Generating Neoislet Cells from Stem Cells of Monocytic Origin by Inkibj4H

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									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 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.
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:
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:
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).
          Weak Signal for ß-
               actin

           Weak Signal for
               Pdx-1




After 7 Days in ICM Culture
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 :
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 On Day 21 After Culture
in ICM




         Insulin                          Glucagon
IV. Results of Insulin and C – 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).
IV. Results of Insulin and C – Peptide After Glucose
Challenge
    7
    6
    5
    4
    3
    2
    1
    0
          50        300       400           Insulin
                                           C-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 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- 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 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:
Autologus implantation of Neoislet Cells generated
from stem cell of monocytic origin in canine.
Methods and Results:
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 :
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 :
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 function
A- 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 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)
                                                                     Normal GTT
                250                                                  Impiared GTT
Glucose Level




                200
                150
                100
                50
                 0
                      0   30       60          90     120      150
                                        Time




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

								
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