adipose derived stem cells based regenerative terapy in
TRANSCRIPT
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Adipose derived stem cells based
regenerative terapy in Wrocław.
Current state and future prospects
Leszek Masłowski, Wojciech Witkiewicz, Maria Paprocka •Regional Specialistic Hospital, Research and Development Centre, Wroclaw, Poland
•Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of
Sciences, Wroclaw, Poland
•WROVASC Integrated Cardiovascular Centre, Wroclaw, Poland
Anna Czarnecka
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Therapeutic angiogenesis
Stem cells:
peripheral blood bone marrow
adipose tissue Wharton Yelly
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Therapeutic angiogenesis
stem cells application
intraarterial intra tissue
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MATERIAL 20 CLI – patients IV Fontaine , Rutheford 5
Age 41 – 61 y 50,3 + 10,1
Duration of CLI: 6 m – 8 y mean 3,7 + 2,7 lat
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Methods • Bone marrow aspiration under general/spinal anaesthesia
550ml/ACD-A 10:1 (multiple aspitration biopsy)
• Separation of the mononuclear cells using the COBE Spectra separator
• Final volume of the preparation 40 - 50ml
Phenotype: CD34+: 15,8%±0.25, CD45-CD34-: 10.8%±0.96, CD45-CD34- CD90+: 0.1%±0.02, CD45-CD34-CD105+: 2.8%±0.4, CD45-CD34-CD73+: 0.07%±0.01, CD45-CD34-CD117+: 0.02%±0.007
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Methods
• Intramuscular implantation by 40-50 injections
• Number of implanted cells: 30.2 x 108 ± 4.5 x 108
• Arteriography prior to implantation and after 3 months
• ABI
• Pain assesment by VAS scale
• Ulcers planimetr
• Follow up 24 months
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Stem cells separation:
COBE Spectra separator. Cleanness 90%
Final volume 40-50ml
Phenotype:
CD34+: 15,8%±0.25, CD45-CD34-:
10.8%±0.96, CD45-CD34-CD90+:
0.1%±0.02, CD45-CD34-CD105+:
2.8%±0.4, CD45-CD34-CD73+:
0.07%±0.01, CD45-CD34-CD117+:
0.02%±0.007
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Intramuscular implantation
40- 50 injections 15 - 20mm depth
Number of implanted cells: 30.2 x 108 ± 4.5 x 108
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Reduction of pain
0
4
8
przed 1 mies 3 mies
6,17
4,625
5,23
4,63
3,66 3,98
VAS
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Healing of ulcers
0
100
200
300
400
500
600
przed 10 dni 1
mies
2
mies
3
mies
502,27
156,53
77,32
32,34
146,53 168,6
cm 2
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Results
Healing of ulcers
Pat No 1
22.12.2003r 10.04.2004r
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Results
Pat No 8
04.03.2004r
10.04.2004r
16.12.2004r
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Angiography
Pat No 2
15.12.2003r 19.03.2004r
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Results
• Reduction of pain 18/20
• Complete healing 9/20
• Partial healing (>50%) 6/20
• Progression of necrosis/ulcers 5/20
• Amputaion 2/20
• Neovascularisation 9/20
Follow-up 24 months
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Adipose
tissue derived mesenchymal stem cells
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Mesenchymal stem cells have now been isolated
from various tissues including: - bone marrow, -
peripheral blood, - cord blood, - muscle, - skin, -
synovium, - adipose tissue
Adipose tissue could be one of the most suitable
cell sources for cell therapy, because of its - easy
accessibility, - minimal morbidity and - abundance
of mesenchymal stem cells.
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Aim of the study
To assess the effectiveness and safety of autologous
transplantation of adipose tissue-derived stem cells
(adipose derived stem cells) in the treatment of
chronic wounds (diabetic foot ulcers and venous leg
ulcers)
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Patients
21 patients with t. 2 DM
7 woman
14 men
Mean age: 60.2 + 5.64 y
Standard follow-up 6 months
diabetic foot ulcers
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Patients
•14 patients
11 woman
3 men
Mean age: 66,6 ±9,5 y
Standard follow-up 6 months
venous leg ulcers
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Methods
Adipose tissue was harvested by aspiration after
infiltration by local anesthetic and tumescent isotonic
solution.
Stem and regenerative cells were separated using a
closed and fully automated system CELLUTION 800
from Cytori Therapeutics USA.
5 ml of a concentrated cell suspension were implanted
into subcutaneous tissue around the wound and the
wound bed.
The phenotype of the cells obtained was determined
immediately after separation and after 7 days of culture
on RPMI with 10% fetal calf serum
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Methods
Harvesting of the adipose tissue by tumescent – aspirations
method
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5 ml of a concentrated cell suspension were implanted into
subcutaneous tissue around the wound and the wound bed.
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Methods
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Results
Preparation - 5ml 5.6 x 106 + 4 x 106 cells
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Results
Phenotype of the cells after separation
CD45
CD31
CD34
CD133
CD146
CD105
CD90
X + SD 25,52
+ 7,49
22,83
+ 13,47
59,33
+ 28,45
0,5 + 0,84 18,83
+ 16,66
28,5
+ 25,99
55
+ 27,84
CD45
CD31
CD34
CD133
CD146
CD105
CD90
X + SD 7,4
+ 10,59
8,4
+ 11,84
34,8
+ 27,69
0,0 16,8
+ 2,17
81,25
+ 34,18
93,8
+ 9,55
Phenotype of the cells after culture
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Clinical results
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Clinical results
No significant side effects of the lipoaspirations or
applications of the stem cells were observed
Clinical improvement was observed in 17 patients
suffering from diabetic foot ulcers (77,3%) including 8
completely healed and 9 with >50% reduction of ulcer
area
Clinical improvement was observed in 10 patients with
venous leg ulcers (71,4%) including 3 completely
healed and 7 with >50% reduction of ulcer area
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Clinical results
No improvement was observed in 4 patients ( with
diabetic foot ulcers), including 2 with progression of
necrotic lesions resulting in amputation
No improvement was observed in 4 patients (with
venous leg ulcers)
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Conclusion
Autotransplantation of adipose tissue stem cells is
a safe and effective treatment method for diabetic
foot ulcers, however with transient effect after the
single application.
The use of stem cells propagated in vitro will
provide a good continuation of the current study
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Projects for the future
ADMSC autotransplantation for patients with scleroderma
and advanced Raynaud phenomenon with acral ulcers
Allogenic ADMSC in autologic plasma gel as a biological
dressing for chronic wounds
Allogenic ADMSC in autologic plasma gel as a biological
dressing for corneal ulcers
Stem cells conditoned medium for chronic wounds
Therapeutic angiogenesis with stem cells conditioned medium
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Thank for your attention