IJMCM Winter 2014, Vol 3, No 1

Original Article

Mesenchymal Stem Cells as a Feeder Layer Can Prevent Apoptosis of Expanded Hematopoietic Stem Cells Derived from Cord Blood Roya Mehrasa1, Hamidreza Vaziri1, Arezoo Oodi2, Mona Khorshidfar2, Mahin Nikogoftar2, Monireh Golpour3, Naser Amirizadeh2∗, 1. University of Guilan, Rasht, Iran. 2. Blood Transfusion Research center, High institute for Research and Education in Transfusion Medicine,Tehran, Iran. 3. Cellular and Molecular Biology Research Center, Babol University of Medical Sciences, Babol, Iran.

Submmited 20 July 2013; Accepted 21 Sep 2013

Umbilical cord blood (UCB) has been used for transplantation in the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). Because of insufficient number of cord blood CD34+ cells, the expansion of these cells seems to be important for clinical application. Mesenchymal stromal cells (MSCs), playing an important role in HSCs maintenance, were used as feeder layer. Apoptosis and cell cycle distribution of expanded cells were analyzed in MSCs co-culture and cytokine conditions and results were compared. Three culture conditions of cord blood HSCs were prepared ex-vivo for 14 days: cytokines (SCF, TPO and Flt3L) with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs without cytokines. Expansion was followed by measuring the total nucleated cells (TNCs), CD34+ cells and colonyforming unit (CFU) output. Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells. Maximum cord blood CD34+ cells expansion was observed in day 10. The mean fold change of TNCs and CD34+ cells at day 10 in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). The highest apoptosis rate and the least number of cells in Go/G1 phase were observed in cytokine culture without feeder layer (p=0.041). The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate. Key words: Cord blood expansion, apoptosis, co-culture, mesenchymal stem cell

U

mbilical cord blood (UCB) has been used as

hematologic diseases. Cryopreserved umbilical cord

a source of hematopoietic stem cells (HSCs)

blood (UCB) has also been applied to reduce the

for complete or partial human leukocyte antigen-

risk of graft versus host disease (GVHD),

matched

treatment-related mortality (TRM) and improve

∗

allogenic

transplantation

to

treat

Corresponding author: Assistant professor of high institute for research and education in transfusion medicine

Email: [email protected]

Mehrasa R et al.

survival. However, there are approximately 107 +

CD34 cells in one unit of cord blood, whereas for a

Materials and Methods Isolation of CD34+Cells

successful transplantation, 2.5×105 / kg CD34+ cells

HSCs were collected from fresh human

are required. Evidently, ex vivo expansion of UCB-

umbilical cord blood after obtaining written consent

HSCs is the most effective method to increase the

from normal full-term pregnant women, at the

+

number of CD34 cells and improve the kinetics of

Iranian

UCB-HSCs

according to the ethics committee guidelines.

engraftments

(1-3).

But

HSCs

Blood

Transfusion

Organization

and

Due to the surplus of red blood cells in cord

differentiation to precursor cells during ex-vivo expansion leads to the reduction of primitive HSCs

blood,

the

RBCs

were

(4, 5). Life, death, self-renewal and differentiation

hydroxyethyl

of HSCs are the major procedures that regulate the

Ottawa, Canada). Mononuclear cells (MNCs) were

numbers and lifespan of the HSCs pool which

isolated by density gradient centrifugation on

defect within these processes can contribute to

Ficoll-Hypaque (Bio sciences, Sweden). The MNCs

haemopoietic insufficiencies and development of

were

haemopoietic malignancies (6). In the normal

conjugated with microbeads (Miltenyi Biotec,

hematopoiesis, the balance between cell loss

USA) for 30 min at 4°C. Then CD34+ cells were

(apoptosis and differentiation) and cell gain

isolated by using a column in a magnetic field.

(proliferation and mitosis) can determine the HSC

Feeder Layer Culture

starch (Stem

then incubated

precipitated

using

cell technologies-

with antihuman

CD34

population size (7). Several factors have been

Mesenchymal stromal cells (MSCs) were

identified as potential mechanisms associated with

isolated from fresh bone marrow after obtaining

self-renewal and proliferation of HSC. Some

informed consent from healthy donors (at Taleghani

external factors including GM-CSF, Epo, Flt3,

Hospital, Tehran, Iran). Briefly, MNCs were

Notch and tumor suppressor genes like p21 affect

collected from 10 ml aspirated bone marrow by

stem cell division and regulation of self-renewal

density gradient centrifugation on Ficoll-Hypaque

(7). Apoptosis is a physiological process that

(Bio sciences, Sweden) and were washed with

controls tissue kinetics and homeostasis.

MSCs culture medium (low glucose DMEM

Apoptosis controls hematpoietic stem cells

supplemented with penicillin and streptomycin).

growth factors thereby preventing them from

The MNCs were cultured in a 75 cm2 culture flask

leukemo-genesis. Growth factors, cell-cell contact

at 37°C. After 2–3 days, non-adherent cells were

and intracellular genes expression are important

removed and adherent cells were left in MSCs

factors

the

culture medium until cells confluence achieved

apoptosis,

80%. MSCs were characterized by flow cytometric

proliferation and maintenance of HSCs in a

analysis using monoclonal antibodies against

primitive state, are growth factors and stromal cells

CD105, CD44, CD166, CD90, CD34 and CD45

in culture media (8).

(Dako, Denmark). Potential Differentiation of

that

significant

control factors

apoptosis. enrolled

Among in

Physiologically, stromal layer cells produce

MSCs

was

investigated

by

osteogenic

soluble factors which maintain the primitive

differentiation kit (Chemicon, USA). As a cell

characteristics of HSCs and the direct interaction

feeder layer, MSCs were harvested with 0·25%

between the HSC and MSC can catalyze this

trypsin-EDTA solution (Stem cell technology,

process (9). In this study we evaluated the rate of

Denmark), and 1×104 cells were plated in 6-well

HSCs proliferation and apoptosis in the presence of

plates. When cells reached more than 90%

mesenchymal stromal cells, Flt3-L, SCF and TPO.

confluence in DMEM, they were washed with PBS

Int J Mol Cell Med Winter 2014; Vol 3 No 1 2

Mesenchymal Stem Cells Prevent Apoptosis of Hematopoietic Stem Cells

and were placed in a serum free medium (Stem

Proliferative and phenotypic analysis Cells viability were determined at 0th, 5th, 10th,

Span) to co-culture with HSCs. MSCs Osteogenic differentiation assay

and

Osteogenic differentiation was performed on 4

14th

day

of

culture

by

counting

of

hematopoietic cells in each well using trypan blue

bone marrow derived MSCs. Thus, 5x10 cells

stain (Stem Cell Technologies, USA) and stem cell

were seeded in 24-well plates coated with

and lineage markers were analyzed by flow

collagen/vitronectin.

confluency

cytometry (Partec, Germany) using monoclonal

DMEM enriched with FBS was replaced by

antibodies against CD2, CD19 (Dako, Denmark) to

medium containing 100 µM l- ascorbic acid-2-

evaluate lymphoid lineage differentiation; CD14,

phosphate, 1M β-glycerophosphate and 1mM

CD15, and CD13 to evaluate myeloid differen-

dexamethasone (Chemicon, USA). On day 14, the

tiation; Glycophorin A to evaluate erythroid

cells were fixed with 70% ethanol for 1 h at 4 °C

differentiation and CD34, CD38 to assess the

and stained for 15 min with alizarin red-S (Sigma)

percentage of stem cells. Flow cytometric analysis

at room temperature (RT). Alkaline phosphatase

was performed by incubating harvested cells with

staining was performed with alkaline phosphatase

different

kit (Sigma-Aldrich). Differentiated cells were

antibodies at 4°C for 30 minutes. Then the cells

fixed with acetone and fast blue RR salt in

were washed in PBS and fixed with 2%

naphthol.

paraformaldehyde (Sigma). Isotype controls were

After

AS-MX

Hematoxylin

were

100%

Alkaline used

and

for

Mayer's

staining

and

counterstaining respectively.

fluorescent

conjugated

monoclonal

used in every experiment. Cell Cycle Distribution Analysis by Flow

+

Ex-vivo expansion of CD34 enriched cells +

Cytometry

5

CD34 enriched cells (1×10 ) were cultured in

Cell cycle distribution was evaluated at 10th

6 well plates (Nunc, Denmark) for 14 days in

day of culture by flow cytometry. Prior to staining,

serum-free

technology,

200 µl of 1×106 cells/ml were washed by phosphate

Denmark) at 37°C under 5% of CO2 humidified air

buffered saline (PBS) and re-suspended in 200 µl of

in three culture conditions : cytokines culture

PBS. Cells were treated with 50 µl of RNase

supplemented with SCF (50 ng/mL), TPO (50

(1 mg/ml) and 100 µl propidium iodide (PI,

ng/mL) and FLT3L (40 ng/mL) (Stem cell

400 µg/ml) (Sigma-Aldrich, Spain) for 30 min at

technology, Denmark), co-culture with MSCs

37 °C in the darkness. The fluorescence of stained

feeder layer and above mentioned cytokines and co-

cells was analyzed by flow cytometry and relative

culture with MSCs without any cytokines.

gated cells in each cell cycle phase were

medium

(Stem

cell

Colony-forming cell assays

determined. Data acquisition and analysis were

+

Fresh CD34 cells and expanded cells at 10

th

3

day of culture (1-2×10 ) were seeded in semisolid culture (Metho Cult GF H4434, Stem Cell

performed using flowmacs 2.0 software. Apoptosis Analysis by Annexin V Apoptosis rate were evaluated at 10th day of

manufacturer's

culture by Apoptosis kit (Bioscience, USA). 1×106

instruction. Cells were mixed with Methylcellulose-

cells were washed by phosphate buffered saline

based media and purred in 35-mm Petri dishes and

(PBS) and re-suspended in 1x binding buffer. The

incubated at 37°C, 5% CO2 in a humidified

cells were treated with 5 µl of fluorochrome

Technology)

following

the

th

incubator. After the 14 day of culture, the number

conjugated Annexin V for 15 min at room

of colony was counted under the inverted

temperature. These cells were washed and re-

microscope.

suspended in 1x binding buffer and then 50µl of

3 Int J Mol Cell Med Winter 2014; Vol 3 No 1

Mehrasa R et al.

propidium

iodide

solution

was

added.

The

were considered as markers for MSCs. The MSCs

fluorescence of stained cells was analyzed by flow

were negative for haematopoietic lineage markers,

cytometry after 4 hours.

CD34 and CD45.

Statistical Analysis

Osteogenic differentiation assay

Results obtained from multiple experiments are

Potential

osteogenic

differentiation

of

expressed as the mean ± standard deviation (SD).

bone marrow derived MSCs was assayed by

The data were analyzed using the t-test. Probability

alizarin red staining and evaluation of alkaline

values