Human Inferior Turbinate : An Alternative Tissue Source of Multipotent Mesenchymal Stromal Cells

Presenter –Madan GuptaGuide –Prof S C Sharma

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)

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.

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

MSC roles in vivo

Immunoregulatory properties in vitro

Therapeutic potential

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

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

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.

Materials and Methods

Cell Isolation 0.0366 g- inferior turbinate tissues-

discarded tissue - 10 patients who underwent septoplasty and partial turbinectomy.

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

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

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

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.

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.

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

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 30seconds, 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.

Results

Multilineage Differentiation Potential

Expression of mRNA

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

HYPOTHESIS

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

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 plasma40 ng/mL FGF-2 20 ng/mL EGF.

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 .

Adipogenic differentiation

For adipogenic differentiation, ITSCs - cultivated in

DMEM 10% FBS 1 mM dexamethasone 2mM insulin 500mM 3-isobutyl-1-methylxanthine 200mM indomethacin

Chondrogenic differentiation

cultivated DMEM 10% FBS 10 ng/ml TGF-b1 0.1mM dexamethasone

Alcian blue - to detect the presence of glycosamino glycans

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 .

Results

ITSCs - glial origin

Aarrowheads-p75 positive Schwann cell-like ITSCs

Arrows - axons asterisk- Schwann cells

ITSCs perform differentiation into ectodermal lineage

Cultivated in neuronal differentiation medium for 4 weeks-

ITSCs are able to differentiate into representativemesodermal cell types

Human Respiratory Epithelial Cells from Nasal TurbinateExpressed Stem Cell Genes Even after Serial Passaging

B H I Ruszymah et al Med J Malaysia December 2011

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

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)

RESULTS

Discussion

The lamina propria—main constituent

loose connective tissue rich network of thin-walled

venous sinusoids fibroblasts,

macrophages, T and B lymphocytes,

plasma cell

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.

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.

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.

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.

The Human Nose Harbors a Nicheof Olfactory Ectomesenchymal Stem Cells

Displaying Neurogenic and Osteogenic Properties STEM CELLS AND DEVELOPMENT

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.