Browsing by Author "Hiba BadrEldin Khalil Ahmed Khalil"

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Successful isolation, culturing, proliferation and transdifferentiation of stem cells obtained from Umbilical Cord blood and Wharton jelly
(Al-Neelain University, 2011) Hiba BadrEldin Khalil Ahmed Khalil
ntroduction Mesenchymal stem cells (MSCs) and Haemopoietic stem cell (CD34) could be isolated from human umbilical cord blood. In addition to that MSCs like, could also be isolated from Wharton jelly which represent a rich source of primitive cells, with the same properties of cord blood and bone marrow MSCs. Mesenchymal stem cells and haemopoietic stem cells have the capacity of self-renewal, proliferation and in viva or ex vivo differentiation. This study was conducted to compare stem cells from umbilical cord blood and Wharton jelly, focusing on isolation techniques, identiﬁcation and proliferation using various medium, (MesenCult and DMEM), and differentiation in vivo (albino rats) and ex viva (MethoCult medium), transdifferentiation into hepatocyte like cells and expression of CD34,CD45 and CD10. Materials and Methods Thirty female albino-rats age 5-6 week-old; weight 40-60 g, 20 were used for ‘human cells engraftment, whereas the remaining l0 served as negative controls. Twenty to fourty ml of 25 cord blood samples and length of 12 cm of 25 umbilical cords for Whaiton jelly samples were obtained from 25 newborns delivered after full tenn following ethical consent. The cord blood samples were grouped according to the time of collection after birth into three groups. Group one (G1) include samples processed after 5 - 7 hours, group (G2) after 3 - 9 hours and 9-13 hours for group three (G3), whereas Wharton jelly samples were grouped as G1 which include samples processed aﬁer 56 - 93 hours, G2 aﬁer 19 - 24 hours and 42 - 48 hours for G3 and time of trypsin digestion was 45 minutes for G1 and 30 minutes for G2 &G3.Ficoll Paque was used to obtain the mononuclear cells from cord blood samples while trypsin enzymatic treatment was used for Wharton jelly samples. Puriﬁcation of CD34 was done using RosetteSep and EasyScp techniques. The expression and count of CD34, CD45 and CD10 was detected by desired monoclonal antibodies and analyzed using tlowcytometric. The ex viva proliferation of MSCs was performed in DMEM at day 7 for G2 and day 14 for G1, whereas for MesenCult media it was performed at day 14 for G3. The differentiation process was done by CFU-F ﬁbroblast assay and hepatocyte growth factor protocol, and checked by monitored microscopic, immunocytochemistry and micro albumin assay. CD34 positive cells were cultured for 16 days in MethoCu]t media aﬁer which viable cells count, colonies count assays, and calculation of fold expansion and plating efﬁciency were done. In vivo engrattment and cell proliferation was tested using CD34 positive cells in experimental irradiated albino rats (n=l0) and CD34 improved by human MSCs (n=l0) infused by intravenous and intraperitoneal modality. Statistical methods The data was analyzed using SPSS 13, p.value considered statistically signiﬁcant below 0.05. Descriptive statistics (mean and standard deviation) obtained for numerical variables while frequency distribution and percent for categorical variables. Mean differences were tested by t-test or ANOVA. Results The results showed no signiﬁcant differences in the count of mononuclear cells isolated from cord blood and Wharton jelly samples based on mother age, weeks of gestation, length of umbilical cord and time of processing. The results also showed that the MesenCult media gave the best MSCs viable cells count and fold expansion compared with DMEM. According to the incubation time, DMEM produced the best viable cells count and fold expansion in 7 days in contrast to the 14 days incubation. A statistical signiﬁcant difference (p-value = 0.04) was observed when compared the MSCs viable cells count and fold expansion of cord blood group 1 and Wharton jelly group 2 in DMEM. The results also showed that the plating efﬁciency and colonies count of MSCs to form CFU-ﬁbroblast from Wharton jelly samples was higher with no signiﬁcant statistical difference (p-value % 0.2) than MSCs from cord blood samples. The activation of hepatocyte cell like obtained from the Wharton jelly samples was higher compared with MSCs obtained from umbilical cord blood. However, the difference turned to be statistically insigniﬁcant. Although CD10 was expressed by MSCs, we observed no signiﬁcant difference between cord blood and Wharton jelly samples. Although EasySep technique gave the best puriﬁcation count of CD34 and haemopoietic colonies count (CPU-E, BFU-E and CPU-GM) in ex vivo methoCult media, R0setteSep gave the best self renewal, proliferation into CD45 and engraﬁment in albino rats. In addition the combination of CD34 and MSCs gave the best engraftment and the best site for administration was the intravenous one. Conclusions According to the results, Wharton jelly was the best source of MSCs when as compared to cord blood. Regarding CD10 expression, the cord blood was the best compared to Wharton jelly. The best media for stem cell research was the MesenCult obtained from Stem Cell Technologies Company. The best technique for CD34 isolation was the positive selection conceming EasySep technique, although the best in vivo engraﬁment can be obtained by R0setteSep technique. MSCs look to have the same properties of bone marrow stroma, when it is combined with the haemopoietic stem cell (CD34) in case of in vivo engraftment.
Successful isolation, culturing, proliferation and transdifferentiation of stem cells obtained from Umbilical Cord blood and Wharton jelly A thesis submitted in fulfillment for the requirements of the PhD degree in medical hematolo
(Al-Neelain University, 2011) Hiba BadrEldin Khalil Ahmed Khalil
Abstract Introduction Mesenchymal s tem cel ls ( MSCs) and Haemopoietic s tem cel l ( CD34) coul d be i solated from human umbilical cord blood. In addition to that MSCs like, could also be isolated from Wharton jelly which represent a rich source of primitive cells, with the same properties of cord blood and bone marrow MSCs. Mesenchymal s tem cel ls and haemopoietic stem cells have the cap acity of self-renewal, proliferation and in vivo or ex vivo differentiation. This s tudy was condu cted to compare s tem cells f rom umbilical cord bl ood a nd W harton j elly, focusing on i solation techniques, identification and proliferation using various m edium, (MesenCult a nd DMEM), and differentiation in vivo (albino r ats) and ex vivo (MethoCult medium), transdifferentiation into hepatocyte like cells and expression of CD34,CD45 and CD10. Materials and Methods Thirty female a lbino rats a ge 5 -6 week-old; weight 40 -60 g, 20 w ere us ed for hum an c ells engraftment, whereas the remaining 10 served as negative controls. Twenty t o f ourty ml of 25 cord blood samples and l ength of 12 cm of 25 umbilical cords for Wharton jelly samples were obtained from 25 newborns delivered after full term following ethical consent. XV The c ord bl ood s amples w ere gr ouped a ccording t o t he t ime of c ollection after bi rth into t hree groups. Group one (G1) include samples processed after 5 - 7 hours, group (G2) after 3 - 9 hours and 9-13 hours for group three (G3), whereas Wharton jelly samples were grouped as G1 which include samples processed after 56 - 93 hours, G2 after 19 - 24 hours and 42 - 48 hours for G3 and time of tr ypsin digestion was 45 m inutes for G 1 and 30 minutes for G 2 &G3.Ficoll P aque w as used to obtain the mononuclear cells from cord blood samples while trypsin enzymatic treatment was used f or W harton jelly s amples. P urification of C D34 was done us ing R osetteSep a nd EasySep techniques. The expression and count of CD34, CD45 and CD10 was detected by desired monoclonal antibodies and analyzed using flowcytometric. The ex vivo proliferation of MSCs was performed in DMEM at da y 7 for G2 and day 14 for G 1, whereas fo r MesenCult media it w as performed at day 14 for G3. The differentiation process was done by CFU-F fibroblast assay and hepatocyte growth factor protocol, and checked by monitored microscopic, immunocytochemistry and micro albumin assay. CD34 positive cells were cultured for 16 days in MethoCult media after which viable cells count, colonies count assays, and calculation of fold expansion and plating efficiency were done. In vivo engraftment and cell proliferation was tested using CD34 positive cells in experimental irradiated albino r ats (n=10) a nd CD34 i mproved b y hu man M SCs ( n=10) infused b y intravenous a nd intraperitoneal modality. XVI Statistical methods The da ta w as analyzed using S PSS 13 , p.value considered s tatistically significant below 0.05. Descriptive s tatistics ( mean and standard de viation) obtained f or num erical va riables w hile frequency distribution and percent for categorical variables. Mean differences were tested by t-test or ANOVA. Results The results showed no significant differences in the count of mononuclear cells isolated from cord blood and Wharton jelly s amples based on mother a ge, w eeks of gestation, length of um bilical cord and t ime of pr ocessing. The r esults also s howed t hat t he M esenCult m edia gave t he best MSCs viable cells count and fold expansion compared with DMEM. According to the incubation time, DMEM produced the best viable cells count and fold expansion in 7 days in contrast to the 14 days incubation. A statistical significant difference (p-value = 0.04) was observed when compared the MSCs viable cells count and fold expansion of cord blood group 1 and Wharton jelly group 2 i n DMEM. The results also showed that the plating efficiency and colonies count of MSCs to form CFU-fibroblast from Wharton jelly samples was higher with no significant s tatistical di fference (p-value = 0.2) than M SCs f rom c ord bl ood s amples. The activation of hepatocyte cell like obtained from the Wharton jelly samples was higher compared with MSCs obtained from umbilical cord blood. However, the difference turned to be statistically insignificant. Although C D10 w as expressed by MSCs, w e obs erved no s ignificant di fference between cord blood and Wharton jelly samples. XVII Although EasySep technique gave the best purification count of CD34 and haemopoietic colonies count (CFU-E, BFU-E and CFU-GM) in ex vivo methoCult media, RosetteSep gave the best self renewal, pr oliferation i nto C D45 a nd e ngraftment in albino r ats. In a ddition t he combination of CD34 and MSCs gave the best engraftment and the best site for administration was the intravenous one. Conclusions According to the results, Wharton jelly was the best source of MSCs when as compared to cord blood. Regarding CD10 expression, the cord blood was the best compared to Wharton jelly. The best m edia f or s tem cel l r esearch was t he M esenCult obtained from Stem C ell T echnologies Company. The best technique for CD34 isolation was the positive selection concerning EasySep technique, although the best in vivo engraftment can be obtained by RosetteSep technique. MSCs look t o ha ve t he s ame pr operties of bone m arrow stroma, w hen it is combined w ith t he haemopoietic stem cell (CD34) in case of in vivo engraftment. ملخص الدراسه المقدمه يمكن عزل الخلايا الجذعية الوسيطه و الخلايا الجذعية المكونه للدم من دم الحبل السري البشري والتي لديها القدره على الانتشار ، وتجديد الذات والتمايز إلى خلايا مختلفة في جسم الانسان, كما يمكن التعرف على هذه الخلايا ومميزاتها بالتجارب و البحوث العلميه داخل الانسان او الحيوان او عبر زراعتها في مزارع متخصصه بأستخدام اوساط متباينه . بالإضافة الى دم الحبل السرى هنالك مصدر اخر لهذه الخلايا و هو النسيج المحيط بالاوعيه الدمويه في الحبل السري ويسمى بالوارتون جلي و يمثل مصدرا غنيا للخلايا الوسيطه والتي لها نفس خصائص الخلايا الجذعيه المعزوله من دم الحبل السري ونخاع العظم. هدفت هذه الدراسة للمقارنة بين الخلايا الجذعية الوسيطه المستخلصه من دم الحبل السري والوارتون جلي باستخدام طرق مختلفه للعزل ومن ثم قياس قدرة هذه الخلايا على الانقسام وتجديد الذات و ذلك باستخدام نوعين من الوسائط هما من دم الحبل السري باستخدام تقنيتين CD كما هدفت الدراسه ايضا" الى عزل الخلايا الجذعيه 34 ، DMEM و MesenCult و من ثم قارنت القدره على الانقسام و التكاثر. RosetteSep و EasySep هما 9العينه و طرق البحث T 9T 9دم الحبل السري لاطفال حديثي الولاده و ذلك بعد اخذ الموافقه T 19T 9من T 19T شملت الدراسه 25 عينه دم تراوحت بين 20 الى 40 مل 9T . 9سم T 19T 9 ادنى بلغ 121 T 19T الاخلاقيه من ذويهم اضافه الى 25 عينه وارتون جل اخذت من حبال سريه لاطفال حديثى الولاده بطول 9T 9ابيض تراوحت اعمارهم بين 5 الى 6 اسابيع بوزن يتراوح بين 40 الى 60 جرام , 20 T كما شملت العينه استخدام ثلاثون فأر 1 29،اما ال 10 المتبقيين كانوا بمثابه ضوابط سلبيه. T 19استخدموا لزراعة الخلايا الجذعيه المكونه للدم المعزوله من الانسان T منهم 9 العينه, كما تم T 19T 29تجهيز T 29اء" علي زم ن T 9بن T 19T 9, المجموعه الاولى والثانيه والثالثه و ذلك T 19T 9تم تقسيم عينات الدم الى ثلاثه مجموعات T 9T 19T 29تجهيز T 29اء" علي زم ن T 9بن T 19T 9, المجموعه الاولى والثانيه والثالثه و ذلك T 19T ايضا" تقسيم عينات الوارتن جل الى ثلاثه مجموعات 9من عينات الدم الماخوذه T 19T 9احادية النواة1 T 19T 19استخدام محلول الفيكول لعزل الخلايا T 9كما تم T . 9 ووقت حفظ العينه في انزيم التربسين 1 T 19T العينه .29T 29 و استخدم انزيم التربسين لعزل الخلايا الشبيهه بالخلايا الوسيطه في عينات الوارتون جل T من الحبل السري XIX 9تم زراعة الخلايا الجذعيه الوسيطه المعزوله مسبقا" ف ي وس ط الدلبكو لفترة حضانه 14 يوم للمجموعه الاولى و 7 ايام T 9وقد تم ايضا" زراعة الخلايا في وسط المزنكلت لفترة حضانها 14 يوما للمجموعه الثالثه و ذلك لمعرفة T.12T 129T للمجموعه الثانيه قدرتها على الانقسام و الانتشار و التحول الى مستعمرات الفايبروبلاست. اما طرق تنقية الخلايا الجذعيه المكونه للدم فهي 9T 1 1T2T.2T 19TEasySep29T و1 RosetteSep 9T 2المخصصه لكل كاشف و تحليلها عبر T 19T 29الاجسام المضادة T 9الكواشف المناعيه ( سي دي 45 و 34 و 10 ) تم استخدام T 19T لمعرفة جهاز الانسياب الخلوي. اما عن معرفة الخلايا الجذعيه المكونه للدم, ومقدرتها على انشاء المستعمرات المكونه للدم فقد تم استخدام مزرعة الميزوكلت لفترة حضانه 16 يوما". 9شملت الدراسه عينة الفئران التي تم حقنها عبر وريد الذيل( 9 فئران) و جدار البطن ( 9 فئران) لاختبارمقدرة الخلايا المكونه للدم T الي الوصول الى النخاع و البدء بالانقسام, كما تم ايضا حقن بعض هذه الفئران بخلايا جذعيه مكونه للدم مدعومه بخلايا جذعيه 9T وسيطه.1 النتائج أسابيع T 9T الأم ،و عدد T 9T وفقا لعمر T 9T او الواتون جلي T 9T دم الحبل السري T 9T عينات T 9T المعزولة من T 9T الخلايا T 9T في عدد T 9T اختلاف كبير T 9T 9لم يكن هنالك T مزرعة المزنكلت اعطت نتائج لاعداد الخلايا الجذعيه الوسيطه مقارنة بمزرعة الدلبكو. أفضل عدد T 9T . معالجة العينه T 9T ووقت T 29T،29T الحمل أن T 9T أظهرت النتائج T 9T، اضافة الى ذلك T 9T. للخلايا الوسيطه تم حصده كان من مزرعة الدلبكو التي كانت فترة حضانتها 7 ايام بنسبه اعلى في وسط مزرعة المزنكلت مقارنة بخلايا دم الحبل T 9T المستعمرات T 29T 29عدد T المعزولة من الوارتون جيلى اعطت T 9T الخلايا 9كما اظهرت النتائج نجاح الخلايا الجذعيه الوسيطه و المكونه للدم في التحول الى خلايا شبيه بخلاي ا الكبد تملك نفس T.2T 29 يT السر دم T 9T 9مقارنة بعينات T لبروتينات الكبد T مقدرات الخلايا الكبديه , حيث اعطت الخلايا الجذعيه المعزوله من الوارتون جلى اعلي نسبه9 طريقه لعزل الخلايا المكونه للدم و اعطاء افضل عدد من T 9T أفضل T 9T 9 هي TEasySep9T 9T 9طريقة T كانت T 9T 9 ومن ناحية أخرى T.2T 29T الحبل السري للفئران كانت انجح من T 9T التي تم حقنها RosetteSep2T 9T 29. كما ان تطعيم الخلايا المكونه للدم المعزوله ب T المكونه للدم T 9T المستعمرات ايضا افضل النتائج كانت بعد الحقن الوريدي للخلايا المدعومه بالخلايا الجذعيه الوسيطه . EasySep خلايا الخلاصه وفقا للنتائج , فأن الوارتون جل يعتبر افضل مصدر للخلايا الجذعيه الوسيطه . في المقابل كانت الخلايا الجذعيه الوسيطه .CD المعزوله من دم الحبل السري قد اعطت افضل ظهور لمستضد ال 10 XX عل ى الرغ م م ن ا ن تقني ة ,(CD هي أفضل تقنية لعزل الخلايا المكونه للدم ( 34 EasySep اثبتت الدراسه ان تقنية قد اعطت افضل النتائج داخل الفئران وفقا لمعدل انتشار الخلايا المكونه للدم و عدد المستعمرات.