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Artur A. Isaev, Alexander V. Prihodko, Sergey L. Kiselev, Maria A. Lagarkova, Ivan V. Potapov, Alexey V. Lundup

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Human Stem Cells Institute, Gemabank, Moscow, Russia

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Cord blood (CB) is a safe and rich source of hematopoietic stem cells (HSC) for oncohematology. The main indicators allowing the assessment of transplant feasibility is a number of nucleated (NC) and CD34+ cells.

A number of HSC is directly related to CB volume, depending on the processing technique and cryopreservation conditions. This study aims to isolate, identify, and quantify NC and CD34+ cells, as well as to evaluate cryopreservation on HSC viability and identify factors affecting HSC numbers in CB.

The average volume of collected CB was 71.48±3.8 ml, NC number was 0.82±0.05x10⁹, viability was 93.47±0.37%. CD34+ and NC numbers corresponded directly to cord blood volume, whereas CB volume is reflected in newborn weight. Cryoconservation has no effect on the number of viable HSC. Immunocytochemical analysis did not reveal any effects of cryoconservation on the number of viable CD34+; although, it might be necessary to apply more accurate methods of analysis. The maximum number of nuclear and CD34+ cells that can be isolated from cord blood volume is 70 ml from a newborn with a weight of 3500 g or more.

Keywords

cord blood, cryoconservation, viability, HSC

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Artur A. Isaev, Alexander V. Prihodko, Sergey L. Kiselev, Maria A. Lagarkova, Ivan V. Potapov, Alexey V. Lundup

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Artur A. Isaev, Alexander V. Prihodko, Sergey L. Kiselev, Maria A. Lagarkova, Ivan V. Potapov, Alexey V. Lundup

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Cord blood (CB) is a safe and rich source of hematopoietic stem cells (HSC) for oncohematology. The main indicators allowing the assessment of transplant feasibility is a number of nucleated (NC) and CD34+ cells.

A number of HSC is directly related to CB volume, depending on the processing technique and cryopreservation conditions. This study aims to isolate, identify, and quantify NC and CD34+ cells, as well as to evaluate cryopreservation on HSC viability and identify factors affecting HSC numbers in CB.

The average volume of collected CB was 71.48±3.8 ml, NC number was 0.82±0.05x10⁹, viability was 93.47±0.37%. CD34+ and NC numbers corresponded directly to cord blood volume, whereas CB volume is reflected in newborn weight. Cryoconservation has no effect on the number of viable HSC. Immunocytochemical analysis did not reveal any effects of cryoconservation on the number of viable CD34+; although, it might be necessary to apply more accurate methods of analysis. The maximum number of nuclear and CD34+ cells that can be isolated from cord blood volume is 70 ml from a newborn with a weight of 3500 g or more.

Keywords

cord blood, cryoconservation, viability, HSC

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Cord blood (CB) is a safe and rich source of hematopoietic stem cells (HSC) for oncohematology. The main indicators allowing the assessment of transplant feasibility is a number of nucleated (NC) and CD34+ cells.

A number of HSC is directly related to CB volume, depending on the processing technique and cryopreservation conditions. This study aims to isolate, identify, and quantify NC and CD34+ cells, as well as to evaluate cryopreservation on HSC viability and identify factors affecting HSC numbers in CB.

The average volume of collected CB was 71.48±3.8 ml, NC number was 0.82±0.05x10⁹, viability was 93.47±0.37%. CD34+ and NC numbers corresponded directly to cord blood volume, whereas CB volume is reflected in newborn weight. Cryoconservation has no effect on the number of viable HSC. Immunocytochemical analysis did not reveal any effects of cryoconservation on the number of viable CD34+; although, it might be necessary to apply more accurate methods of analysis. The maximum number of nuclear and CD34+ cells that can be isolated from cord blood volume is 70 ml from a newborn with a weight of 3500 g or more.

Keywords

cord blood, cryoconservation, viability, HSC

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Human Stem Cells Institute, Gemabank, Moscow, Russia

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Human Stem Cells Institute, Gemabank, Moscow, Russia

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Isaev¹, Alexander V. Prihodko¹, Irina N. Saburina², Sergey L. Kiselev¹, Varvara S. Melihova², Ivan V. Potapov¹, Alexey V. Lundup¹</p>" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(222) "

Artur A. Isaev¹, Alexander V. Prihodko¹, Irina N. Saburina², Sergey L. Kiselev¹, Varvara S. Melihova², Ivan V. Potapov¹, Alexey V. Lundup¹

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¹Human Stem Cells Institute, Moscow, Russia; ²Laboratory of Cell Technology, Moscow, Russia

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Rejection, immunosuppression, and tissue and organ compatibility selection are crucial problems in current transplantology. One promising approach is autologous cell transplantation. Therefore investigators and clinicians at present time focus their attention on autologous cell and tissue banking and their storage.

The umbilical cord is the structure containing mesenchymal cells, e.g. fibroblast-like cells (FLC) and umbilical vein endothelial cells (HUVEC). FLC are known as a source for skin burns and scars treatment, while HUVEC can be used for ischemic tissues revascularization. Therefore, developing new isolation protocols, cell culture expansion, and cell banking methods from the umbilical cord are important issues for immunologically compatible transplants in contemporary clinical use.

Here we present our method of consecutive isolation, cultivation, and cryopreservation of two cell types – HUVEC and FLC – from one umbilical cord sample. Plastic-adherent cell cultures that contained diploid cells with high proliferative activity were observed for 4–6 passages. FLC expressed vimentin, collagen types 1 and 4, alfa-actin, possessed low MHC expression, and did not express CD45, CD11b, or CD14. Cultivated HUVEC demonstrated endotheliocyte-like phenotype, expressed the Von Willebrand factor, and formed tubular structures. Both FLC and HUVEC were cryopreserved for a long period and thawed without a lack of morphofunctional properties.

Thus, the proposed method of consecutive isolation provides the possibility to isolate and preserve two cell types from one umbilical cord sample without changing traditional cord blood banking protocols. These cells can be used for autotransplantation in the treatment of numerous diseases and expand the role of personal cell banking as a “biological insurance.”

Keywords

umbilical cord, fibroblast-like cells, HUVEC, banking

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Artur A. Isaev¹, Alexander V. Prihodko¹, Irina N. Saburina², Sergey L. Kiselev¹, Varvara S. Melihova², Ivan V. Potapov¹, Alexey V. Lundup¹

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Artur A. Isaev¹, Alexander V. Prihodko¹, Irina N. Saburina², Sergey L. Kiselev¹, Varvara S. Melihova², Ivan V. Potapov¹, Alexey V. Lundup¹

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Rejection, immunosuppression, and tissue and organ compatibility selection are crucial problems in current transplantology. One promising approach is autologous cell transplantation. Therefore investigators and clinicians at present time focus their attention on autologous cell and tissue banking and their storage.

The umbilical cord is the structure containing mesenchymal cells, e.g. fibroblast-like cells (FLC) and umbilical vein endothelial cells (HUVEC). FLC are known as a source for skin burns and scars treatment, while HUVEC can be used for ischemic tissues revascularization. Therefore, developing new isolation protocols, cell culture expansion, and cell banking methods from the umbilical cord are important issues for immunologically compatible transplants in contemporary clinical use.

Here we present our method of consecutive isolation, cultivation, and cryopreservation of two cell types – HUVEC and FLC – from one umbilical cord sample. Plastic-adherent cell cultures that contained diploid cells with high proliferative activity were observed for 4–6 passages. FLC expressed vimentin, collagen types 1 and 4, alfa-actin, possessed low MHC expression, and did not express CD45, CD11b, or CD14. Cultivated HUVEC demonstrated endotheliocyte-like phenotype, expressed the Von Willebrand factor, and formed tubular structures. Both FLC and HUVEC were cryopreserved for a long period and thawed without a lack of morphofunctional properties.

Thus, the proposed method of consecutive isolation provides the possibility to isolate and preserve two cell types from one umbilical cord sample without changing traditional cord blood banking protocols. These cells can be used for autotransplantation in the treatment of numerous diseases and expand the role of personal cell banking as a “biological insurance.”

Keywords

umbilical cord, fibroblast-like cells, HUVEC, banking

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Rejection, immunosuppression, and tissue and organ compatibility selection are crucial problems in current transplantology. One promising approach is autologous cell transplantation. Therefore investigators and clinicians at present time focus their attention on autologous cell and tissue banking and their storage.

The umbilical cord is the structure containing mesenchymal cells, e.g. fibroblast-like cells (FLC) and umbilical vein endothelial cells (HUVEC). FLC are known as a source for skin burns and scars treatment, while HUVEC can be used for ischemic tissues revascularization. Therefore, developing new isolation protocols, cell culture expansion, and cell banking methods from the umbilical cord are important issues for immunologically compatible transplants in contemporary clinical use.

Here we present our method of consecutive isolation, cultivation, and cryopreservation of two cell types – HUVEC and FLC – from one umbilical cord sample. Plastic-adherent cell cultures that contained diploid cells with high proliferative activity were observed for 4–6 passages. FLC expressed vimentin, collagen types 1 and 4, alfa-actin, possessed low MHC expression, and did not express CD45, CD11b, or CD14. Cultivated HUVEC demonstrated endotheliocyte-like phenotype, expressed the Von Willebrand factor, and formed tubular structures. Both FLC and HUVEC were cryopreserved for a long period and thawed without a lack of morphofunctional properties.

Thus, the proposed method of consecutive isolation provides the possibility to isolate and preserve two cell types from one umbilical cord sample without changing traditional cord blood banking protocols. These cells can be used for autotransplantation in the treatment of numerous diseases and expand the role of personal cell banking as a “biological insurance.”

Keywords

umbilical cord, fibroblast-like cells, HUVEC, banking

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¹Human Stem Cells Institute, Moscow, Russia; ²Laboratory of Cell Technology, Moscow, Russia

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¹Human Stem Cells Institute, Moscow, Russia; ²Laboratory of Cell Technology, Moscow, Russia

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Yael Margolin, David Snyder, Tony Peled

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Gamida Cell, Ltd., Tel Aviv, Israel

" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(12) "Organization" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } } ["SUMMARY_EN"]=> array(36) { ["ID"]=> string(2) "39" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:02:59" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(21) "Description / Summary" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(10) "SUMMARY_EN" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "39" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "17562" ["VALUE"]=> array(2) { ["TEXT"]=> string(2201) "<p class="bodytext">Cord blood transplantation (CBT) is an acceptable treatment for patients with hematological malignancies who have no matched bone marrow graft. However, low cell dose has limited the use of CBT. </p> <p class="bodytext">To overcome cell dose limitation we have developed a novel epigenetic technology for the ex vivo expansion of HPC based on the finding that cellular copper content modulates self­–renewal and differentiation. The lead molecule of our technology is the copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA’s biological activity was attributed to its effect on overall cellular Cu levels as: (a) a treatment with TEPA resulted in the reduction of cellular Cu followed by the inhibition of HPC differentiation, and (b) the excess Cu reversed TEPA's activity and accelerated differentiation. </p> <p class="bodytext">We applied this technology to develop a product called StemEx. A robust manufacturing process under GMP was developed. CD133 progenitor cells are selected from the small portion of a CBU and cultured for 21 days in media containing TEPA and cytokines. The cultured product is transplanted one day after the transplantation of the larger non-cultured portion containing a minimal safety cell dose of 1x10⁷ cells/kg. The processing of clinical batches demonstrates a robust expansion of progenitor cells, with a mean CD34⁺–fold expansion in culture of 76 (range 9–149). Safety and feasibility of transplanting StemEx were demonstrated in a phase I/II trial. StemEx is now developed in an international pivotal registration study. One hundred patients with hematological malignancies – indicated for BMT with no family related donor match – will be transplanted with StemEx and compared to a historical cohort. The primary endpoint is overall survival at 100 days, with a follow-up of 180 days. </p> <h3>Keywords</h3><p> cord blood transplantation, CBT, hematological malignancies, ex vivo expansion, copper chelator, tetraethylenepentamine, TEPA, progenitor cells, CD34+, CD133 </p>" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(2087) "

Cord blood transplantation (CBT) is an acceptable treatment for patients with hematological malignancies who have no matched bone marrow graft. However, low cell dose has limited the use of CBT.

To overcome cell dose limitation we have developed a novel epigenetic technology for the ex vivo expansion of HPC based on the finding that cellular copper content modulates self­–renewal and differentiation. The lead molecule of our technology is the copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA’s biological activity was attributed to its effect on overall cellular Cu levels as: (a) a treatment with TEPA resulted in the reduction of cellular Cu followed by the inhibition of HPC differentiation, and (b) the excess Cu reversed TEPA's activity and accelerated differentiation.

We applied this technology to develop a product called StemEx. A robust manufacturing process under GMP was developed. CD133 progenitor cells are selected from the small portion of a CBU and cultured for 21 days in media containing TEPA and cytokines. The cultured product is transplanted one day after the transplantation of the larger non-cultured portion containing a minimal safety cell dose of 1x10⁷ cells/kg. The processing of clinical batches demonstrates a robust expansion of progenitor cells, with a mean CD34⁺–fold expansion in culture of 76 (range 9–149). Safety and feasibility of transplanting StemEx were demonstrated in a phase I/II trial. StemEx is now developed in an international pivotal registration study. One hundred patients with hematological malignancies – indicated for BMT with no family related donor match – will be transplanted with StemEx and compared to a historical cohort. The primary endpoint is overall survival at 100 days, with a follow-up of 180 days.

Keywords

cord blood transplantation, CBT, hematological malignancies, ex vivo expansion, copper chelator, tetraethylenepentamine, TEPA, progenitor cells, CD34+, CD133

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Yael Margolin, David Snyder, Tony Peled

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Yael Margolin, David Snyder, Tony Peled

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Cord blood transplantation (CBT) is an acceptable treatment for patients with hematological malignancies who have no matched bone marrow graft. However, low cell dose has limited the use of CBT.

To overcome cell dose limitation we have developed a novel epigenetic technology for the ex vivo expansion of HPC based on the finding that cellular copper content modulates self­–renewal and differentiation. The lead molecule of our technology is the copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA’s biological activity was attributed to its effect on overall cellular Cu levels as: (a) a treatment with TEPA resulted in the reduction of cellular Cu followed by the inhibition of HPC differentiation, and (b) the excess Cu reversed TEPA's activity and accelerated differentiation.

We applied this technology to develop a product called StemEx. A robust manufacturing process under GMP was developed. CD133 progenitor cells are selected from the small portion of a CBU and cultured for 21 days in media containing TEPA and cytokines. The cultured product is transplanted one day after the transplantation of the larger non-cultured portion containing a minimal safety cell dose of 1x10⁷ cells/kg. The processing of clinical batches demonstrates a robust expansion of progenitor cells, with a mean CD34⁺–fold expansion in culture of 76 (range 9–149). Safety and feasibility of transplanting StemEx were demonstrated in a phase I/II trial. StemEx is now developed in an international pivotal registration study. One hundred patients with hematological malignancies – indicated for BMT with no family related donor match – will be transplanted with StemEx and compared to a historical cohort. The primary endpoint is overall survival at 100 days, with a follow-up of 180 days.

Keywords

cord blood transplantation, CBT, hematological malignancies, ex vivo expansion, copper chelator, tetraethylenepentamine, TEPA, progenitor cells, CD34+, CD133

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Cord blood transplantation (CBT) is an acceptable treatment for patients with hematological malignancies who have no matched bone marrow graft. However, low cell dose has limited the use of CBT.

To overcome cell dose limitation we have developed a novel epigenetic technology for the ex vivo expansion of HPC based on the finding that cellular copper content modulates self­–renewal and differentiation. The lead molecule of our technology is the copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA’s biological activity was attributed to its effect on overall cellular Cu levels as: (a) a treatment with TEPA resulted in the reduction of cellular Cu followed by the inhibition of HPC differentiation, and (b) the excess Cu reversed TEPA's activity and accelerated differentiation.

We applied this technology to develop a product called StemEx. A robust manufacturing process under GMP was developed. CD133 progenitor cells are selected from the small portion of a CBU and cultured for 21 days in media containing TEPA and cytokines. The cultured product is transplanted one day after the transplantation of the larger non-cultured portion containing a minimal safety cell dose of 1x10⁷ cells/kg. The processing of clinical batches demonstrates a robust expansion of progenitor cells, with a mean CD34⁺–fold expansion in culture of 76 (range 9–149). Safety and feasibility of transplanting StemEx were demonstrated in a phase I/II trial. StemEx is now developed in an international pivotal registration study. One hundred patients with hematological malignancies – indicated for BMT with no family related donor match – will be transplanted with StemEx and compared to a historical cohort. The primary endpoint is overall survival at 100 days, with a follow-up of 180 days.

Keywords

cord blood transplantation, CBT, hematological malignancies, ex vivo expansion, copper chelator, tetraethylenepentamine, TEPA, progenitor cells, CD34+, CD133

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Gamida Cell, Ltd., Tel Aviv, Israel

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Gamida Cell, Ltd., Tel Aviv, Israel

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Paina, Yulia A. Stankevich, Ilya V. Kazantsev, Natalia V. Stancheva, Alla A. Golovacheva, Elena V. Babenko, Alexander L. Alyanskiy, Natalia E. Ivanova, Elena V. Semenova, Petr V. Krugliakov, Dmitriy G. Polyntsev, Liudmila S. Zubarovskaya, Boris V. Afanasyev</p>" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(274) "

Olesya V. Paina, Yulia A. Stankevich, Ilya V. Kazantsev, Natalia V. Stancheva, Alla A. Golovacheva, Elena V. Babenko, Alexander L. Alyanskiy, Natalia E. Ivanova, Elena V. Semenova, Petr V. Krugliakov, Dmitriy G. Polyntsev, Liudmila S. Zubarovskaya, Boris V. Afanasyev

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Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Correspondence
Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia, Phone: +7 (812) 232-81-20
E-mail: bmt-registry@spmu.rssi.ru

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Background

The usage of haploidentical donors is one curative option for patients (pts) with acute leukemias without a donor – related or unrelated – match.

Patients and methods

Twenty-four very high-risk pts underwent haploidentical SCT: ALL –10 (42%) pts, AML – 11 (44%) pts, JMML – 1pt, CML – 1pt, and resistant neuroblastoma – 1pt. The total number of resistant/in progression pts was 16 (66%), while 8 (33%) pts were in remission. Nineteen (79%) pts were children (aged 1–18), and 5 (21%) were adults (aged 21–47). In each case reduced intensity conditioning regimens (RIC) were used: Flu and ATG with the addition of different alkylating agents (busulphan, melphalan or thiotepa). Sources of HSC-PBSC and bone marrow. For PBSC CD34+ positive selection was performed with CliniMACS. The mean CD34+ count was 12.8x10⁶ /kg (1.6–30.7). In 20 pts aGVHD prophylaxis consisted of CsA and a short course of MTX with or without MMF. In 4 pts tacrolimus and MMF were used. In 2 pts in D-1, mesenchymal stem cells (MSC) from third–party donors were used to prevent aGVHD; while in 3pts, MSC was used for the treatment of aGVHD.

Results

The incidence and severity of aGVHD were no greater than in other types of allo-HSCT: 6 (25%) pts had grade III–IV aGVHD with skin and gut involvement and 1 pt died. In the case of MSC usage in the conditioning regimen only aGVHD stage I was observed. The treatment of aGVHD with MSC was successful: of 3pts, 2 achieved CR. The toxicity of the conditioning regimen was acceptable: 6 (25%) developed grade II–III organ toxicity, 5 (21%) pts had invasive aspergillosis, and 8 (33%) pts had CMV reactivation. The 1-year OS was 62%, with median observation terms of 4.6 months (1 to 12 months). Five pts died of relapse and 3 in CR (1pt from infection and another from engraftment and aGVHD of the gut).

Conclusions

Haploidentical HSCT with RIC is characterized by acceptable toxicity, aGVHD control, and stable engraftment. It proved to be a good option for the group of pts with poor prognosis. Randomized clinical trials are necessary for an estimation of the therapeutic effect of MSCs in haploidentical HSCT pts.

Keywords

haplo-SCT, alternative donor source, salvage therapy

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Olesya V. Paina, Yulia A. Stankevich, Ilya V. Kazantsev, Natalia V. Stancheva, Alla A. Golovacheva, Elena V. Babenko, Alexander L. Alyanskiy, Natalia E. Ivanova, Elena V. Semenova, Petr V. Krugliakov, Dmitriy G. Polyntsev, Liudmila S. Zubarovskaya, Boris V. Afanasyev

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Olesya V. Paina, Yulia A. Stankevich, Ilya V. Kazantsev, Natalia V. Stancheva, Alla A. Golovacheva, Elena V. Babenko, Alexander L. Alyanskiy, Natalia E. Ivanova, Elena V. Semenova, Petr V. Krugliakov, Dmitriy G. Polyntsev, Liudmila S. Zubarovskaya, Boris V. Afanasyev

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Background

The usage of haploidentical donors is one curative option for patients (pts) with acute leukemias without a donor – related or unrelated – match.

Patients and methods

Twenty-four very high-risk pts underwent haploidentical SCT: ALL –10 (42%) pts, AML – 11 (44%) pts, JMML – 1pt, CML – 1pt, and resistant neuroblastoma – 1pt. The total number of resistant/in progression pts was 16 (66%), while 8 (33%) pts were in remission. Nineteen (79%) pts were children (aged 1–18), and 5 (21%) were adults (aged 21–47). In each case reduced intensity conditioning regimens (RIC) were used: Flu and ATG with the addition of different alkylating agents (busulphan, melphalan or thiotepa). Sources of HSC-PBSC and bone marrow. For PBSC CD34+ positive selection was performed with CliniMACS. The mean CD34+ count was 12.8x10⁶ /kg (1.6–30.7). In 20 pts aGVHD prophylaxis consisted of CsA and a short course of MTX with or without MMF. In 4 pts tacrolimus and MMF were used. In 2 pts in D-1, mesenchymal stem cells (MSC) from third–party donors were used to prevent aGVHD; while in 3pts, MSC was used for the treatment of aGVHD.

Results

The incidence and severity of aGVHD were no greater than in other types of allo-HSCT: 6 (25%) pts had grade III–IV aGVHD with skin and gut involvement and 1 pt died. In the case of MSC usage in the conditioning regimen only aGVHD stage I was observed. The treatment of aGVHD with MSC was successful: of 3pts, 2 achieved CR. The toxicity of the conditioning regimen was acceptable: 6 (25%) developed grade II–III organ toxicity, 5 (21%) pts had invasive aspergillosis, and 8 (33%) pts had CMV reactivation. The 1-year OS was 62%, with median observation terms of 4.6 months (1 to 12 months). Five pts died of relapse and 3 in CR (1pt from infection and another from engraftment and aGVHD of the gut).

Conclusions

Haploidentical HSCT with RIC is characterized by acceptable toxicity, aGVHD control, and stable engraftment. It proved to be a good option for the group of pts with poor prognosis. Randomized clinical trials are necessary for an estimation of the therapeutic effect of MSCs in haploidentical HSCT pts.

Keywords

haplo-SCT, alternative donor source, salvage therapy

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Background

The usage of haploidentical donors is one curative option for patients (pts) with acute leukemias without a donor – related or unrelated – match.

Patients and methods

Twenty-four very high-risk pts underwent haploidentical SCT: ALL –10 (42%) pts, AML – 11 (44%) pts, JMML – 1pt, CML – 1pt, and resistant neuroblastoma – 1pt. The total number of resistant/in progression pts was 16 (66%), while 8 (33%) pts were in remission. Nineteen (79%) pts were children (aged 1–18), and 5 (21%) were adults (aged 21–47). In each case reduced intensity conditioning regimens (RIC) were used: Flu and ATG with the addition of different alkylating agents (busulphan, melphalan or thiotepa). Sources of HSC-PBSC and bone marrow. For PBSC CD34+ positive selection was performed with CliniMACS. The mean CD34+ count was 12.8x10⁶ /kg (1.6–30.7). In 20 pts aGVHD prophylaxis consisted of CsA and a short course of MTX with or without MMF. In 4 pts tacrolimus and MMF were used. In 2 pts in D-1, mesenchymal stem cells (MSC) from third–party donors were used to prevent aGVHD; while in 3pts, MSC was used for the treatment of aGVHD.

Results

The incidence and severity of aGVHD were no greater than in other types of allo-HSCT: 6 (25%) pts had grade III–IV aGVHD with skin and gut involvement and 1 pt died. In the case of MSC usage in the conditioning regimen only aGVHD stage I was observed. The treatment of aGVHD with MSC was successful: of 3pts, 2 achieved CR. The toxicity of the conditioning regimen was acceptable: 6 (25%) developed grade II–III organ toxicity, 5 (21%) pts had invasive aspergillosis, and 8 (33%) pts had CMV reactivation. The 1-year OS was 62%, with median observation terms of 4.6 months (1 to 12 months). Five pts died of relapse and 3 in CR (1pt from infection and another from engraftment and aGVHD of the gut).

Conclusions

Haploidentical HSCT with RIC is characterized by acceptable toxicity, aGVHD control, and stable engraftment. It proved to be a good option for the group of pts with poor prognosis. Randomized clinical trials are necessary for an estimation of the therapeutic effect of MSCs in haploidentical HSCT pts.

Keywords

haplo-SCT, alternative donor source, salvage therapy

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Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Correspondence
Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia, Phone: +7 (812) 232-81-20
E-mail: bmt-registry@spmu.rssi.ru

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Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
St. Petersburg, Russia

Correspondence
Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia, Phone: +7 (812) 232-81-20
E-mail: bmt-registry@spmu.rssi.ru

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Subbotina, Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, George L. Mentkevich</p>" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(111) "

Natalia S. Subbotina, Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, George L. Mentkevich

" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(6) "Author" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } } ["ORGANIZATION_EN"]=> array(36) { ["ID"]=> string(2) "38" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:02:59" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(12) "Organization" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(15) "ORGANIZATION_EN" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "38" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "17621" ["VALUE"]=> array(2) { ["TEXT"]=> string(469) "<p class="bodytext">Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia<br /><br /><b>Correspondence</b><br> Natalia N. Subbotina, Institute of Pediatric Oncology and Hematology, 24, Kashirskoye shosse, 115476, Moscow, Russia <br> Phone: +7 (903) 1990666 <br>E-mail: <a href="mailto:irdolg@rambler.ru">irdolg@rambler.ru</a> </p>" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(383) "

Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia

Correspondence
Natalia N. Subbotina, Institute of Pediatric Oncology and Hematology, 24, Kashirskoye shosse, 115476, Moscow, Russia
Phone: +7 (903) 1990666
E-mail: irdolg@rambler.ru

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The main purpose of this study was to evaluate the potential outcomes of haploidentical hematopoietic stem cell transplantation (HSCT) with reduced intensity conditioning regimen (RIC) in the management of pediatric solid and hematological malignancies.

Our protocol, based (mostly) on fludarabine, ATG, and busulfan, was used in 40 pediatric patients with refractory hematological (n=28) or solid (n=12) malignancies. Diagnoses were: AML: 10, ALL: 4, CML: 4, JMML: 5, MDS: 1, NHL: 4, NB: 7, Ewing's S: 4, and melanoma: 1. The median age of patients (pts) was 8.5 yrs (1–18). HLA compatibility was: 3/6:  62.5%, 4/6: 27.5%, and 5/6: 10%. In vitro graft T-depletion with vincristine and methylprednisolone was the only procedure performed.

Three pts with leukemia progression at the time of transplantation didn’t recover and died. Four pts with JMML/MDS rejected and relapsed less than 2 months after transplantation. Thirty-three pts (82.5%) recovered, and achieved full donor chimeras after transplantation. Toxicity was mild in all but one case. Incidence of acute GVHD II–IV in first 100 days was 53%
(gr IV=0). Incidence of chronic GVHD was 52%. Relapse rate 1 yr after transplantation was 75% and 33% for solid tumors and hemoblastoses respectively. In two pts with hemoblastoses the second transplantation was successful. In the entire group the results were as follows: 9 pts (22.5%) now alive, with median follow-up of 41 months (8.1–88.5); 20 (50%) pts died due to relapse; and 11 (27.5%) died of other causes: 2 pts (5%) of acute GVHD, 7pts (17.5%) of chronic GVHD, and 2 pts (5%) of infection.

Haploidentical HSCT after RIC in children can provide long-term anti-leukemia/lymphoma effect without significant complications. Patients with JMML/MDS and solid tumors need additional therapeutic modalities.

Keywords

anti-tumor effect, reduced intensity, haploidentical hematopoietic stem cell transplantation, children

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Subbotina, Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, George L. Mentkevich</p>" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(111) "

Natalia S. Subbotina, Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, George L. Mentkevich

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Natalia S. Subbotina, Igor S. Dolgopolov, Roman I. Pimenov, Vasilii K. Boyarshinov, George L. Mentkevich

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The main purpose of this study was to evaluate the potential outcomes of haploidentical hematopoietic stem cell transplantation (HSCT) with reduced intensity conditioning regimen (RIC) in the management of pediatric solid and hematological malignancies.

Our protocol, based (mostly) on fludarabine, ATG, and busulfan, was used in 40 pediatric patients with refractory hematological (n=28) or solid (n=12) malignancies. Diagnoses were: AML: 10, ALL: 4, CML: 4, JMML: 5, MDS: 1, NHL: 4, NB: 7, Ewing's S: 4, and melanoma: 1. The median age of patients (pts) was 8.5 yrs (1–18). HLA compatibility was: 3/6:  62.5%, 4/6: 27.5%, and 5/6: 10%. In vitro graft T-depletion with vincristine and methylprednisolone was the only procedure performed.

Three pts with leukemia progression at the time of transplantation didn’t recover and died. Four pts with JMML/MDS rejected and relapsed less than 2 months after transplantation. Thirty-three pts (82.5%) recovered, and achieved full donor chimeras after transplantation. Toxicity was mild in all but one case. Incidence of acute GVHD II–IV in first 100 days was 53%
(gr IV=0). Incidence of chronic GVHD was 52%. Relapse rate 1 yr after transplantation was 75% and 33% for solid tumors and hemoblastoses respectively. In two pts with hemoblastoses the second transplantation was successful. In the entire group the results were as follows: 9 pts (22.5%) now alive, with median follow-up of 41 months (8.1–88.5); 20 (50%) pts died due to relapse; and 11 (27.5%) died of other causes: 2 pts (5%) of acute GVHD, 7pts (17.5%) of chronic GVHD, and 2 pts (5%) of infection.

Haploidentical HSCT after RIC in children can provide long-term anti-leukemia/lymphoma effect without significant complications. Patients with JMML/MDS and solid tumors need additional therapeutic modalities.

Keywords

anti-tumor effect, reduced intensity, haploidentical hematopoietic stem cell transplantation, children

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The main purpose of this study was to evaluate the potential outcomes of haploidentical hematopoietic stem cell transplantation (HSCT) with reduced intensity conditioning regimen (RIC) in the management of pediatric solid and hematological malignancies.

Our protocol, based (mostly) on fludarabine, ATG, and busulfan, was used in 40 pediatric patients with refractory hematological (n=28) or solid (n=12) malignancies. Diagnoses were: AML: 10, ALL: 4, CML: 4, JMML: 5, MDS: 1, NHL: 4, NB: 7, Ewing's S: 4, and melanoma: 1. The median age of patients (pts) was 8.5 yrs (1–18). HLA compatibility was: 3/6:  62.5%, 4/6: 27.5%, and 5/6: 10%. In vitro graft T-depletion with vincristine and methylprednisolone was the only procedure performed.

Three pts with leukemia progression at the time of transplantation didn’t recover and died. Four pts with JMML/MDS rejected and relapsed less than 2 months after transplantation. Thirty-three pts (82.5%) recovered, and achieved full donor chimeras after transplantation. Toxicity was mild in all but one case. Incidence of acute GVHD II–IV in first 100 days was 53%
(gr IV=0). Incidence of chronic GVHD was 52%. Relapse rate 1 yr after transplantation was 75% and 33% for solid tumors and hemoblastoses respectively. In two pts with hemoblastoses the second transplantation was successful. In the entire group the results were as follows: 9 pts (22.5%) now alive, with median follow-up of 41 months (8.1–88.5); 20 (50%) pts died due to relapse; and 11 (27.5%) died of other causes: 2 pts (5%) of acute GVHD, 7pts (17.5%) of chronic GVHD, and 2 pts (5%) of infection.

Haploidentical HSCT after RIC in children can provide long-term anti-leukemia/lymphoma effect without significant complications. Patients with JMML/MDS and solid tumors need additional therapeutic modalities.

Keywords

anti-tumor effect, reduced intensity, haploidentical hematopoietic stem cell transplantation, children

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Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia

Correspondence
Natalia N. Subbotina, Institute of Pediatric Oncology and Hematology, 24, Kashirskoye shosse, 115476, Moscow, Russia
Phone: +7 (903) 1990666
E-mail: irdolg@rambler.ru

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Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia

Correspondence
Natalia N. Subbotina, Institute of Pediatric Oncology and Hematology, 24, Kashirskoye shosse, 115476, Moscow, Russia
Phone: +7 (903) 1990666
E-mail: irdolg@rambler.ru

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array(2) { ["TEXT"]=> string(109) "<p>Vladimir S. Tolmachev, Elizaveta I. Borovitskaya, Natalia O. Kudrina, Tatiana G. Kravtsova</p>" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(97) "

Vladimir S. Tolmachev, Elizaveta I. Borovitskaya, Natalia O. Kudrina, Tatiana G. Kravtsova

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Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan

Correspondence
Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan
E-mail: lab_cyto_kz@mail.ru

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We conducted a cytochemical analysis of the structure and function of chromatin found in cord blood mononuclear cells (MNC). In the cellular cycle, 97.9% MNC were in stage G₀-G₁, 1.1% in the phase of synthesis, 0.9% in G₂-M, and proliferation 1.9%. The expression of receptors CD 34⁺ and CD 45⁺ was changed by the cultivation of MNC: chromatin was destabilized according to the data of histone and non-histone proteins. The growth factors were approved in the culture of MNC. The level of MNC with apoptosis in an unfrozen sample increased. The changes of the supramolecular structure of MNC chromatin were displayed in acridine orange fluorochromes and rivanol SO₂ with DNA phosphate groups binding diminishing, and in an increase of gistons primulin and durable green coloration. The condensation of MNC chromatin in cord blood was twice as high as in peripheral blood lymphocytes. The reduced ability of MNC chromatin to bind to an ethidium bromide and the acridine orange was saved through deproteinization, before affecting 0.45 M NaCl on cells. The methods of erythrocytes’ predecessors receipt was studied from the culture of cord and peripheral blood MNC for the treatment of newborns with low weight, anemia, and hypoxia. The bases of fetal hypoxia diagnostics and prognosis of the early postnatal period were developed by specific markers of apoptosis: complex DNA-Ca2⁺ and DNA fragmentation by lymphocytes of the mother.

Keywords

mononuclear cells, cord blood, cellular cycle, apoptosis, ethidium bromide, acridine orange, complex DNA-Ca2+, DNA fragmentation

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Vladimir S. Tolmachev, Elizaveta I. Borovitskaya, Natalia O. Kudrina, Tatiana G. Kravtsova

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Vladimir S. Tolmachev, Elizaveta I. Borovitskaya, Natalia O. Kudrina, Tatiana G. Kravtsova

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We conducted a cytochemical analysis of the structure and function of chromatin found in cord blood mononuclear cells (MNC). In the cellular cycle, 97.9% MNC were in stage G₀-G₁, 1.1% in the phase of synthesis, 0.9% in G₂-M, and proliferation 1.9%. The expression of receptors CD 34⁺ and CD 45⁺ was changed by the cultivation of MNC: chromatin was destabilized according to the data of histone and non-histone proteins. The growth factors were approved in the culture of MNC. The level of MNC with apoptosis in an unfrozen sample increased. The changes of the supramolecular structure of MNC chromatin were displayed in acridine orange fluorochromes and rivanol SO₂ with DNA phosphate groups binding diminishing, and in an increase of gistons primulin and durable green coloration. The condensation of MNC chromatin in cord blood was twice as high as in peripheral blood lymphocytes. The reduced ability of MNC chromatin to bind to an ethidium bromide and the acridine orange was saved through deproteinization, before affecting 0.45 M NaCl on cells. The methods of erythrocytes’ predecessors receipt was studied from the culture of cord and peripheral blood MNC for the treatment of newborns with low weight, anemia, and hypoxia. The bases of fetal hypoxia diagnostics and prognosis of the early postnatal period were developed by specific markers of apoptosis: complex DNA-Ca2⁺ and DNA fragmentation by lymphocytes of the mother.

Keywords

mononuclear cells, cord blood, cellular cycle, apoptosis, ethidium bromide, acridine orange, complex DNA-Ca2+, DNA fragmentation

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We conducted a cytochemical analysis of the structure and function of chromatin found in cord blood mononuclear cells (MNC). In the cellular cycle, 97.9% MNC were in stage G₀-G₁, 1.1% in the phase of synthesis, 0.9% in G₂-M, and proliferation 1.9%. The expression of receptors CD 34⁺ and CD 45⁺ was changed by the cultivation of MNC: chromatin was destabilized according to the data of histone and non-histone proteins. The growth factors were approved in the culture of MNC. The level of MNC with apoptosis in an unfrozen sample increased. The changes of the supramolecular structure of MNC chromatin were displayed in acridine orange fluorochromes and rivanol SO₂ with DNA phosphate groups binding diminishing, and in an increase of gistons primulin and durable green coloration. The condensation of MNC chromatin in cord blood was twice as high as in peripheral blood lymphocytes. The reduced ability of MNC chromatin to bind to an ethidium bromide and the acridine orange was saved through deproteinization, before affecting 0.45 M NaCl on cells. The methods of erythrocytes’ predecessors receipt was studied from the culture of cord and peripheral blood MNC for the treatment of newborns with low weight, anemia, and hypoxia. The bases of fetal hypoxia diagnostics and prognosis of the early postnatal period were developed by specific markers of apoptosis: complex DNA-Ca2⁺ and DNA fragmentation by lymphocytes of the mother.

Keywords

mononuclear cells, cord blood, cellular cycle, apoptosis, ethidium bromide, acridine orange, complex DNA-Ca2+, DNA fragmentation

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Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan

Correspondence
Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan
E-mail: lab_cyto_kz@mail.ru

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Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan

Correspondence
Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan
E-mail: lab_cyto_kz@mail.ru

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Artur A. Isaev, Alexander V. Prihodko, Sergey L. Kiselev, Maria A. Lagarkova, Ivan V. Potapov, Alexey V. Lundup
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Human Stem Cells Institute, Gemabank, Moscow, Russia
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                    [TEXT] => <p class="bodytext"> Cord blood (CB) is a safe and rich source of hematopoietic stem cells (HSC) for oncohematology. The main indicators allowing the assessment of transplant feasibility is a number of nucleated (NC) and CD34+ cells.<br /><br />A number of HSC is directly related to CB volume, depending on the processing technique and cryopreservation conditions. This study aims to isolate, identify, and quantify NC and CD34+ cells, as well as to evaluate cryopreservation on HSC viability and identify factors affecting HSC numbers in CB.<br /><br />The average volume of collected CB was 71.48±3.8 ml, NC number was 0.82±0.05x10<sup>9</sup>, viability was 93.47±0.37%. CD34+ and NC numbers corresponded directly to cord blood volume, whereas CB volume is reflected in newborn weight. Cryoconservation has no effect on the number of viable HSC. Immunocytochemical analysis did not reveal any effects of cryoconservation on the number of viable CD34+; although, it might be necessary to apply more accurate methods of analysis. The maximum number of nuclear and CD34+ cells that can be isolated from cord blood volume is 70 ml from a newborn with a weight of 3500 g or more. </p>
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 Cord blood (CB) is a safe and rich source of hematopoietic stem cells (HSC) for oncohematology. The main indicators allowing the assessment of transplant feasibility is a number of nucleated (NC) and CD34+ cells.

A number of HSC is directly related to CB volume, depending on the processing technique and cryopreservation conditions. This study aims to isolate, identify, and quantify NC and CD34+ cells, as well as to evaluate cryopreservation on HSC viability and identify factors affecting HSC numbers in CB.

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Keywords
 cord blood, cryoconservation, viability, HSC
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Artur A. Isaev1, Alexander V. Prihodko1, Irina N. Saburina2, Sergey L. Kiselev1, Varvara S. Melihova2, Ivan V. Potapov1, Alexey V. Lundup1
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1Human Stem Cells Institute, Moscow, Russia; 2Laboratory of Cell Technology, Moscow, Russia
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                    [TEXT] => <p class="bodytext"> Rejection, immunosuppression, and tissue and organ compatibility selection are crucial problems in current transplantology. One promising approach is autologous cell transplantation. Therefore investigators and clinicians at present time focus their attention on autologous cell and tissue banking and their storage.<br /><br />The umbilical cord is the structure containing mesenchymal cells, e.g. fibroblast-like cells (FLC) and umbilical vein endothelial cells (HUVEC). FLC are known as a source for skin burns and scars treatment, while HUVEC can be used for ischemic tissues revascularization. Therefore, developing new isolation protocols, cell culture expansion, and cell banking methods from the umbilical cord are important issues for immunologically compatible transplants in contemporary clinical use.<br /><br />Here we present our method of consecutive isolation, cultivation, and cryopreservation of two cell types – HUVEC and FLC – from one umbilical cord sample. Plastic-adherent cell cultures that contained diploid cells with high proliferative activity were observed for 4–6 passages. FLC expressed vimentin, collagen types 1 and 4, alfa-actin, possessed low MHC expression, and did not express CD45, CD11b, or CD14. Cultivated HUVEC demonstrated endotheliocyte-like phenotype, expressed the Von Willebrand factor, and formed tubular structures. Both FLC and HUVEC were cryopreserved for a long period and thawed without a lack of morphofunctional properties. <br /><br />Thus, the proposed method of consecutive isolation provides the possibility to isolate and preserve two cell types from one umbilical cord sample without changing traditional cord blood banking protocols. These cells can be used for autotransplantation in the treatment of numerous diseases and expand the role of personal cell banking as a “biological insurance.”</p>
<h3>Keywords</h3><p> umbilical cord, fibroblast-like cells, HUVEC, banking</p>
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 Rejection, immunosuppression, and tissue and organ compatibility selection are crucial problems in current transplantology. One promising approach is autologous cell transplantation. Therefore investigators and clinicians at present time focus their attention on autologous cell and tissue banking and their storage.

The umbilical cord is the structure containing mesenchymal cells, e.g. fibroblast-like cells (FLC) and umbilical vein endothelial cells (HUVEC). FLC are known as a source for skin burns and scars treatment, while HUVEC can be used for ischemic tissues revascularization. Therefore, developing new isolation protocols, cell culture expansion, and cell banking methods from the umbilical cord are important issues for immunologically compatible transplants in contemporary clinical use.

Here we present our method of consecutive isolation, cultivation, and cryopreservation of two cell types – HUVEC and FLC – from one umbilical cord sample. Plastic-adherent cell cultures that contained diploid cells with high proliferative activity were observed for 4–6 passages. FLC expressed vimentin, collagen types 1 and 4, alfa-actin, possessed low MHC expression, and did not express CD45, CD11b, or CD14. Cultivated HUVEC demonstrated endotheliocyte-like phenotype, expressed the Von Willebrand factor, and formed tubular structures. Both FLC and HUVEC were cryopreserved for a long period and thawed without a lack of morphofunctional properties. 

Thus, the proposed method of consecutive isolation provides the possibility to isolate and preserve two cell types from one umbilical cord sample without changing traditional cord blood banking protocols. These cells can be used for autotransplantation in the treatment of numerous diseases and expand the role of personal cell banking as a “biological insurance.”
Keywords
 umbilical cord, fibroblast-like cells, HUVEC, banking
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</p>
<p class="bodytext">To overcome cell dose limitation we have developed a novel epigenetic technology for the ex vivo expansion of HPC based on the finding that cellular copper content modulates self­–renewal and differentiation. The lead molecule of our technology is the copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA’s biological activity was attributed to its effect on overall cellular Cu levels as: (a) a treatment with TEPA resulted in the reduction of cellular Cu followed by the inhibition of HPC differentiation, and (b) the excess Cu reversed TEPA's activity and accelerated differentiation.
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<p class="bodytext">We applied this technology to develop a product called StemEx. A robust manufacturing process under GMP was developed. CD133 progenitor cells are selected from the small portion of a CBU and cultured for 21 days in media containing TEPA and cytokines. The cultured product is transplanted one day after the transplantation of the larger non-cultured portion containing a minimal safety cell dose of 1x10<sup>7</sup> cells/kg. The processing of clinical batches demonstrates a robust expansion of progenitor cells, with a mean CD34<sup>+</sup>–fold expansion in culture of 76 (range 9–149).  Safety and feasibility of transplanting StemEx were demonstrated in a phase I/II trial. StemEx is now developed in an international pivotal registration study.  One hundred patients with hematological malignancies – indicated for BMT with no family related donor match – will be transplanted with StemEx and compared to a historical cohort. The primary endpoint is overall survival at 100 days, with a follow-up of 180 days.  
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<h3>Keywords</h3><p> cord blood transplantation, CBT, hematological malignancies, ex vivo expansion, copper chelator, tetraethylenepentamine, TEPA, progenitor cells, CD34+, CD133 
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Cord blood transplantation (CBT) is an acceptable treatment for patients with hematological malignancies who have no matched bone marrow graft. However, low cell dose has limited the use of CBT.   

To overcome cell dose limitation we have developed a novel epigenetic technology for the ex vivo expansion of HPC based on the finding that cellular copper content modulates self­–renewal and differentiation. The lead molecule of our technology is the copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA’s biological activity was attributed to its effect on overall cellular Cu levels as: (a) a treatment with TEPA resulted in the reduction of cellular Cu followed by the inhibition of HPC differentiation, and (b) the excess Cu reversed TEPA's activity and accelerated differentiation.

We applied this technology to develop a product called StemEx. A robust manufacturing process under GMP was developed. CD133 progenitor cells are selected from the small portion of a CBU and cultured for 21 days in media containing TEPA and cytokines. The cultured product is transplanted one day after the transplantation of the larger non-cultured portion containing a minimal safety cell dose of 1x107 cells/kg. The processing of clinical batches demonstrates a robust expansion of progenitor cells, with a mean CD34+–fold expansion in culture of 76 (range 9–149).  Safety and feasibility of transplanting StemEx were demonstrated in a phase I/II trial. StemEx is now developed in an international pivotal registration study.  One hundred patients with hematological malignancies – indicated for BMT with no family related donor match – will be transplanted with StemEx and compared to a historical cohort. The primary endpoint is overall survival at 100 days, with a follow-up of 180 days.  

Keywords
 cord blood transplantation, CBT, hematological malignancies, ex vivo expansion, copper chelator, tetraethylenepentamine, TEPA, progenitor cells, CD34+, CD133 

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Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University,
 St. Petersburg, Russia

Correspondence
 Memorial R. M. Gorbacheva Institute of Children Hematology and Transplantation, St. Petersburg Pavlov State Medical University, 6/8, Tolstoy str., St. Petersburg, 199044, Russia, Phone: +7 (812) 232-81-20

 E-mail: bmt-registry@spmu.rssi.ru  

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                    [TEXT] => <h3>Background</h3>
<p> The usage of haploidentical donors is one curative option for patients (pts) with acute leukemias without a donor – related or unrelated – match. </p>
<h3>Patients and methods</h3>
<p> Twenty-four very high-risk pts underwent haploidentical SCT: ALL –10 (42%) pts, AML – 11 (44%) pts, JMML – 1pt, CML – 1pt, and resistant neuroblastoma – 1pt. The total number of resistant/in progression pts was 16 (66%), while 8 (33%) pts were in remission. Nineteen (79%) pts were children (aged 1–18), and 5 (21%) were adults (aged 21–47). In each case reduced intensity conditioning regimens (RIC) were used: Flu and ATG with the addition of different alkylating agents (busulphan, melphalan or thiotepa). Sources of HSC-PBSC and bone marrow. For PBSC CD34+ positive selection was performed with CliniMACS. The mean CD34+ count was 12.8x10<sup>6</sup> /kg (1.6–30.7). In 20 pts aGVHD prophylaxis consisted of CsA and a short course of MTX with or without MMF. In 4 pts tacrolimus and MMF were used. In 2 pts in D-1, mesenchymal stem cells (MSC) from third–party donors were used to prevent aGVHD; while in 3pts, MSC was used for the treatment of aGVHD. </p>
<h3>Results</h3>
<p> The incidence and severity of aGVHD were no greater than in other types of allo-HSCT: 6 (25%) pts had grade III–IV aGVHD with skin and gut involvement and 1 pt died. In the case of MSC usage in the conditioning regimen only aGVHD stage I was observed. The treatment of aGVHD with MSC was successful: of 3pts, 2 achieved CR. The toxicity of the conditioning regimen was acceptable: 6 (25%) developed grade II–III organ toxicity, 5 (21%) pts had invasive aspergillosis, and 8 (33%) pts had CMV reactivation. The 1-year OS was 62%, with median observation terms of 4.6 months (1 to 12 months). Five pts died of relapse and 3 in CR (1pt from infection and another from engraftment and aGVHD of the gut). </p>
<h3>Conclusions</h3>
<p> Haploidentical HSCT with RIC is characterized by acceptable toxicity, aGVHD control, and stable engraftment. It proved to be a good option for the group of pts with poor prognosis. Randomized clinical trials are necessary for an estimation of the therapeutic effect of MSCs in haploidentical HSCT pts.</p>
<h3>Keywords</h3><p> haplo-SCT, alternative donor source, salvage therapy</p>
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Background
 The usage of haploidentical donors is one curative option for patients (pts) with acute leukemias without a donor – related or unrelated – match. 
Patients and methods
 Twenty-four very high-risk pts underwent haploidentical SCT: ALL –10 (42%) pts, AML – 11 (44%) pts, JMML – 1pt, CML – 1pt, and resistant neuroblastoma – 1pt. The total number of resistant/in progression pts was 16 (66%), while 8 (33%) pts were in remission. Nineteen (79%) pts were children (aged 1–18), and 5 (21%) were adults (aged 21–47). In each case reduced intensity conditioning regimens (RIC) were used: Flu and ATG with the addition of different alkylating agents (busulphan, melphalan or thiotepa). Sources of HSC-PBSC and bone marrow. For PBSC CD34+ positive selection was performed with CliniMACS. The mean CD34+ count was 12.8x106 /kg (1.6–30.7). In 20 pts aGVHD prophylaxis consisted of CsA and a short course of MTX with or without MMF. In 4 pts tacrolimus and MMF were used. In 2 pts in D-1, mesenchymal stem cells (MSC) from third–party donors were used to prevent aGVHD; while in 3pts, MSC was used for the treatment of aGVHD. 
Results
 The incidence and severity of aGVHD were no greater than in other types of allo-HSCT: 6 (25%) pts had grade III–IV aGVHD with skin and gut involvement and 1 pt died. In the case of MSC usage in the conditioning regimen only aGVHD stage I was observed. The treatment of aGVHD with MSC was successful: of 3pts, 2 achieved CR. The toxicity of the conditioning regimen was acceptable: 6 (25%) developed grade II–III organ toxicity, 5 (21%) pts had invasive aspergillosis, and 8 (33%) pts had CMV reactivation. The 1-year OS was 62%, with median observation terms of 4.6 months (1 to 12 months). Five pts died of relapse and 3 in CR (1pt from infection and another from engraftment and aGVHD of the gut). 
Conclusions
 Haploidentical HSCT with RIC is characterized by acceptable toxicity, aGVHD control, and stable engraftment. It proved to be a good option for the group of pts with poor prognosis. Randomized clinical trials are necessary for an estimation of the therapeutic effect of MSCs in haploidentical HSCT pts.
Keywords
 haplo-SCT, alternative donor source, salvage therapy
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                    [TEXT] => <p class="bodytext">Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia<br /><br /><b>Correspondence</b><br> Natalia N. Subbotina, Institute of Pediatric Oncology and Hematology, 24, Kashirskoye shosse, 115476, Moscow, Russia
<br> Phone: +7 (903) 1990666
<br>E-mail: <a href="mailto:irdolg@rambler.ru">irdolg@rambler.ru</a> 
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Institute of Pediatric Oncology and Hematology, Bone Marrow Transplantation Department, Moscow, Russia

Correspondence
 Natalia N. Subbotina, Institute of Pediatric Oncology and Hematology, 24, Kashirskoye shosse, 115476, Moscow, Russia

 Phone: +7 (903) 1990666

E-mail: irdolg@rambler.ru 

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                    [TEXT] => <p class="bodytext">The main purpose of this study was to evaluate the potential outcomes of haploidentical hematopoietic stem cell transplantation (HSCT) with reduced intensity conditioning regimen (RIC) in the management of pediatric solid and hematological malignancies. <br /><br />Our protocol, based (mostly) on fludarabine, ATG, and busulfan, was used in 40 pediatric patients with refractory hematological (n=28) or solid (n=12) malignancies. Diagnoses were: AML: 10, ALL: 4, CML: 4, JMML: 5, MDS: 1, NHL: 4, NB: 7, Ewing's S: 4, and melanoma: 1. The median age of patients (pts) was 8.5 yrs (1–18). HLA compatibility was: 3/6:  62.5%, 4/6: 27.5%, and 5/6: 10%. In vitro graft T-depletion with vincristine and methylprednisolone was the only procedure performed. <br /><br />Three pts with leukemia progression at the time of transplantation didn’t recover and died. Four pts with JMML/MDS rejected and relapsed less than 2 months after transplantation. Thirty-three pts (82.5%) recovered, and achieved full donor chimeras after transplantation. Toxicity was mild in all but one case. Incidence of acute GVHD II–IV in first 100 days was 53% <br>(gr IV=0). Incidence of chronic GVHD was 52%. Relapse rate 1 yr after transplantation was 75% and 33% for solid tumors and hemoblastoses respectively. In two pts with hemoblastoses the second transplantation was successful. In the entire group the results were as follows: 9 pts (22.5%) now alive, with median follow-up of 41 months (8.1–88.5); 20 (50%) pts died due to relapse; and 11 (27.5%) died of other causes: 2 pts (5%) of acute GVHD, 7pts (17.5%) of chronic GVHD, and 2 pts (5%) of infection. <br /><br />Haploidentical HSCT after RIC in children can provide long-term anti-leukemia/lymphoma effect without significant complications. Patients with JMML/MDS and solid tumors need additional therapeutic modalities.
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<h3>Keywords</h3><p> anti-tumor effect, reduced intensity, haploidentical hematopoietic stem cell transplantation, children
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The main purpose of this study was to evaluate the potential outcomes of haploidentical hematopoietic stem cell transplantation (HSCT) with reduced intensity conditioning regimen (RIC) in the management of pediatric solid and hematological malignancies. 

Our protocol, based (mostly) on fludarabine, ATG, and busulfan, was used in 40 pediatric patients with refractory hematological (n=28) or solid (n=12) malignancies. Diagnoses were: AML: 10, ALL: 4, CML: 4, JMML: 5, MDS: 1, NHL: 4, NB: 7, Ewing's S: 4, and melanoma: 1. The median age of patients (pts) was 8.5 yrs (1–18). HLA compatibility was: 3/6:  62.5%, 4/6: 27.5%, and 5/6: 10%. In vitro graft T-depletion with vincristine and methylprednisolone was the only procedure performed. 

Three pts with leukemia progression at the time of transplantation didn’t recover and died. Four pts with JMML/MDS rejected and relapsed less than 2 months after transplantation. Thirty-three pts (82.5%) recovered, and achieved full donor chimeras after transplantation. Toxicity was mild in all but one case. Incidence of acute GVHD II–IV in first 100 days was 53% 
(gr IV=0). Incidence of chronic GVHD was 52%. Relapse rate 1 yr after transplantation was 75% and 33% for solid tumors and hemoblastoses respectively. In two pts with hemoblastoses the second transplantation was successful. In the entire group the results were as follows: 9 pts (22.5%) now alive, with median follow-up of 41 months (8.1–88.5); 20 (50%) pts died due to relapse; and 11 (27.5%) died of other causes: 2 pts (5%) of acute GVHD, 7pts (17.5%) of chronic GVHD, and 2 pts (5%) of infection. 

Haploidentical HSCT after RIC in children can provide long-term anti-leukemia/lymphoma effect without significant complications. Patients with JMML/MDS and solid tumors need additional therapeutic modalities.

Keywords
 anti-tumor effect, reduced intensity, haploidentical hematopoietic stem cell transplantation, children

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                    [TEXT] => <p class="bodytext">Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan<br /><br /><b>Correspondence</b><br> Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan
<br> E-mail: <a href="mailto:lab_cyto_kz@mail.ru">lab_cyto_kz@mail.ru</a> 
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Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan

Correspondence
 Scientific Center of Obstetrics, Gynecology, and Perinatology, Almaty, MH Kazakhstan

 E-mail: lab_cyto_kz@mail.ru 

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                    [TEXT] => <p class="bodytext">We conducted a cytochemical analysis of the structure and function of chromatin found in cord blood mononuclear cells (MNC). In the cellular cycle, 97.9% MNC were in stage G<sub>0</sub>-G<sub>1</sub>, 1.1% in the phase of synthesis, 0.9% in G<sub>2</sub>-M, and proliferation 1.9%. The expression of receptors CD 34<sup>+</sup> and CD 45<sup>+</sup> was changed by the cultivation of MNC: chromatin was destabilized according to the data of histone and non-histone proteins. The growth factors were approved in the culture of MNC. The level of MNC with apoptosis in an unfrozen sample increased. The changes of the supramolecular structure of MNC chromatin were displayed in acridine orange fluorochromes and rivanol SO<sub>2</sub> with DNA phosphate groups binding diminishing, and in an increase of gistons primulin and durable green coloration. The condensation of MNC chromatin in cord blood was twice as high as in peripheral blood lymphocytes. The reduced ability of MNC chromatin to bind to an ethidium bromide and the acridine orange was saved through deproteinization, before affecting 0.45 M NaCl on cells. The methods of erythrocytes’ predecessors receipt was studied from the culture of cord and peripheral blood MNC for the treatment of newborns with low weight, anemia, and hypoxia. The bases of fetal hypoxia diagnostics and prognosis of the early postnatal period were developed by specific markers of apoptosis: complex DNA-Ca2<sup>+</sup> and DNA fragmentation by lymphocytes of the mother.</p>
<h3>Keywords</h3><p> mononuclear cells, cord blood, cellular cycle, apoptosis, ethidium bromide, acridine orange, complex DNA-Ca2+, DNA fragmentation</p>
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We conducted a cytochemical analysis of the structure and function of chromatin found in cord blood mononuclear cells (MNC). In the cellular cycle, 97.9% MNC were in stage G0-G1, 1.1% in the phase of synthesis, 0.9% in G2-M, and proliferation 1.9%. The expression of receptors CD 34+ and CD 45+ was changed by the cultivation of MNC: chromatin was destabilized according to the data of histone and non-histone proteins. The growth factors were approved in the culture of MNC. The level of MNC with apoptosis in an unfrozen sample increased. The changes of the supramolecular structure of MNC chromatin were displayed in acridine orange fluorochromes and rivanol SO2 with DNA phosphate groups binding diminishing, and in an increase of gistons primulin and durable green coloration. The condensation of MNC chromatin in cord blood was twice as high as in peripheral blood lymphocytes. The reduced ability of MNC chromatin to bind to an ethidium bromide and the acridine orange was saved through deproteinization, before affecting 0.45 M NaCl on cells. The methods of erythrocytes’ predecessors receipt was studied from the culture of cord and peripheral blood MNC for the treatment of newborns with low weight, anemia, and hypoxia. The bases of fetal hypoxia diagnostics and prognosis of the early postnatal period were developed by specific markers of apoptosis: complex DNA-Ca2+ and DNA fragmentation by lymphocytes of the mother.
Keywords
 mononuclear cells, cord blood, cellular cycle, apoptosis, ethidium bromide, acridine orange, complex DNA-Ca2+, DNA fragmentation
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