or
CD19 and HLA-DR surface molecules.
(3)MSCs must have the capacity to differentiate into
osteoblasts, adipocytes, and chondroblasts under in vitro
conditions.

4. Biological characteristics of
MSCs
 The ability to home to sites of inflammation
following tissue injury when injected intravenously
 The ability to differentiate into various cell types
 The ability to secrete multiple bioactive molecules
capable of stimulating recovery of injured cells and
inhibiting inflammation
 The lack of immunogenicity and the ability to
perform immunomodulatory functions

5. MSC roles in vivo

6. Immunoregulatory properties
in vitro

7. Therapeutic potential

8. Clinical applications of MSCs
 Le Blanc K et al- haploidentical MSCs-
aGVHD of the gut and liver.
 Ringdén O et al-6 gut,1 skin, 1 liver

10.  The best-characterized MSC population -
originating from bone marrow.
 Alternative sources.
 preliminary studies- human turbinate
fibroblasts - growth in a chondrogenic
medium acquire a chondrogenic
phenotype

11. HYPOTHESIS
 In a previous investigation- fibroblasts from inferior
turbinate tissue were plastic-adherent and
differentiated into chondroid cells when grown in a
chondrogenic medium- expressing the chondrogenic
markers aggrecan and type II collagen as assessed by
RT-PCR
 Fibroblasts isolated from the inferior turbinate are
multipotent MSCs- testing their capacity to
differentiate into chondrogenic, osteogenic,
adipogenic, and neurogenic cells.

12. Materials and Methods
Cell Isolation
 0.0366 g- inferior turbinate tissues-
discarded tissue - 10 patients who
underwent septoplasty and partial
turbinectomy.

13. Washing of tissue & culturte
 three times with antibiotic-antimycotic solution.
 twice with phosphate buffered saline (PBS) solution.
 75-mm2 cell culture disc(20mL of DMEM (Dulbecco’s Modified Eagle
Media, Gibco& 10 percent FBS was added)
 incubated in a 37°C, 5 percent CO2 incubator for 3 weeks,
 The fibroblasts attached to the bottom of the culture dishes were
obtained.
test for chracterization of MSCs

15. Multilineage Differentiation Potential
of hTMSCs
 Chondrogenesis
 seeding cells onto polycaprolactone sponges.
 Chondrogenic supplements
TGF-b1
IGF-1
 DMEM supplemented with 10% fetal bovine serum (FBS)
 100 nM dexamethasone
 50 mM ascorbic acid-2 phosphate
 50 mg/mL insulin transferrin sodium selenite
 1 mM sodium pyruvate
 40 mM proline
 2 mM Lglutamine

16. osteogenic differentiation
 low-glucose DMEM supplemented with 10%
FBS.
 100 U/mL penicillin.
 100 mg/mL streptomycin.
 0.1 mM dexamethasone.
 50 mM ascorbate-2 phosphate
 10 mM b-glycerophosphate

17. adipogenic differentiation
 maintained for 3 days in a-MEM (Gibco)
supplemented with 10% FBS and antibiotics.
 incubated in a-MEM supplemented with
 10 mg/mLinsulin.
 200 mM indomethacin.
 0.1 mM dexamethasone.
 0.5mM 3-isobutyl-1-methylxanthine .
 10%FBS.

18. neurogenic differentiation
 neurobasal medium with neurogenic
supplement.
 10 ng/mL glial-derived neurotrophin factor.
 10 ng/mL brain-derived neurotrophin factor.
 10 ng/mL neurotrophin.
 1 mL B-27 supplement.
 2 mML-glutamine.
 1% penicillin-streptomycin.

19. RNA Extraction and RT-PCR of
hTMSCs
 Total RNA was extracted from the cells cultured differentiation
usingTRIzol reagent .
 Total RNA from each sponge- diluted to a volume of 10 mL in
DEPC/DDW and used in a reaction (10 mL) containing
 first-strand buffer ,
 DTT
 dNTP
 oligo (dT) 15 primer
 Rnase inhibitor
 SuperScript II
 Incubated at 45C for 60minutes.
 The cDNA was stored at 220C until neede

20. Amplification of dna
 PCR reactions (50 mL) contained
 Flexi DNApolymerase
 MgCl2
 dNTPs
 cDNA template,
 and primers specific : b-actin
 aggrecan
 type I collagen
 type II collagen
 osteocalcin
 bone sialoprotein (BSP)
 osterix
 bone morphogenetic protein-2 (BMP-2)

The thermal conditions for PCR
amplification were initially
denaturation at 94C for 3
minutes, followed by 35 cycles
of 94C for 30
seconds, 53C for 30 seconds,
and 72C for 30 seconds,with a
final extension at 72C for 5
minutes. PCR products were
separated in 2% agarose
(Sigma) gels in 13 Trisacetate-
EDTA buffer containing
ethidium bromide.

21. Results

22. Multilineage Differentiation Potential

23. Expression of mRNA

24.  Isolation of Novel Multipotent Neural Crest-
Derived Stem Cells from Adult Human
Inferior Turbinate
 Stefan Hauser et al
 STEM CELLS AND DEVELOPMENT
Volume 21, Number 5, 2012

25. HYPOTHESIS
 The respiratory mucosa of adult human
inferior turbinate may contain -NCSC
population.

26. Material and Methods
 Cell isolation and culture of ITSCs
 Biopsy tissues - immediately placed in PBS on ice- mechanical
mincing using the Mcllwain TissueChopper.
 Afterward, the tissue was dissociated using CollagenaseI.
 The primary cultures were grown for at least 72h in serum-free
medium consisting of Dulbecco’s modified Eagle’s medium/Ham
F-12 (DMEM/F-12; Invitrogen) supplemented with
 basic fibroblast growth factor-2
 epidermal growth factor
 Heparin
For rapid expansion
cultivated using DMEM/F-12
supplemented with:
10%human blood plasma
40 ng/mL FGF-2
20 ng/mL EGF.

27. neuronal differentiation
 For induced neuronal differentiation- re-suspended in DMEM
containing
 10% FBS
 1 mM dexamethasone
 2mM insulin
 500mM 3-isobutyl-1-methylxanthine
 200mMindomethacin
 200mM ethanol
 After 28 days, RNA isolation, immunocytochemical stainings
and detection of synaptic vesicle .

28. Adipogenic differentiation
 For adipogenic differentiation, ITSCs -
cultivated in
 DMEM
 10% FBS
 1 mM dexamethasone
 2mM insulin
 500mM 3-isobutyl-1-methylxanthine
 200mM indomethacin

29. Chondrogenic differentiation
 cultivated DMEM
 10% FBS
 10 ng/ml TGF-b1
 0.1mM dexamethasone
 Alcian blue - to detect the presence of
glycosamino glycans

30. Osteogenic differentiation
 cultivated in DMEM
 10% FBS
 100 nM dexamethasone
 10mM b-glycerophosphate
 0.05mM L-ascorbic acid-2-phosphate
 ALP activity was measured in differentiated
ITSCs using the Alkaline Phosphatase
Detection Kit
 To detect biological mineralization cells were
stained with Von Kossa and Alizarin Red S .

31. Results
 ITSCs - glial origin
 Aarrowheads-p75 positive Schwann cell-like ITSCs
 Arrows - axons
 asterisk- Schwann cells

32. ITSCs perform differentiation into
ectodermal lineage
 Cultivated in neuronal differentiation
medium for 4 weeks-

34. ITSCs are able to differentiate into representative
mesodermal cell types

35. Human Respiratory Epithelial
Cells from Nasal Turbinate
Expressed Stem Cell Genes
Even after Serial Passaging
 B H I Ruszymah et al
 Med J Malaysia December 2011

36. HYPOTHESIS
 To ensure the capability of
proliferation, the stem cell property
of RE cells from the nasal turbinate.
 FZD-9 and BST-1 were chosen as the
stem cell marker

37. MATERIALS AND METHODS
 Six human nasal turbinate specimens
under aseptic conditions, the specimens were
digested in 0.3% Collagenase type I solution - 4-12 hours.
 The cell suspension containing fibroblasts and
respiratory epithelial cells - centrifuged at 6,500 rpm - 5
minutes.
 resuspended in 5-10ml trypsin EDTA and kept in
shaker incubator - 5 minutes at 370C.
 same volume of Trypsin Inhibitor (Gibco/BRL, USA)
 cultured - Defined Keratinocytes Serum Free Medium
(DKSFM) +Modified Eagle’s Medium (DMEM)
supplemented with 5% Fetal Bovine Serum (FBS)

38. RESULTS

40. Discussion
 The lamina propria—main constituent
loose connective tissue
rich network of thin-walled
venous sinusoids
fibroblasts,
macrophages,
T and B lymphocytes,
plasma cell

41.  All 3 study strongly suggest that these cells
are MSCs and demonstrate the possibility of
using human inferior turbinate tissues
removed during turbinate surgery as a
source of stem cells
 In culture, even after multiple passage no
change in characteristic of these cells.

43.  The ability to easily isolate MSCs - tissues
discarded during surgical procedures
 reduce pain and morbidity in MSC donors
increase donor participation
 In all avobe 3 study dicarded tissue used
for cultivation of MSCs.

44.  Corporation of Korea--- 134,619 turbinate
surgeries performed in 2009 to treat enlarged
inferior turbinates.
 Sufficient turbinate tissue should be available
to support seed cell or individual cell banks.
 Future use of tissue that is normally discarded
during surgery may enable the establishment of
custom-made tissue and cell banks for
patients.

45. Limitation
To compare proliferation and differentiation
between hTMSCs and MSCs from bone
marrow and adipose tissue.
 bone marrow–derived MSCs exhibit an
age-related decline in life span, proliferation,
and osteogenic potential-our study did not
explianed about this.
 No compare of hypertrophic turbinate -
with normal turbinate tissue.

46. The Human Nose Harbors a Niche
of Olfactory Ectomesenchymal Stem Cells
Displaying Neurogenic and Osteogenic Properties
STEM CELLS AND DEVELOPMENT

47. Conclusion
 Above studies proved that hTMSCs can
differentiate into other types of adult cells.
 This will allow us to develop an effective
tissue-regenerating method with the use of
adult cells.
 Cell banks can be established from
stromal-cell-rich turbinate tissues that would
otherwise be discarded after nasal surgery.