Generation of Insulin Producing Cells using Mesenchymal...

The Canadian Journal of Clinical Nutrition, Volume 1, Issue 1, January 2013 ISSN 1927-8950 (Online Edition)

Canad J Clin Nutr is published by Global Science Heritage, (http://www.globalscienceheritage.org), a registered publisher

by the Library and Archives/Government of Canada, (www.collectionscanada.gc.ca)

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Generation of Insulin Producing Cells using Mesenchymal Stem Cells

Derived from Bone Marrow of New-Zealand White Rabbits

Abdelaziz A. Mohamed1*

, Manal M. Saad 1

, Sherif H. Abdeen 2

, Mona K. Marei 1

1Tissue Engineering Laboratories, Faculty of Dentistry, Alexandria University, Egypt.

2Department of

Zoology, Faculty of Science, Mansoura University, Egypt.

*Corresponding Author: Dr. Abdelaziz Mohamed. Email: [email protected]

ABSTRACT

Background: The utilization of stem cell trans-differentiation into insulin-producing cells

(IPCs) would provide potential promising therapy for diabetes mellitus (DM). Objective:

The study was aimed to investigate the differentiation potential of rabbit’s bone marrow-

derived cells into insulin producing cells. Methods: Bone marrow-derived mesenchymal

stem cells (MSCs) were obtained from three New-Zealand male white rabbits and propagated

in a primary culture for 14 days in low glucose Dulbecco's Modified Eagle's Medium

(DMEM), harvested and subjected to another three passages encompassing 21 days. After

that, MSCs were functionally defined by their ability to differentiate into osteoblasts. This

was achieved by incubation with the DMEM containing 10-7

M dexamethasone, 10 mM β-

glycerophosphate and 50 µl/ml of ascorbic acid. Osteogenic differentiation was followed up

at days 2, 4, 7 and 9 by staining cells with alizarin red S and vonKossa. Results: It was found

that the onset of differentiation was at day 7 and continued at day 9. On the other hand,

another patch of MSCs were induced into insulin-producing cells by two step incubation. The

first with high glucose serum-free DMEM containing 0.5 mmol/L β-mercaptoethanol for

three days and the second follows by incubation with the same medium containing 10

mmol/L nicotinamideinstead of β-mercaptoethanolfor 18 days. Trans-differentiation was

followed up at days 4, 9, 14 and 21. It was found that the cells have trans-differentiated into

insulin-producing cells starting from day 14 and continued in the subsequent days as judged

by their affinity to stain with diphenylthiocarbazone (dithizone or DTZ) at days 14 and 21.

Conclusion: The results would provide some insights of using rabbit's bone marrow as a

source of MSCs with their differentiation potentials. This might help for the development of a

future stem cell therapy for diabetes as well as several other human diseases.

Key words: Mesenchymal Stem Cells, Insulin-producing Cells, Diabetes Mellitus

Mohamed AA, Saad MM, Abdeen SH, Marei MK. Generation of Insulin Producing Cells using

Mesenchymal Stem Cells Derived from Bone Marrow of New-Zealand White Rabbits. Canad J

Clin Nutr 2013; 1(1): 47-66.

DOI: http://dx.doi.org/10.14206/canad.j.clin.nutr.2013.01.06

Page 47-66

mailto:[email protected]

http://dx.doi.org/10.14206/canad.j.clin.nutr.2013.01.06


Canad J Clin Nutr is published by Global Science Heritage, (http://www.globalscienceheritage.org), a registered

publisher by the Library and Archives/Government of Canada, (www.collectionscanada.gc.ca)

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INTRODUCTION

Stem cells are undifferentiated cells which have the ability to renew themselves through

mitotic cell division and differentiate into a diverse range of specialized cell types (1). Of

these cells, mesenchymal stem cells (MSCs) have a large capacity for self-renewal while

maintaining their multipotency. MSCs display multiline age differentiation potentials into

a variety of cells such as osteoblasts, adipocytes and chondrocytes as well as myocytes

and possibly neuron-like cells (2). They are capable of suppressing immune responses

and offer therapeutic potential for achieving transplantation tolerance (3). Accordingly,

MSCs have been considered as an appropriate source for cell and gene therapy tools for

treatment in a number of injuries, congenital and degenerative diseases including; spinal

cord injury (4), osteogenesis imperfecta (5), stroke (6), parkinsonism (7) and diabetes

mellitus (8).

Diabetes mellitus refers to a group of chronic metabolic diseases characterized by

hyperglycemia due to defects in insulin secretion, insulin action, or both (9).Type I,

insulin-dependent, diabetes is the result of autoimmune destruction of insulin-producing

pancreatic β-cells. On the other hand, in type two or non-insulin-dependent diabetes,

insulin production is inadequate mainly because of the peripheral insulin resistance and

subsequent β-cell apoptosis. Diabetes and its devastating effects, which include

retinopathy, nephropathy, stroke and heart attack, afflict more than 194 million people

worldwide. According to the World Health Organization, these numbers will be more

than double by 2030 (10, 11).

Survival of patients with insulin-dependent diabetes relies on recurring insulin delivery,

which does not cure the disease or prevent diabetes-associated maladies. Although, Type

2 diabetes can be managed through a combination of diet, exercise and prescription of

drugs, almost 30% of the affected people will require frequent administration of insulin.

Therefore, the development of therapies to replace insulin regimens is highly desirable.

To that end, islet transplantation has afforded promising results, with some patients

experiencing insulin independence for more than 5 years after the initial procedure (12).

Ongoing procedural improvements are underway to increase the time span of liberation

from insulin and reducing the side effects due to immunosuppressant. However, the

scarcity of available donor tissues hinders wide application of pancreas/islet

transplantation. Thus, the hope stands in finding renewable sources of islet β-cells such as

pancreatic β-cell lines, embryonic stem cells (ESCs), adult progenitor cells (APCs),

regenerating native islet cells (13) and MSCs (14).

From all of the previously mentioned literatures, it was therefore of interest to carry out

experiments investigating the differentiation potential of rabbit's bone marrow-derived

stem cells into insulin-producing cells as a first step for their future use in islet

transplantation purposes.




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MATERIALS AND METHODS

Abbreviations

The following abbreviations are used; Adult progenitor cells (APCs), deionized distilled

water (ddH2O), diabetes mellitus (DM), dimethyl sulfoxide (DMSO),

diphenylthiocarbazone (dithizone or DTZ), Dulbecco's Modified Eagle's Medium

(DMEM), embryonic stem cells (ESCs), fetal bovine serum (FBS), hours (hr), insulin I

and II, glucose transporter-2 (Glut-2), insulin-producing cells (IPCs), intra-muscular

(IM), mesenchymal stem cells (MSCs), minutes (min), mononuclear cells (MNCs),

pancreatic duodenal homeobox-1 (Pdx-1) and phosphate buffer saline (PBS).

Animal work

All animal experiments were performed according to the guidelines of the National

Institutes of Health (NIH) for laboratory animal use (15). All procedures applied in this

study were of no pain category (category C).

Aspiration bone marrow-derived cells

Bone marrow aspiration was performed according to the method of Horan et al.

(16).Three male New-Zealand white rabbits (obtained from the research farm, Faculty of

Agriculture, Alexandria University) were anesthetized by intra-muscular (IM) injection

with 5 mg/kg xylazine as a preanaesthetic medication. After 5 min, animals were intra-

muscularly injected with 30 mg/kg ketamine-HCl. All surgical procedures were

performed in a biological safety cabinet (Nuaire biological safety class II, T-Telestar,

Spain) with surgery room disinfected overnight by UV light. The fur of the thigh was

shaved and skin disinfected. Skin incision was made to expose the rabbit's femur. A hole

was drilled in the femur using a surgical rose-head bur. A sterile plastic aspiration needle

containing 8 µl heparin/ml sample was used. The marrow sample was kept in a sterile

package till transferred to the cell culture laboratory. The surgical wound was sutured

and the rabbit was taken back to the animal care unit. All animals tolerated the surgical

procedures for bone marrow aspiration. No significant changes in body weight, no

postoperative infections were observed.

Cultivation media

All buffers, media and their supplements were purchased from BioWhittkers® Lonza,

Verviers, Belgium unless otherwise noted. These media included: Basal medium

[Low glucose Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 mmol glucose/L

and supplemented with10% (v/v) fetal bovine serum (FBS),1% L-glutamine,10 IU/ml

penicillin/streptomycin and 20 mM HEPES]. Ostogenic differentiation medium [Low

glucose DMEM with4.5 mmol glucose/L and supplemented with 10% FBS, 1% L-

glutamine, 1% penicillin/streptomycin, 20 mM HEPES,10 mM β-




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glycerophosphate(Sigma, USA),50 µg/ml ascorbic acid (Sigma, USA) and10-7

M

dexamethasone (Sigma, USA)].

First step trans-differentiation medium: Serum-free high glucose DMEM with 25 mmol

glucose/L and supplemented with 20 mM HEPES,1% L-glutamine,1%

penicillin/streptomycin and 0.5 mmol/L β-mercaptoethanol (Sigma Aldrich Co.,

Germany). Second step trans-differentiation medium: Serum-freehigh glucose DMEM

with 25 mmol glucose/L and supplemented with 20 mM HEPES buffer,1% L-

glutamine,1% penicillin/streptomycin and10 mmol/L nicotinamide (Sigma Aldrich Co.,

Germany).

Media preparation

The powder supplements of the media were dissolved in deionized distilled water

(ddH2O) obtained from Barnstead water deionization system (Barnstead-Thermolyne,

USA). In case of using another dissolvent, it was sterilized by membrane filtration

through 0.22 μm microbiological filter system (TPP-Europe, Switzerland). After adding

the supplements, the pH of the medium was adjusted at 7 and medium was then sterilized

by membrane filtration. A sterility test was performed for 3 days at 37C. Medium

contamination was detected depending on the color change of the phenol red, which gives

a yellow-orange color through the change in pH to acidic value as a function of the

microbial growth. A sample was also examined microscopically after passing the sterility

test. All media were stored at 4C until being used.

Isolation of rabbit's bone marrow-derived mesenchymal stem cells

The method of Sun et al., (17) was used with minor modifications. Bone marrow samples

of about 1.5 to 2 ml were aspirated from each rabbit and transferred to the cell culture

laboratory in a sterile package. Inside a biological safety cabinet class II (Thermo

electron corporation, Germany) samples were diluted with a double volume of the basal

medium, collected by centrifugation at 390 g for 7 min at room temperature and washed

twice with phosphate buffer saline, pH 7.4 (0.0067 M PBS without calcium and

magnesium).

The samples which have clots or bone marrow tissue were filtered through 200 µm filter

(Sigma Aldrich Co., Germany) using PBS for washing and liberation of the trapped cells

in the tissue. Bone marrow mononuclear cells (MNCs) were then collected by

centrifugation, washed twice with the basal medium and resuspended in one ml of the

same basal medium, counted (10 l of cells + 10 µl of 0.4% methyl violet + 180 µl

glacial acetic acid) and tested for viability using hemocytometer. MNCs were then seeded

in polystyrene coated T-flasks at a density of 5.5 x 106cells/cm

2and incubated at 37

oC,

5% Co2 and 95% humidity. Except for the first four days which were required for cell

adhesion, the cells were fed twice a week by washing with pre-wormed PBS then adding

fresh medium. The cells were allowed to proliferate till reaching the 100% confluence in

14 days (the primary culture).




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Only the cells which attach to the polystyrene surface were allowed to grow while

suspended ones were discarded during washing. Cell morphology was followed up daily

using a phase contrast inverted microscope (NIKON-Japan) using (SIS) software system

for capturing and analysis of images. All cultures were independently repeated for three

times.

Confluent monolayer of cells were harvested by trypsinization using trypsin/EDTA

buffered solution 6 ml of 0.5% trypsin, 200 mg/L versene EDTA (Gibco/Invitrogen,

Germany) at 37oC for 10 min. The suspended cells were rapidly neutralized with a double

volume of the basal medium to stop the action of trypsin. The cells were then collected

by centrifugation, washed twice with pre-wormed PBS, counted and tested for viability.

The cells were then reseeded in fresh medium at a density of 1400 cells per cm2 in a new

cell culture T-flasks. The cells were allowed to grow for three subsequent passages of 7

days each. After passage three, MSCs were subjected to osteogenic differentiation and

trans-differentiation into insulin-producing cells (18).

Estimation of viable cell concentration

Viable cell concentration was determined using trypan blue exclusion method (19). Ten

l of cells were diluted to 1:2 in 0.4% trypan blue stain. The hydrophilic trypan blue

diffuses through cell membrane of the dead cells. Hence, the viable cells appear bright

while the unviable ones appear blue. Cell viability was determined according to the

following equation: Cell viability = Total number of viable cells/Total cell count ×100

Cryopreservation of bone marrow-derived cells

At the end of each culture, a separate patch of cells were trypsinized and collected for

cryopreservation (20). The cells were cryopreserved at a density of 1×106 cells/ml in

freezing medium; basal medium with 25% FBS and 5% v/v dimethyl sulfoxide (DMSO).

The cells were then stored at -156ºC in cryo-vials (Nunc, Wiesbaden, Germany) in the

vapor phase of a liquid nitrogen locator (Cryo Biological Storage System Locator 4 plus

thermolyne, USA).After several pilot trials, cryopreservation of cells was optimized in

5% DMSO as a cryoprotectant. This protocol yielded the highest cell viability of 69%

upon revitalization. In order to revitalize the cells, cryogenic-vials were warmed in a

water bath at 37oC for 2 min and resuspended rapidly in FBS, centrifuged for 5 min,

washed twice with PBS, counted, tested for viability and resuspended in the basal

medium. Counting and testing viability were done before and after cryopreservation as a

routine procedure.

Characterization of rabbit's bone marrow-derived MSCs

Rabbit's bone marrow-derived MSCs were characterized by their induction into

osteogenic differentiation using the method of Nadriet al.,(21). The cells were incubated

with the osteogenic differentiation medium at the end of passage three, especially, when

they have reached 80-90% confluency. The cultures, in 6-well plates, last for two weeks

with cells fed twice a week. Cell morphology was followed up daily. To detect the onset

of osteogenic differentiation, cells were stained with alizarin red S and von Kossa at days

2, 4, 7 and 9.




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Alizarin red S staining of the differentiated osteoblasts

Calcium depositions of osteoblasts were detected through its reaction with alizarin

sulphate with scarlet to red color indicating positive results (22). For staining, the cells

were washed with pre-warmed PBS, fixed in 4% formaldehyde for 30 min. The fixative

was then removed and the cells were washed using distilled water. The alizarin red S

stain pH 4, (2% alizarinsulphonate; LobachemieCo., India) was added to cover all the

cells for approximately 5 min at room temperature. The stain was then discarded and the

cells were washed again with distilled water to remove the excess of stain then air dried.

Red patches in the tested cultures were examined microscopically. Cells propagated in

the basal medium were included as control cultures.

vonKossa staining of the differentiated osteoblasts

vonKossa staining was applied to confirm the osteogenic differentiation of MSCs (23).

Like the staining protocol of alizarin red S, the cells were washed with pre-warmed PBS,

fixed for with 4% formaldehyde 30 min and washed with distilled water. The cells were

then incubated with 1% aqueous silver nitrate solution (Fisher scientific-UK) in a clear

glass coplin jar placed under a 60-100 watt light bulb for 1 hr. After that, the cells were

carefully washed with distilled water to eliminate all the debris of silver nitrate and

incubated with 5% sodium thiosulfate for 5 min. The solution was discarded and the cells

were washed again with distilled water and air dried. The progress of the reaction was

followed up visually. The staining intensity of the brown to black color was a reference

for the presence of calcium produced by the differentiated osteoblasts.

Trans-differentiation of MSCs into insulin-producing cells

At the end of passage three, especially when MSCs have reached 80-90% confluence, the

cells were induced into insulin producing cells through two steps. The first step included

the incubation of the cells with the 1ststep trans-differentiation medium for three days.

This was followed by a careful wash with pre-warmed PBS and incubation with the

2nd

step trans-differentiation medium for 18 days. All over the two steps, the cells were

fed twice a week (17).

Staining of insulin-producing cells with dithizone

Dithizone (DTZ), a zinc-chelating agent, is known to selectively stain pancreatic β cells

by crimson red color. In the secretory granule of a mature insulin-producing cell, every 6

insulin molecules are coordinated by a Zn atom giving the red color. For staining, the

cells were incubated with DTZ stain (Lobachemie Co., India; 10 mg/ml DTZ in DMSO

diluted to 1:100 in the basal medium) for 30 min. The cells were then carefully washed

with pre-wormed PBS for three times. The staining result was monitored under an

inverted microscope. After 5 hours, the stain was completely disappeared and the cells

were able to grow successfully (24).

Data analysis

Statistical analyses were performed with one-way ANOVA followed by SPSS software

version 10 (SPSS Science, Chicago, IL, USA) analysis. Data (mean ± SD) were




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considered statistically significant at a value of P < 0.05. ANOVAtest was applied to

compare the growth kinetics during the different time points of the expansion period i.e.

the primary culture and the first two passages.

RESULTS

Isolation of bone marrow-derived mesenchymal stem cells

Isolation of bone marrow-derived MSCs required a primary culture of 14 days and three

subsequent passages of 21 days. This can be summarized as follows; bone marrow

samples of 1.5 - 2 ml were processed for isolation of mononuclear cells (MNCs) giving

rise to the mean numbers of cells 3.9×107. MNCs were then seeded in a primary culture

at a density of 5.5×105 cells/cm

2 giving a yield of 15.6×10

3 cells/cm

2 at the end the

primary culture (day 14). Whereas, the number of cells seeded in passage one and two

was 1.4×103 cells/cm

2 and the number of cells yielded was 8.4×10

3 and 7.2×10

3cells/cm

2

for passages one and two, respectively. The cells were allowed to grow in passage three

similar to that of passages one and two with one difference of being differentiated or

trans-differentiated at the 100% confluency of passage three without transfer to a next

passage (Table 1). Except for day zero of the primary culture, comparison of the growth

kinetics of all culture during the expansion period (the first 5 weeks) revealed that all

cultures were significantly grow (P < 0.05) starting from day 2 after reseeding.

In the primary culture, MSCs were allowed to adhere to the polystyrene tissue culture

flask surface in the first four days without media change. Whereas, at day five all of the

co-cultured red blood corpuscles (RBCs) and cell debris were discarded with the washing

step prior to the media change. At day five, MSCs showed an anchorage-dependent

growth with their attachment confirmed via the emergence of expanded cytoskeleton

processes. In the following days, cells proliferate with increased cell to cell contact

giving rise to cell colonies. By the end of the primary culture, at day 14, the cell

proliferation reached about 90% confluence. Thus, the cells were trypsinized and

reseeded in another culture (Figure 1).

In passage one and unlike the primary culture, the cells adhere in few hours and overnight

incubation was sufficient to make the cells to completely adhere in day 1. However in

day 2, the cells started to contact with each other. In the subsequent days, the cells

proliferated, forming colonies and their morphology changed to the spindle-shape. The

cells reached the 100% confluence in only 7 days (Figure 2). After that, the cells were

collected by trypsinization and reseeded in a new cell culture flask for another passage.

The number of cells at the end of passage one was 8.2×103 cells/cm

2 with doubling time

of 21.6 hr. The viability after harvesting was 98%.

However in passage two, the cells showed similar manner of cell growth. They showed

substratum adherence, migration, cell-cell contact, colony formation and confluence. The

cell yield of passage two was 7.2×103 cells/cm

2 and doubling time was 17.63 hr.




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Characterization of MSCs by osteogenic differentiation

Cells were incubated in conditioned medium of dexamethasone, β-glycerophosphate and

ascorbic acid. They were following up daily with an inverted microscope. The cells were

stained with alizarin red S and von Kossa stains in days 2, 4, 7 and 9 to determine the

onset of osteogenic differentiation. All tested cultures showed positive results starting

from day 7. However, controls showed negative staining all over the four time points.

On the other hand, the morphology of the cells under the stress of osteogenic

differentiation showed the spindle-shape in the tested cultures. The controls showed some

aspect of over confluent appearance. The difference in cell morphology between the

tested and control cultures during the first few days till day 7 was not clear but with

staining the discrimination was possible (Figure 3).

Trans-differentiation of MSCs into insulin-producing cells

MSCs were induced into insulin-producing cells via two steps. The first step included the

incubation of cells with high glucose serum-free DMEM containing β-mercaptoethanol.

Upon trans-differentiation, the cells showed aggregated colonies. The first step of

differentiation was extended to 3 days then the second step follows at day 4 by replacing

β-mercaptoethanol with nicotinamide. At day 4, the colonies were formed and developed

in the subsequent days. At day 14, the morphology of cells was changed to the rounded to

oval shape and continued with this morphology in days 19 and 21 (Figure 4).

The dithizone (DTZ) staining was applied to test the production of insulin at different

time points including days 4, 9, 14 and 21. While, the tested cultures showed an affinity

to the DTZ stain (crimson red color) starting from day 14 and continued in day 21, the

controls showed negative results all over the time of the experiment (Figure 5).

DISCUSSION Mesenchymal stem cells are multipotentstem cells that can differentiate into a variety of

cell types (25). Bone marrow-derived MSCs, also named colony-forming fibroblastic

cells (26), marrow stromal stem cells (27) and mesenchymal progenitor cells (17), have

been isolated and characterized from many species including: rats, cats, dogs, baboons

and rhesus monkeys, rabbits, pigs, goats and sheeps (21). MSCs have many advantages

including: the ease of isolation, the extensive multi-lineage differentiation potential (18,

28), the hypo-immunogenic property which modulate the lymphocytic functions, the

possibility of allogenic transplantation, the capability of systemic transplantation for

generalized diseases as well as the local implantation for the local tissue defects. MSCs

can also be used as a vehicle for genes in gene therapy protocols or to generate

transplantable tissues and organs in tissue engineering protocols. Accordingly, MSCs are

among the first stem cell types to be introduced in the clinic and several encouraging

clinical trials are under way to study the efficacy and long-term safety of therapeutics

based on MSCs (29, 30).

Several recent studies have demonstrated the feasibility of generating insulin-producing

cells obtained from progenitor cells of various cellular sources, including the pancreas,

http://en.wikipedia.org/wiki/Multipotent

http://en.wikipedia.org/wiki/Multipotent

http://en.wikipedia.org/wiki/Cellular_differentiation




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liver and intestinal epithelium, as well as, the pluripotent embryonic stem cells of mouse

and human origin. However, even with the conceptual advances offered by these

findings, some obstacles, such as the immune rejection and autoimmunity against newly

formed cells derived from pancreatic stem cells, still remain. Despite their promising

potential, it may also prove difficult to obtain enough autologous adult stem cells from

these organs. To overcome these limitations, the possibility of using rabbit bone marrow

derived cells was explored as a source of insulin-producing cells under specific in vitro

culture conditions. Bone marrow has been known for years to represent a safe and

abundant source for large quantities of adult stem cells (13).

In the present study, the source of MSCs was the bone marrow of New-Zealand white

rabbits. This animal model was chosen due its availability, the ease of handling and the

ease of choice between in and out-bred. In case of aspiration of bone marrow samples

from mice or rats, the animal is often killed before aspiration. Contrarily, there is no need

to kill the rabbit to aspirate a bone marrow sample with the advantage of repeating

aspiration within 8 weeks (16).

Several techniques have been developed to obtain pure cultures of MSCs by the reduction

or elimination of non-MSCs from bone marrow cultures (31). This was achieved, in the

present study, by the application of a prolonged expansion step through which all of the

bone marrow-derived cells pass in a primary culture for 14 days and three subsequent

passages of 21 days. During this time, only MSCs, due to their adherent property, were

retained in the culture while all of the other non-adherent cells were eliminated by

washing during media change. Moreover, the selective medium and the polystyrene-

coated tissue culture flasks enhanced the adhesion properties of the MSCs (21).

Cultures of MSCs is usually contaminated by different types of non-MSCs adherent cells

like monocytes and macrophage (32) and as MSCs are more responsive to trypsin, they

were purified in the present study with an optimized concentration of trypsin/EDTA (17).

Unlike the primary culture when the cells needed 14 day to reach the 100% confluency,

the behavior of the cells changed in the subsequent passages as they reached the 100%

confluency in only 7 days. This could be owing to the gradual selection of a learned

mesenchymal cell clones with proceeding passages (33).

One of the hallmarks of MSCs is their multi-potency, defined as the ability to

differentiate into several mesenchymal lineages including; bone, cartilage, tendon,

muscle, marrow stroma and adipose tissue. Usually, the tri-lineage differentiation into

bone, adipose tissue and cartilage is taken as a criterion for the multi-potentiality of these

cells (34). In the current study, MSCs were differentiated into osteoblasts using a

conditioned medium containing dexametasone, ascorbic acid and β-glycerophosphate.

The differentiation was followed up at different time points including days 2, 4, 7 and 9

through staining with alizarin red S and von Kossa stains. The onset of osteogenic

differentiation was at day 7 as judged by the positive staining affinity of the calcium

depositions of the osteoblasts with the two stains. These results confirmed the

mesenchymal stem lineage of the processed cells

(35).




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Upon trans-differentiation, the presence of insulin was evidenced using the dithizone

stain (DTZ) which is a zinc chelating agent. It does not require prior fixation and stains

insulin molecules either in the cells or in the media.DTZ disappears after 5 hr leaving live

cells able to complete their life cycle (24, 36). The onset of trans-differentiation was

followed up at different time points including; days 4, 9, 14 and 21. It was found that a

complete trans-differentiation was obvious in day 21 while partial trans-differentiation

was observed in day 14. In contrast, there were no signs of trans-differentiation at days 4

and 9. Despite DTZ is a vital stain, the staining intervals were chosen minimal to

decrease the stress applied on the cells at every staining time.

On the other hand, the microscopic examination for the trans-differentiation revealed

aggregations like clusters of insulin producing cells with a diameter ranging from 100 to

150µm. These clusters appeared from day 2 of differentiation and continued to develop

till day 12 where the cells acquired the spindle-shape. This morphology was changed to

the rounded and oval shapes at day 14 which may be considered as a sign for the meso-

endodermal differentiation (37).

There are two key steps in culture conditions that appear important for inducing the trans-

differentiation of MSCs cells into insulin-producing islet-like cells. First, the transfer of

cells from an expansion period in low-glucose medium to differentiation in high-glucose

medium until certain genes, such as pancreatic duodenal homeobox-1 (Pdx-1), insulin I

and II, glucose transporter-2 (Glut-2), and islet amyloid polypeptide, become detectable.

Second, in order for the MSCs cells to become glucose responsive, further differentiation

and maturation are required through either in vitro culture with cell promoting factors,

such as nicotinamide, mercaptoethanol or transplantation of the cells into diabetic

animals (17, 38). It is well known that glucose is a growth factor for cells. It promotes

cell replication in vitro and in vivo and increases insulin content of the cell (39).

Nicotinamide is a poly (ADP-ribose) synthetase inhibitor known to differentiate and

increase cell mass in the cultured human fetal pancreatic cells (40). It protects the cells

from desensitization induced by the prolonged exposure to large amounts of glucose. It

have also been demonstrated that nicotinamide promoted the formation of fetal porcine

islet-like cell clusters and increased the rates of pro-insulin biosynthesis in these clusters.

Moreover, the stimulatory effects of nicotinamide on insulin production and content by

fetal porcine islet-like cell clusters result from neo-formation of cells through

differentiation (41). Another study described how nicotinamide-treated islets derived

from the pancreatic progenitor cell had more insulin and secreted significantly more

insulin than cultures treated with glucose alone (42).

The present study used the low glucose medium for cell expansion and for induction to

insulin-producing cells the medium was changed to high glucose and this was the first

key step in trans-differentiation. The second key step was achieved through culturing

with the media containing β-mecaptoethanol then nicotinamide as cell promoting factors.




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In conclusion, the present data suggest potential uses of MSCs from rabbit's bone marrow

in induction into insulin-producing cells. This provides hope for a future development a

stem cell therapy for diabetes as well as several other human diseases and injuries.

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Table 1. Bone marrow-derived cell growth during culture

Total number of living cells

(Mean cells / cm2 ± SD) × 10

3

Primary

culture

Day 0 Day 5 Day 7* Day 9* Day 14*

550 ± 88.8 5.1 ± 0.8 7.77 ± 0.76 11.37 ± 1.01 15.67 ±

0.67

Passage

one


1.4± 0 1.67 ±

0.21 2.93 ± 0.15 5.23 ± 0.31 8.4 ± 0.2

Passage

two


1.4± 0 1.63 ± 0.15 2.3 ± 0.53 4.23 ± 0.31 7.2 ± 0.6

Passage

three

Day 0 Reached the 100% confluency after 7 days.

Characterization by osteogenic differentiation.

Trans-differentiation into insulin-producing cells. 1.4± 0

* Significant cell growth (P < 0.05) compared to the beginning of culture with the

exception of day zero of the primary culture where cultures were compared to the

day 5.




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Figure 1. Photomicrographs of MSCs during the primary culture. Days of follow up

included; (A) day 4, (B) day 5, (C) day 6, (D) day 7, (E) day 9 and (F) day 14. Black

arrow in A refers to a red blood corpuscle and whites arrows in A and B refer to the

adherent MSCs.




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Figure 2. Photomicrographs of MSCs during passage one. Days of follow up

included. (A) Day one and (B) day three, with arrows referring to cell-cell contact.

(C)and (D) are representing the culture at days 4 and 6, respectively. (E) represents the

morphology of the mesenchymal stem cell colony and (F) represents two proliferating

colonies in their way to adhere with arrows representing cell movement.




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Figure 3. Photomicrographs of MSCs at day seven during osteogenic differentiation. (A)and(B) are representing the control and tested cultures, respectively, stained with

alizarin red S. (C) and (D) are representing similar control and tested cultures,

respectively, stained with von Kossa. The orange to red patches in B and the brown to

black patches in D indicate the osteogenic differentiation.




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Figure 4.Photomicrographs of MSCs during the trans-differentiation into insulin-

producing cells. Days of follow up included; (A, B) day 2, (C, D) day 14 and (E, F) day

19. (A, C) are representing the control cultures and (B, D, E, F) are representing the

tested cultures. The control cultures showed an over confluency (arrows). The change in

cell morphology of the tested cultures from the spindle-shape to the oval or rounded-

shape was noted from day 14 and continued to day 19.

Figure 5. Photomicrographs of dithizone (DTZ) stained MSCs during the trans-

differentiation into insulin-producing cells at day 14 (A, B) and day 21 (C, D). (A, C)

are representing the control cultures and (B, D) are representing the tested cultures. The

control cultures showed an over confluency with negative staining. While, the tested ones

showed the positive staining (crimson red patches).

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