The demand for islet cells for treatment of

diabetes and lack of pancreata and the

problem of immunosuppression with the

allogenic transplantation directed our thoughts

towards stem cell therapy. Stem cell therapy

can be defined as group of new techniques or

technologies that relay on replacing diseased

or disfunctioning cells with healthy functioning

one.

Stem cells could be obtained from human fetuses,

umbilical cords, or embryonic tissues derived from

fertilized eggs. However these sources of stem cells

raise ethical and legal questions, pose a risk of

transmitting infections, and/or may be ineffective

because of immune rejection.

Aim of The WorkAim of The Work

In the present study, we have described the

characterization and differentiation in vitro of a

subset of human peripheral blood monocytes that

behave as stem cell. The ability to obtain these stem

cells from an easily accessible source such as

peripheral blood should make them valuable

candidates for autologus transplantation.

I- Generation of Neoislet Cells .

Mononuclear layer was obtained from peripheral blood

of healthy donors by density gradient centrifugation

and further purified by MACS technology to obtain

monocytes [Miltenyi Biotic - Germany].

The purity of the monocytes was tested by flow

cytometry analysis of CD14 , also CD34 was done.

Monocytes are then cultured for 6 days in dedifferentiation culture media; RPMI 1640 based medium containing 10% fetal calf serum,L-glutamine, penicillin , streptomycin and specific growth factor: macrophage colony stimulating factor (M-CSF), human interleukin 3 (IL3).

These cells are now termed programmable cells of monocytic origin (PCMO) and flow cytometry analysis for CD14 and CD34 was performed again. PCMO were then cultured in islet cell conditioning medium [ICM] containing epidermal growth factor, hepatocyte growth factor, nicotinamide and glucose for 21 days to be differentiated into neoislet cells.

II- Analysis of Neoislet Cells.II- Analysis of Neoislet Cells.

After 7,14 and 21 days differentiated PCMO were

subjected to real time RT-PCR and

immunohistochemistry. Glucose challenge test was

done on day 21 and insulin and C-peptide were

assayed by radioimmunoassay .

I. Flow Cytometry Analysis:I. Flow Cytometry Analysis:

Monocytes were tested by flow cytometry before

culturing for CD14 and CD34. They showed that

CD14 (specific for monocytes) was (82% + 6%) and

CD34 ( marker of stem cell) was (0% + 3%) .

II. Neoislet Cell Markers by RT-PCR:II. Neoislet Cell Markers by RT-PCR:

We confirmed neoislet differentiation by real time RT-

PCR at day 7, 14 and 21 of culturing in ICM for

common islet cell markers; β - actin, Pdx-1, insulin

and glucagon.

At day 7 (as shown in fig 1), weak amplification signal

for β - actin and Pdx-1 was detected. No insulin or

glucagon expression was found.

After 14 days we got good amplification signals for

Pdx-1, insulin and glucagon gene.

While after 21 days (as show in fig2) maximum

expression of gene Pdx-1, β - actin, insulin and

glucagon was found. ( We found no amplification

signals for Pdx-1, insulin , glucagon in PCMO which

was used as control).

After 7 Days in ICM Culture

Weak Signal for ß-actin

Weak Signal for Pdx-1

Pdx-1 gene expressed in our Total Reference RNA, HUMAN

Insulin gene expressed in our Total Reference RNA, HUMAN

Glucagon gene expressed in our Total Reference RNA, HUMAN

ß-actin gene expressed in our Total Reference RNA, HUMAN

After 21 Days in ICM Culture

III. Immunohistochemistry :III. Immunohistochemistry :

Neoislets cells were subjected to

immunohistochemistry against insulin and glucagon.

In day 7 positive staining for glucagon

(as shown in fig 3) and negative for insulin was found

while positive for both insulin and glucagon in day 21

(as shown in fig 4a & 4b). Immunohistochemistry

staining was found to be negative for both insulin and

glucagon in PCMO.

On Day 7 After Culture in ICM Positive Staining for Glucagon and Negative for Insulin

Positive For Both Insulin and Glucagon Positive For Both Insulin and Glucagon On Day 21 After Culture in ICM On Day 21 After Culture in ICM

InsulinInsulin GlucagonGlucagon

IV. Results of Insulin and C – IV. Results of Insulin and C –

Peptide:Peptide:

Supernatant collected from the glucose challenge

performed to the neoislet cells was assayed by RIA

for insulin and C-peptide. The results showed that

insulin was (3.2, 5.7 and 6.8 µIU/ ml) and C-peptide

results were (0.8, 1.4 and 1.5 ng/ml) for glucose

concentrations: 50, 300 and 400 mg/dl respectively.

(as shown in fig 5).

0

1

2

3

4

5

6

7

50 300 400

IV. Results of Insulin and C – IV. Results of Insulin and C – Peptide After Glucose ChallengePeptide After Glucose Challenge

InsulinInsulin

C-peptideC-peptide

In the present study, the in-vitro differentiated

monocytes derived neo-islet cells resembles primary

human counterpart in several aspects:

1- Genetic Markers of Neoislet Cells1- Genetic Markers of Neoislet Cells

by real time RT-PCR showed expression of Pdx-1

which is a regulatory gene important for β-cell function

detected early after 1 week of culture in ICM.

Real time RT-PCR analysis of our results revealed

endogenous de novo expression of insulin and

glucagon at high amplification signals at day 21 of ICM

culture.

2- Immunohistochemichal2- Immunohistochemichal assay in our

work provides positive glucagon granules at day 7 in

ICM while at day 21, it provides positive results for

both insulin and glucagon which mimics the normal

embryological development of the islet cells. As during

embryonic life, β-cells are recognized 2 weeks after

alpha cells development.

3- Metabolic Function3- Metabolic Function by performing glucose

challenge test , Neoislet cells appears to resemble

pancreatic islet cells in metabolic activity by secreting

insulin and C-peptide in a glucose dependent fashion.

Aim of The Work:Aim of The Work:Autologus implantation of Neoislet Cells generated

from stem cell of monocytic origin in canine.

Methods and Results:Methods and Results:Preparation of Dogs Preparation of Dogs Seven male mongrel dogs were chosen. 6 dogs

were prepared for implantation and 1 for control.

Each dog was subjected to single IV injection of

alloxan to induce diabetes.

Generation of Neoislet Cells :Generation of Neoislet Cells :30 ml of peripheral blood was taken from each dog

separately . The peripheral blood monocytes were

cultured for 28 days by the above technique to

generate neoislets cells.

Implantation :Implantation : Six dogs were injected by neoislet cells suspended in

1 ml ICM in the deltoid muscle and injected between

the muscle fibers. The seventh dog (control dog)

was injected by 1 ml ICM only. All dogs were off of

insulin at the day of implantation.

Evaluation of graft functionEvaluation of graft functionA- Fasting Blood Glucose ofA- Fasting Blood Glucose of (FBG) of 3

dogs were normalized. The FBG of 4th and 5th dogs

were ameliorated; the 4th didn’t need insulin (135 + 5

mg/dl) while the 5th dog need a small dose of insulin

(200 + 5 mg/dl). The 6th dog died one day after

implantation and the cause of death was unexplained.

FBG of the control dog remained high.

B- To Assess The Ability ofB- To Assess The Ability of the implanted

dogs for glucose load disposal, an oral glucose

tolerance test was performed for the first 4 dogs.

Oral Glucose Tolerance Test (OGTT)

0

50

100

150

200

250

0 30 60 90 120 150

Time

Glu

cose

Lev

el

Normal GTT

Impiared GTT

3 dogs showed normal pattern of oral glucose tolerance curve. The 4th dog showed a pattern of impaired glucose tolerance.

C- ImmunohistochemistryC- Immunohistochemistry

After 50 days, the grafts were removed from all

dogs for immunohistochemical study. In the 5

recipient dogs, the cells were viable and showed

the presence of positive insulin and glucagon

granules between the muscle fibers as shown in fig

(7a and 7b).

The graft of the control dog was negative for both

insulin and glucagon granules.

( Fig. 7-b) ms Bx Insulin Positive x 400

( Fig. 7-a) ms bx Glucagon Positive x 400

DISCUSSION DISCUSSION In our study, autologus implantation of neo-islet

cells into diabetic dogs was capable of

regulating or ameliorating blood glucose level

post implantation up to 50 days

Despite that the intramuscular implantation

might not be the ideal site for islet

implantation, it has been chosen for its high

neovascularization capability and its easy

accessability (*).

(*) Mahgoub M, Ammar A., Ashmawi H., Akl M., Hammam O. The deltoid muscle could be a suitable site for islet

transplantation 2005; poster presentation in the 10th IPITA congress, Geneva, Switzerland.

After removal of the graft from the deltoid

muscle, the dog recurred to hyperglycemic

state as pre implantation compared to the

control diabetic dog which remained

hyperglycemic throughout the study.

The most exciting perspective in this study is the

potential use of stem cell of monocytic origin for

treatment of diabetes mellitus.

In possible future clinical applications, islet cells may

first be generated in vitro and upon autologus

transplantation into patients may substitute for their

endogenous counterparts.

Thank YouThank You