Daniela Piroth

Synergistic effects of growth factors and mesenchymal stromal cells for expansion of hematopoietic stem and progenitor cells

OBJECTIVE The number of hematopoietic stem and progenitor cells (HPCs) per cord blood unit is limited, and this can result in delayed engraftment or graft failure. In vitro expansion of HPCs provides a perspective to overcome these limitations. Cytokines as well as mesenchymal stromal cells (MSCs) have been shown to support HPCs ex vivo expansion, but a systematic analysis of their interplay remains elusive. MATERIALS AND METHODS Twenty different combinations of growth factors (stem cell factor [SCF], thrombopoietin [TPO], fibroblast growth factor-1 [FGF-1], angiopoietin-like 5, and insulin-like growth factor-binding protein 2), either with or without MSC coculture were systematically compared for their ability to support HPC expansion. CD34(+) cells were stained with carboxyfluorescein diacetate N-succinimidyl ester to monitor cell division history in conjunction with immunophenotype. Colony-forming unit frequencies and hematopoietic reconstitution of nonobese diabetic severe combined immunodeficient mice were also assessed. RESULTS Proliferation of HPCs was stimulated by coculture with MSCs. This was further enhanced in combination with SCF, TPO, and FGF-1. Moreover, these conditions maintained expression of primitive surface markers for more than four cell divisions. Colony-forming unit-initiating cells were not expanded without stromal support, whereas an eightfold increase was reached by simultaneous cytokine-treatment and MSC coculture. Importantly, in comparison to expansion without stromal support, coculture with MSCs significantly enhanced hematopoietic chimerism in a murine transplantation model. CONCLUSIONS The supportive effect of MSCs on hematopoiesis can be significantly increased by addition of specific recombinant growth factors; especially in combination with SCF, TPO, and FGF-1.

Maternal and fetal cord blood lipids in intrauterine growth restriction.

Abstract Aim: Small for gestational age neonates (SGA) could be subdivided into two groups according to the underlying causes leading to low birth weight. Intrauterine growth restriction (IUGR) is a pathologic condition with diminished growth velocity and fetal compromised well-being, while non-growth restricted SGA neonates are constitutionally (genetically determined) small. Antenatal sonographic measurements are used to differentiate these two subgroups. Maternal metabolic changes contribute to the pathogenesis of IUGR. A disturbed lipid metabolism and cholesterol supply might affect the fetus, with consequences for fetal programming of cardiovascular diseases. We evaluated fetal serum lipids and hypothesized a more atherogenic lipoprotein profile in IUGR fetuses. Methods: Umbilical cord serum lipids and oxidative modified, low-density lipoprotein (oxLDL) concentrations were measured by colorimetric enzymatic measurements, or by ELISA. Values of IUGR (n=36) and constitutionally small for gestational age neonates (SGA, n=22) were compared with those of healthy, adequate for gestational age, born neonates (CN, n=97). SAS-statistic software was used and two-way ANOVA was adjusted for gestational age at delivery. Results: Fetal high-density lipoprotein cholesterol (HDL-C) and total cholesterol (TC) concentrations were found to be lower in the IUGR compared to the CN and SGA groups (HDL-C: P<0.001, TC: P<0.01). Atherogenic indices, including the oxLDL/LDL-C ratio, were increased in the IUGR compared to the CN group (oxLDL/LDL-C ratio: P<0.001). Conclusion: Our results support the hypothesis of a disturbed cholesterol supply in IUGR fetuses. Born SGA has been shown to be a risk factor for developing cardiovascular disease later in life. Since HDL-C has anti-inflammatory properties, a reduced HDL-C during fetal development, and an increase in atherogenic indices, might provide a link to this observation in IUGR fetuses.

The Evaluation of the Oxidative State of Low-Density Lipoproteins in Intrauterine Growth Restriction and Preeclampsia

Objective. To evaluate the oxidative state of lipoproteins in pregnancies complicated by intrauterine growth restriction (IUGR) in comparison to preeclampsia (PE) and healthy pregnant control subjects (CN). Methods. Maternal serum of 20 PE, 29 IUGR, and 29 gestational age-matched CN were analyzed. Total cholesterol (TC), low-density lipoprotein (LDL)-bound cholesterol (LDL-C), and oxidized LDL (oxLDL) concentration were measured once between 25 and 34 weeks of gestation. Statistical estimates were performed by Students t-test. Results. Serum concentrations of LDL-C and TC were significantly reduced in IUGR [LDL-C: CN – mean = 146 mg/dL, SD = ± 40.1; IUGR – mean = 102 mg/dL, SD = ± 27.3 (p < 0.0001); PE – mean = 130 mg/dL, SD = 38.8 mg/dL; TC: CN – mean = 259/dL, SD = ± 46.8; IUGR – mean = 218 mg/dL, SD = ± 35.0 (p < 0.001); PE – mean = 244 mg/dL, SD = 48.2]. There was no significant difference in oxLDL/LDL-C ratio within the three groups (CN: mean = 0.76, SD = 0.24; IUGR: mean = 0.74, SD = 0.12; PE: mean = 0.77, SD = 0.22). Conclusion. Our results show a lower maternal LDL-C and TC concentration in IUGR pregnancies. These data contribute to the hypothesis of a decreased cholesterol supply to the fetus in IUGR. However, we could not confirm the hypothesis of an altered oxidative state in neither IUGR nor PE.

Immediate reconstruction with an expander/implant following ablatio mammae because of breast cancer : side effects and cosmetic results after adjuvant chest wall radiotherapy.

Background and Purpose:Timing and sequencing of radiotherapy in the context of allogenous breast reconstruction have not been standardized. The aim of the present study was to assess the influence of adjuvant radiotherapy on morbidity and patient satisfaction after allogenous breast reconstruction.Patients and Methods:33 patients underwent mastectomy between 1999 and 2008 and had immediate breast reconstruction with an expander placement in subpectoral/epipectoral location. 24 patients had adjuvant chemotherapy. Adjuvant external-beam radiotherapy with a median dose of 50.4 Gy was given after expander filling and on average 5.2 months prior to placement of the definitive implant. 22 patients with the definitive implant were considered for analysis of capsular fibrosis rate. Questionnaires were sent to all patients to assess cosmetic outcome and satisfaction.Results:Acute adverse effects were comparable to adjuvant radiotherapy after breast-conserving surgery, resulting in an erythema rate grade 1/2/3 in 21.2%/66.7%/6.1% of patients, respectively. After a mean follow-up of 24.9 months, 9.1%/18.2%/15.2%/9.1% of patients presented a capsular fibrosis grade 1/2/3/4, respectively. Severe deformation/asymmetry of the reconstructed breast was seen in 27.3%/33.3% of patients, respectively. Of the 22 patients with definitive implant, five (22.7%) lost the implant due to painful capsular fibrosis. Of these 22 patients, 50% were very satisfied or satisfied with the reconstruction result. Overall, 81% of patients would request breast reconstruction again.Conclusion:Adjuvant radiotherapy with the use of a subtotally filled expander prior to definitive allogenous breast reconstruction is feasible with acceptable morbidity. An interdisciplinary consultation concerning the cosmetic outcome and potential side effects is absolutely necessary.Hintergrund und Ziel:Das optimale Timing und die Sequenz einer Strahlentherapie im Kontext einer allogenen Brustrekonstruktion sind unklar. Ziel der vorliegenden Arbeit war die Beurteilung des Einflusses der adjuvanten Radiotherapie auf Morbiditat und Zufriedenheit der Patientinnen nach allogener Brustrekonstruktion.Patienten und Methodik:33 Patientinnen erhielten zwischen 1999 und 2008 eine Mastektomie mit subpektoraler/epipektoraler Expanderplatzierung. 24 Patientinnen wurden adjuvant chemotherapiert. Die perkutane Strahlentherapie wurde nach Expanderfullung im Mittel 5,2 Monate vor der Implantateinlage mit median 50,4 Gy durchgefuhrt. 22 Patientinnen mit endgultigem Implantat gingen in die Analyse der Kapselfibroserate ein. An alle Patientinnen wurden Fragebogen zur Beurteilung des kosmetischen Ergebnisses und der Zufriedenheit versandt.Ergebnisse:Die Akutnebenwirkungen waren mit denen der adjuvanten Strahlentherapie nach brusterhaltender Therapie vergleichbar. Es zeigte sich ein Erythem Grad 1/2/3 in 21,2%/66,7%/6,1% der Falle. Nach einem mittleren Nachbeobachtungszeitraum von 24,9 Monaten zeigten 9,1%/18,2%/15,2%/9,1% der Patientinnen eine Kapselfibrose Grad 1/2/3/4. Eine hochgradige Verformung/Asymmetrie der rekonstruierten Brust wurde in 27,3%/33,3% der Falle gesehen. Bei funf der 22 Patientinnen (22,7%) mit endgultigem Implantat kam es zum Implantatverlust aufgrund schmerzhafter Kapselfibrosierung. Von diesen 22 Patienten waren 50% sehr zufrieden oder zufrieden mit dem Ergebnis des Wiederaufbaus. Insgesamt 81% der Patientinnen wurden wieder eine Brustrekonstruktion durchfuhren lassen.Schlussfolgerung:Eine adjuvante Strahlentherapie ist bei weitgehend gefulltem Expander im Rahmen der allogenen Brustrekonstruktion mit akzeptabler Morbiditat moglich. Eine interdisziplinare Beratung hinsichtlich des zu erwartenden kosmetischen Ergebnisses und moglicher Nebenwirkungen ist jedoch unabdingbar.

Immediate Reconstruction with an Expander/Implant Following Ablatio Mammae because of Breast Cancer

Background and Purpose:Timing and sequencing of radiotherapy in the context of allogenous breast reconstruction have not been standardized. The aim of the present study was to assess the influence of adjuvant radiotherapy on morbidity and patient satisfaction after allogenous breast reconstruction.Patients and Methods:33 patients underwent mastectomy between 1999 and 2008 and had immediate breast reconstruction with an expander placement in subpectoral/epipectoral location. 24 patients had adjuvant chemotherapy. Adjuvant external-beam radiotherapy with a median dose of 50.4 Gy was given after expander filling and on average 5.2 months prior to placement of the definitive implant. 22 patients with the definitive implant were considered for analysis of capsular fibrosis rate. Questionnaires were sent to all patients to assess cosmetic outcome and satisfaction.Results:Acute adverse effects were comparable to adjuvant radiotherapy after breast-conserving surgery, resulting in an erythema rate grade 1/2/3 in 21.2%/66.7%/6.1% of patients, respectively. After a mean follow-up of 24.9 months, 9.1%/18.2%/15.2%/9.1% of patients presented a capsular fibrosis grade 1/2/3/4, respectively. Severe deformation/asymmetry of the reconstructed breast was seen in 27.3%/33.3% of patients, respectively. Of the 22 patients with definitive implant, five (22.7%) lost the implant due to painful capsular fibrosis. Of these 22 patients, 50% were very satisfied or satisfied with the reconstruction result. Overall, 81% of patients would request breast reconstruction again.Conclusion:Adjuvant radiotherapy with the use of a subtotally filled expander prior to definitive allogenous breast reconstruction is feasible with acceptable morbidity. An interdisciplinary consultation concerning the cosmetic outcome and potential side effects is absolutely necessary.Hintergrund und Ziel:Das optimale Timing und die Sequenz einer Strahlentherapie im Kontext einer allogenen Brustrekonstruktion sind unklar. Ziel der vorliegenden Arbeit war die Beurteilung des Einflusses der adjuvanten Radiotherapie auf Morbiditat und Zufriedenheit der Patientinnen nach allogener Brustrekonstruktion.Patienten und Methodik:33 Patientinnen erhielten zwischen 1999 und 2008 eine Mastektomie mit subpektoraler/epipektoraler Expanderplatzierung. 24 Patientinnen wurden adjuvant chemotherapiert. Die perkutane Strahlentherapie wurde nach Expanderfullung im Mittel 5,2 Monate vor der Implantateinlage mit median 50,4 Gy durchgefuhrt. 22 Patientinnen mit endgultigem Implantat gingen in die Analyse der Kapselfibroserate ein. An alle Patientinnen wurden Fragebogen zur Beurteilung des kosmetischen Ergebnisses und der Zufriedenheit versandt.Ergebnisse:Die Akutnebenwirkungen waren mit denen der adjuvanten Strahlentherapie nach brusterhaltender Therapie vergleichbar. Es zeigte sich ein Erythem Grad 1/2/3 in 21,2%/66,7%/6,1% der Falle. Nach einem mittleren Nachbeobachtungszeitraum von 24,9 Monaten zeigten 9,1%/18,2%/15,2%/9,1% der Patientinnen eine Kapselfibrose Grad 1/2/3/4. Eine hochgradige Verformung/Asymmetrie der rekonstruierten Brust wurde in 27,3%/33,3% der Falle gesehen. Bei funf der 22 Patientinnen (22,7%) mit endgultigem Implantat kam es zum Implantatverlust aufgrund schmerzhafter Kapselfibrosierung. Von diesen 22 Patienten waren 50% sehr zufrieden oder zufrieden mit dem Ergebnis des Wiederaufbaus. Insgesamt 81% der Patientinnen wurden wieder eine Brustrekonstruktion durchfuhren lassen.Schlussfolgerung:Eine adjuvante Strahlentherapie ist bei weitgehend gefulltem Expander im Rahmen der allogenen Brustrekonstruktion mit akzeptabler Morbiditat moglich. Eine interdisziplinare Beratung hinsichtlich des zu erwartenden kosmetischen Ergebnisses und moglicher Nebenwirkungen ist jedoch unabdingbar.

Serum after Autologous Transplantation Stimulates Proliferation and Expansion of Human Hematopoietic Progenitor Cells

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34+ cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34+, CD133+, CD45−) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool.

GAR22: A novel target gene of thyroid hormone receptor causes growth inhibition in human erythroid cells

OBJECTIVE Thyroid hormone receptors (TRs) are ligand-dependent transcription factors with a major impact on erythroid cell development. Here we investigated TR activity on red cell gene expression and identified TR target genes. The impact of the TR target gene GAR22 (growth arrest-specific 2 [GAS2]-related gene on chromosome 22) on red cell differentiation was determined. MATERIALS AND METHODS Stem cell factor/erythropoietin (SCF/EPO)-dependent red cell progenitors were differentiated in vitro in the presence or absence of thyroid hormone. Hormone-induced changes in gene expression were measured by a genome-wide approach with DNA microarrays. Ectopic expression of the TR target gene GAR22 was used to determine its impact on red cell differentiation. RESULTS Ligand-activated TR effectively accelerated red cell progenitor differentiation in vitro concomitantly with inducing growth arrest. We demonstrate that activated TR-induced specific gene expression patterns of up- or downregulated genes, including distinct clusters associated with accelerated differentiation in response to treatment. Mining for T3-induced genes identified basic transcription element binding protein 1/Krüppel-like factor 9 (BTEB1/KLF9) and GAR22 as TR target genes. BTEB1/KLF9 is a known TR target gene while GAR22, initially identified as a putative tumor suppressor, represents a novel TR target gene. We demonstrate that ectopic GAR22 expression in red cell progenitors lengthens the cell cycle and causes growth inhibition, but leaves red cell gene expression unaffected. CONCLUSION This study identifies GAR22 as a novel and direct TR target gene. Our results suggest that hormone-induced GAR22 might represent an important trigger of growth inhibition induced by thyroid hormone in red cell progenitors.

Compatibility of different polymers for cord blood-derived hematopoietic progenitor cells.

The low yield of hematopoietic progenitor cells (HPC) present in cord blood grafts limits their application in clinics. A reliable strategy for ex vivo expansion of functional HPC is a present goal in regenerative medicine. Here we evaluate the capacity of several two-dimensional polymers to support HPC proliferation. Basic compatibility was tested by measuring cell viability, cytotoxicity and apoptosis of CD34+ progenitors that were short and long-term exposed to sixteen bio and synthetic polymers. Resomer® RG503, PCL and Fibrin might be good alternatives to tissue culture plastic for culture of CB-derived CD34+ progenitors. Further, these polymers will be produced in three-dimensional structures and tested for their cytocompatibility.

Ex vivo expansion of cord blood-CD34+ cells using IGFBP2 and Angptl-5 impairs short-term lymphoid repopulation in vivo

Cord blood‐derived haematopoietic stem cells (CB‐HSCs) are an attractive source for transplantation in haematopoietic disorders. However, the yield of CB‐HSCs per graft is limited and often insufficient, particularly for the treatment of adult patients. Here we compare the capacity of three cytokine cocktails to expand CB‐CD34+ cells. Cells were cultured for 5 or 14 days in media supplemented with: (a) SCF, FL, IL‐3 and IL‐6 (SFLIL3/6); (b) SCF, TPO, FGF‐1 and IL‐6 (STFIL6); and (c) SCF, TPO, FGF‐1, IGFBP2 and Angptl‐5 (STFAI). We observed that STFAI‐culture expansion sustained the most vigorous cell proliferation, maintenance of CD34+ phenotype and colony‐forming unit counts. In addition, STFAI‐cultured cells had a potent ex vivo migration activity. STFAI‐expanded cells were able to engraft NSG mice. However, no significant difference in overall engraftment was observed among the expansion cocktails. Assessment of short‐term reconstitution using multilineage markers demonstrated that the STFAI cocktail for HSCs expansion greatly improved total cell expansion but may impair short‐term lymphoid repopulation. Copyright

Immediate Reconstruction with an Expander/Implant Following Ablatio Mammae because of Breast Cancer@@@Sofortrekonstruktion mittels Expander/Implantat nach Ablatio aufgrund eines Mammakarzinoms. Nebenwirkungen und kosmetische Ergebnisse nach adjuvanter Brustwandbestrahlung: Side Effects and Cosmetic Results after Adjuvant Chest Wall Radiotherapy

Background and Purpose:Timing and sequencing of radiotherapy in the context of allogenous breast reconstruction have not been standardized. The aim of the present study was to assess the influence of adjuvant radiotherapy on morbidity and patient satisfaction after allogenous breast reconstruction.Patients and Methods:33 patients underwent mastectomy between 1999 and 2008 and had immediate breast reconstruction with an expander placement in subpectoral/epipectoral location. 24 patients had adjuvant chemotherapy. Adjuvant external-beam radiotherapy with a median dose of 50.4 Gy was given after expander filling and on average 5.2 months prior to placement of the definitive implant. 22 patients with the definitive implant were considered for analysis of capsular fibrosis rate. Questionnaires were sent to all patients to assess cosmetic outcome and satisfaction.Results:Acute adverse effects were comparable to adjuvant radiotherapy after breast-conserving surgery, resulting in an erythema rate grade 1/2/3 in 21.2%/66.7%/6.1% of patients, respectively. After a mean follow-up of 24.9 months, 9.1%/18.2%/15.2%/9.1% of patients presented a capsular fibrosis grade 1/2/3/4, respectively. Severe deformation/asymmetry of the reconstructed breast was seen in 27.3%/33.3% of patients, respectively. Of the 22 patients with definitive implant, five (22.7%) lost the implant due to painful capsular fibrosis. Of these 22 patients, 50% were very satisfied or satisfied with the reconstruction result. Overall, 81% of patients would request breast reconstruction again.Conclusion:Adjuvant radiotherapy with the use of a subtotally filled expander prior to definitive allogenous breast reconstruction is feasible with acceptable morbidity. An interdisciplinary consultation concerning the cosmetic outcome and potential side effects is absolutely necessary.Hintergrund und Ziel:Das optimale Timing und die Sequenz einer Strahlentherapie im Kontext einer allogenen Brustrekonstruktion sind unklar. Ziel der vorliegenden Arbeit war die Beurteilung des Einflusses der adjuvanten Radiotherapie auf Morbiditat und Zufriedenheit der Patientinnen nach allogener Brustrekonstruktion.Patienten und Methodik:33 Patientinnen erhielten zwischen 1999 und 2008 eine Mastektomie mit subpektoraler/epipektoraler Expanderplatzierung. 24 Patientinnen wurden adjuvant chemotherapiert. Die perkutane Strahlentherapie wurde nach Expanderfullung im Mittel 5,2 Monate vor der Implantateinlage mit median 50,4 Gy durchgefuhrt. 22 Patientinnen mit endgultigem Implantat gingen in die Analyse der Kapselfibroserate ein. An alle Patientinnen wurden Fragebogen zur Beurteilung des kosmetischen Ergebnisses und der Zufriedenheit versandt.Ergebnisse:Die Akutnebenwirkungen waren mit denen der adjuvanten Strahlentherapie nach brusterhaltender Therapie vergleichbar. Es zeigte sich ein Erythem Grad 1/2/3 in 21,2%/66,7%/6,1% der Falle. Nach einem mittleren Nachbeobachtungszeitraum von 24,9 Monaten zeigten 9,1%/18,2%/15,2%/9,1% der Patientinnen eine Kapselfibrose Grad 1/2/3/4. Eine hochgradige Verformung/Asymmetrie der rekonstruierten Brust wurde in 27,3%/33,3% der Falle gesehen. Bei funf der 22 Patientinnen (22,7%) mit endgultigem Implantat kam es zum Implantatverlust aufgrund schmerzhafter Kapselfibrosierung. Von diesen 22 Patienten waren 50% sehr zufrieden oder zufrieden mit dem Ergebnis des Wiederaufbaus. Insgesamt 81% der Patientinnen wurden wieder eine Brustrekonstruktion durchfuhren lassen.Schlussfolgerung:Eine adjuvante Strahlentherapie ist bei weitgehend gefulltem Expander im Rahmen der allogenen Brustrekonstruktion mit akzeptabler Morbiditat moglich. Eine interdisziplinare Beratung hinsichtlich des zu erwartenden kosmetischen Ergebnisses und moglicher Nebenwirkungen ist jedoch unabdingbar.