Biomedical subjects

Hermann Eichler

Publications and source records attributed to Hermann Eichler.

11 recordsLinked to original sources

PubMed2006

Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.

Mesenchymal stem cells (MSCs) represent a promising tool for new clinical concepts in supporting cellular therapy. Bone marrow (BM) was the first source reported to contain MSCs. However, for clinical use, BM may be detrimental due to the highly invasive donation procedure and the decline in MSC number and differentiation potential with increasing age. More recently, umbilical cord blood (UCB), attainable by a less invasive method, was introduced as an alternative source for MSCs. Another promising source is adipose tissue (AT). We compared MSCs derived from these sources regarding morphology, the success rate of isolating MSCs, colony frequency, expansion potential, multiple differentiation capacity, and immune phenotype. No significant differences concerning the morphology and immune phenotype of the MSCs derived from these sources were obvious. Differences could be observed concerning the success rate of isolating MSCs, which was 100% for BM and AT, but only 63% for UCB. The colony frequency was lowest in UCB, whereas it was highest in AT. However, UCB-MSCs could be cultured longest and showed the highest proliferation capacity, whereas BM-MSCs possessed the shortest culture period and the lowest proliferation capacity. Most strikingly, UCB-MSCs showed no adipogenic differentiation capacity, in contrast to BM- and AT-MSCs. Both UCB and AT are attractive alternatives to BM in isolating MSC: AT as it contains MSCs at the highest frequency and UCB as it seems to be expandable to higher numbers.

Adipose Tissue↗

PubMed2006

Multiple-laboratory comparison of in vitro assays utilized to characterize hematopoietic cells in cord blood.

BACKGROUND: Understanding the variability in results obtained by multiple laboratories is important because cord blood units are distributed worldwide for transplantation. STUDY DESIGN AND METHODS: Four exercises were conducted by multiple laboratories to assess assay variability on nucleated cell (NC), mononuclear cell (MNC) by hematology analyzers [HAs], and CD34+ cell (flow cytometry) measurements. Exercise 1 was an intralaboratory exercise in which the reproducibility of cell measurements was determined. Exercises 2 and 3 involved the shipment of identical processed cord blood samples. In Exercise 2, laboratory-specific methods were utilized. In Exercise 3, two commercial CD34+ cell methods (Stem-Kit and TruCOUNT) were used. In Exercise 4, CD34+ cell levels were determined on repetitive regating of identical list-mode files. RESULTS: Intralaboratory reproducibility was highest for NC measurements and lowest for CD34+ cell measurements. In Exercise 2, all laboratories except one utilized HA with an impedance technology and determined comparable results for NC and MNC levels, whereas the other laboratory utilized a HA with an optical counting method. Substantial variation was observed on measuring CD34+ cells with ranges of 32 to 141, 32 to 66, and 25 to 116 CD34+ cells per microL for the three identical samples. In Exercise 3, on the use of one specific commercial assay, the ranges of CD34+ levels were 214 to 411 and 62 to 178 cells per microL for the two identical samples. Nearly all participating laboratories determined comparable CD34+ levels on the use of identical list-mode files. CONCLUSION: These studies indicate that substantial variability in CD34+ cell levels were determined with flow cytometry. The variability in NC and MNC levels was minimal with HA methodology.

Antigens, CD↗

PubMed2005

Stem cell proteomes: a profile of human mesenchymal stem cells derived from umbilical cord blood.

Multipotent mesenchymal stem cells (MSCs) derived from human umbilical cord blood (UCB) represent promising candidates for the development of future strategies in cellular therapy. To create a comprehensive protein expression profile for UCB-MSCs, one UCB unit from a full-term delivery was isolated from the unborn placenta, transferred into culture, and their whole-cell protein fraction was subjected to two-dimensional electrophoresis (2-DE). Unambiguous protein identification was achieved with peptide mass fingerprinting matrix-assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS), peptide sequencing (MALDI LIFT-TOF/TOF MS), as well as gel-matching with previously identified databases. In overall five replicate 2-DE runs, a total of 2037 +/- 437 protein spots were detected of which 205 were identified representing 145 different proteins and 60 isoforms or post-translational modifications. The identified proteins could be grouped into several functional categories, such as metabolism, folding, cytoskeleton, transcription, signal transduction, protein degradation, detoxification, vesicle/protein transport, cell cycle regulation, apoptosis, and calcium homeostasis. The acquired proteome map of nondifferentiated UCB-MSCs is a useful inventory which facilitates the identification of the normal proteomic pattern as well as its changes due to activated or suppressed pathways of cytosolic signal transduction which occur during proliferation, differentiation, or other experimental conditions.

Databases, Protein↗

PubMed2004

Fully automated processing of buffy-coat-derived pooled platelet concentrates.

BACKGROUND: The OrbiSac device, which was developed to automate the manufacture of buffy-coat PLT concentrates (BC-PCs), was evaluated. STUDY DESIGN AND METHODS: In-vitro characteristics of BC-PC preparations using the OrbiSac device were compared with manually prepared BC-PCs. For standard processing (Std-PC, n = 20), four BC-PCs were pooled using 300 mL of PLT AS (PAS) followed by soft-spin centrifugation and WBC filtration. The OrbiSac preparation (OS-PC, n = 20) was performed by automated pooling of four BC-PCs with 300 mL PAS followed by centrifugation and inline WBC filtration. All PCs were stored at 22 degrees C. Samples were withdrawn on Day 1, 5, and 7 evaluating PTL count, blood gas analysis, glucose, lactate, LDH, beta-thromboglobulin, hypotonic shock response, and CD62p expression. RESULTS: A PLT content of 3.1 +/- 0.4 x 10(11) (OS-PCs) versus 2.7 +/- 0.5 x 10(11) (Std-PCs, p < 0.05) was found. A CV of 19 percent (Std-PC) versus 14 percent (OS-PC) suggests more standardization in the OS group. At Day 7, the Std-PCs versus OS-PCs showed a glucose consumption of 1.03 +/- 0.32 micro mol per 10(9) PLT versus 0.75 +/- 0.25 micro mol per 10(9) PLT (p < 0.001), and a lactate production of 1.50 +/- 0.86 micro mol per 10(9) versus 1.11 +/- 0.61 micro mol per 10(9) (p < 0.001). The pH (7.00 +/- 0.19 vs. 7.23 +/- 0.06; p < 0.001), pO(2) (45.3 +/- 18 vs. 31.3 +/- 10.4 mmHg; p < 0.01), and HCO(3) levels (4.91 +/- 1.49 vs. 7.14 +/- 0.95 mmol/L; p < 0.001) suggest a slightly better aerobic metabolism within the OS group. Only small differences in CD62p expression was observed (37.3 +/- 12.9% Std-PC vs. 44.8 +/- 6.6% OS-PC; p < 0.05). CONCLUSION: The OrbiSac device allows an improved PLT yield without affecting PLT in-vitro characteristics and may enable an improved consistency in product volume and yield.

Blood Gas Analysis↗

PubMed2004

Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood.

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However, attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria. Using optimized isolation and culture conditions, in up to 63% of 59 low-volume UCB units, cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 x 10(8) mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition, osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However, in contrast to MSCs, MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than 15 hours, a net volume of more than 33 ml, and an MNC count of more than 1 x 10(8) MNCs. Because MSC-like cells can be isolated at high efficacy from full-term UCB donations, we regard UCB as an additional stem cell source for experimental and potentially clinical purposes.

Adult↗

PubMed2003

Flow cytometric analysis of T cell proliferation in a mixed lymphocyte reaction with dendritic cells.

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells. They can be generated in vitro from CD14+ cells, and also from CD34+ progenitor cells. Although T cell proliferation using [3H] thymidine incorporation assay has been used widely to check DC function, this technique only provides limited information about the T cell proliferation. Here, we describe a novel method for quantitative analysis of T cell proliferation using flow cytometry. MATERIALS AND METHODS: DCs were generated from CD14+ cells from six healthy blood donors. Monocytes were isolated using positive selection with magnetic cell sorting (MACS) and then cultured with IL-4, GM-CSF, IL-1beta, IL-6, TNF-alpha and PGE(2) to yield fully mature DCs. Allogeneic naive T lymphocytes with known mismatches in HLA classes I and II were cocultured with DCs. Naive T cells without DC stimulation served as negative controls. T cells were harvested on days 0, 3, 5, 7, 9, 11 and analysed by flow cytometry. CD3-ECD and CD4-fluorescein isothiocyanate (FITC) or CD8-FITC antibodies were used to distinguish T cell subsets, whereas T cell activation was measured by assessment of HLA-DR, CD45RO, CD25 and CD71 expression. For T cell quantification, fluorescent microparticles were used. Dead cells were excluded with 7-AAD. The bromdeoxyridine (BrdU)-incorporation ELISA procedure was also performed in order to compare with the T cell proliferation assay with regard to absolute cell counts and CD71 expression. RESULTS: The initial T cell concentration on day 1 was 203.9+/-39.7 (173-265) CD3+/CD4+ cells/micro l and 184.5+/-41.6 (148-260) CD3+/CD8+ cells/micro l. The maximal T cell proliferation was recorded on day 7 with a five- to tenfold T cell expansion which resulted in 1994.9+/-383 (1446-2404) CD3+/CD4+ cells/micro l and 944+/-303.7 (560-1483) CD3+/CD8+ cells/micro l. Furthermore, activation markers of both cell lineages were upregulated and reached maxima on days 7 (CD71) and 9 (CD25, HLA-DR). T cell count/micro l as well as CD71 expression both correlated significantly with BrdU incorporation. CONCLUSION: Flow cytometric analysis permits simple, precise and rapid quantification of T cell proliferation in a mixed lymphocyte reaction with DCs. Activation, proliferation and cell viability can be simultaneously determined. CD71 is particularly well suited as an activation marker for the simultaneous measurement of T cell proliferation. Thus, specific T cell subsets involved in antigen-specific proliferation can be evaluated in detail.

Bromodeoxyuridine↗

PubMed2003

Engraftment capacity of umbilical cord blood cells processed by either whole blood preparation or filtration.

Umbilical cord blood (UCB) preparation needs to be optimized in order to develop more simplified procedures for volume reduction, as well as to reduce the amount of contaminating cells within the final stem cell transplant. We evaluated a novel filter device (StemQuick((TM))E) and compared it with our routine buffy coat (BC) preparation procedure for the enrichment of hematopoietic progenitor cells (HPCs). Two groups of single or pooled UCB units were filtered (each n = 6), or equally divided in two halves and processed by filtration and BC preparation in parallel (n = 10). The engraftment capacity of UCB samples processed by whole blood (WB) preparation was compared with paired samples processed by filtration in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse animal model. Filtration of UCB units in the two groups with a mean volume of 87.8 and 120.7 ml, respectively, and nucleated cell (NC) content of 9.7 and 23.8 x 10(8) resulted in a sufficient mean cell recovery for mononucleated cells ([MNCs] 74.2%-77.5%), CD34(+) cells (76.3%-79.0%), and colony-forming cells (64.1%-86.3%). Moreover, we detected a relevant depletion of the transplants for RBCs (89.2%-90.0%) and platelets ([PLTs] 77.5%-86.1%). In contrast, the mean depletion rate using BC processing proved to be significantly different for PLTs (10%, p = 0.03) and RBCs (39.6%, p < 0.01). The NC composition showed a highly significant increase in MNCs and a decrease in granulocytes after filtration (p < 0.01), compared with a less significant MNC increase in the BC group (p < 0.05). For mice transplanted with WB-derived progenitors, we observed a mean of 15.3% +/- 15.5% of human CD45(+) cells within the BM compared with 19.9% +/- 16.8% for mice transplanted with filter samples (p = 0.03). The mean percentage of human CD34(+) cells was 4.2% +/- 3.1% for WB samples and 4.5% +/- 3.2% for filter samples (p = 0.68). As the data of NOD/SCID mice transplantation demonstrated a significant engraftment capacity of HPCs processed by filtration, no negative effect on the engraftment potential of filtered UCB cells versus non-volume-reduced cells from WB transplants was found. The StemQuick((TM))E filter devices proved to be a useful tool for Good Manufacturing Practices conform enrichment of HPCs and MNCs out of UCB. Filtration enables a quick and standardized preparation of a volume-reduced UCB transplant, including a partial depletion of granulocytes, RBCs, and PLTs without the need for centrifugation. Therefore, it seems very probable that filter-processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans.

Animals↗

PubMed2003

Messenger RNA profiling of human platelets by microarray hybridization.

Platelets are generally believed to be inactive in terms of de novo protein synthesis. On the other hand, the presence of ribosomes and mRNA molecules is well established. Many studies have used reverse transcriptase (RT) -PCR for detection of gene transcripts in platelets. As RT-PCR is a very sensitive method, any leukocyte contamination of platelet preparations can lead to false results. We performed three filtration procedures to minimize leukocyte contamination of pooled buffy-coat platelet concentrates prior to RNA isolation. Furthermore, by applying a genomic PCR approach with 50 amplification cycles we demonstrated that nucleated cells were not detectable. Microarray hybridization was used to analyze 9,850 individual human genes in RNA from purified platelets. In total we identified 1,526 (15.5%) positive genes. The data were confirmed in six individual experiments each performed on a PC pooled from four individual blood donations. Genes specific for nucleated blood cells such as CD4, CD83 and others were negative and verified the purity of PC. Overrepresentation of positive genes was found in the functional categories of glycoproteins/integrins (22.6% vs. 15.5%, p=0.029) and receptors (20.7% vs. 15.5%, p<0.001). Gene transcripts encoding RANTES, GRO-alpha, MIP-1alpha, MIP-1beta, and others were found at high levels of signal intensity and confirmed literature data. This work provides a mRNA profile of human platelets and a complete list of results can be downloaded from the website of our institute www.ma.uni-heidelberg.de/inst/iti/plt_array.xls. The knowledge about gene transcripts may have an impact on the characterization of novel proteins and their functions in platelets.

Adult↗

PubMed2002

Collection of autologous monocytes for dendritic cell vaccination therapy in metastatic melanoma patients.

BACKGROUND: Dendritic cells (DCs) for immunotherapy of malignant melanoma can be generated from partially enriched monocytes prepared from PBMNCs. The feasibility of a single steady-state leukapheresis procedure to enrich monocytes for a complete vaccination series with up to 10 vaccinations was investigated. STUDY DESIGN AND METHODS: Thirty-eight patients (27 males and 11 females) with metastatic melanoma were enrolled in the study. All leukapheresis procedures were performed by a continuous flow method (Spectra, Cobe BCT) with a standard MNC program. RESULTS: An average of 11.7 L (range, 8-14 L) of whole blood was processed within 197.3 +/- 23.7 minutes, and a mean of 13.5 +/- 5.7 x 109 WBCs in a final volume of 191.0 +/- 24.2 mL was collected. The MNC purity in the apheresis component was 81.5 +/- 15.1 percent, from which 29.8 +/- 14.7 percent were monocytes. Thus, 11.0 +/- 5.0 x 109 MNCs and 3.2 +/- 2.0 x 109 monocytes were collected per procedure. Linear regression analysis revealed a high correlation between the absolute number of monocytes in peripheral blood before the apheresis procedure and the number of monocytes in the collected component (r=0.74, p < 0.0001). For the generation of DCs, 1.6 +/- 0.8 x 109 MNCs were plated into culture dishes; 3.2 +/- 1.8 percent of the cultured cells matured to DCs, which resulted in 56.5 +/- 49.4 x 106 DCs (range, 6.3-178) per patient for the complete vaccination series. CONCLUSION: A target dose of monocytes for the complete vaccination series could be obtained by a single convenient, safe, steady-state leukapheresis procedure in each patient without the need for G-CSF mobilization. The absolute number of monocytes in peripheral blood before the apheresis procedure is the best predictive variable for the yield of monocytes in the apheresis component.

Adult↗

PubMed2002

Nonobese diabetic-severe combined immunodeficient mice transplantation of volume-reduced and thawed umbilical cord blood transplants following closed-system immunomagnetic cell selection.

BACKGROUND: Protocols for the expansion of human umbilical cord blood (UCB) progenitors begin with the selection of CD34+ cells from stored frozen and thawed units. Use of an immunomagnetic selection procedure within a closed blood bag system for volume-reduced UCB transplants was evaluated, and the influence of CD34 cell selection on in vivo engraftment potential was studied. STUDY DESIGN AND METHODS: Eleven thawed buffy coat-processed UCB units were processed within a standard blood bag with a washing solution. In six independent experiments, the same dosage of 2 x 104 CD34+ cells from paired selected and nonselected samples was transplanted into NOD-SCID mice. In two experiments, cells from the negative fraction were also transplanted. RESULTS: The purity of CD34+ cells after selection was correlated with the removal of supernatant after the first washing step and therefore with adequate removal of damaged or dead cells (r=0.86, p < 0.01). Mice transplanted with unselected UCB cells had more human cells within their marrow than animals transplanted with selected cells (8.6 +/- 5.9% selected group vs. 19.8 +/- 14.2% unselected group; p=0.04), whereas no engraftment could be observed transplanting cells from the two negative fractions. A higher percentage of human CD45+ cells in the unselected group were found to be positive for CD38, CD14, CD33, and CD19, indicating a higher potential for these unselected progenitors to differentiate into myeloid cells and B cells. CONCLUSIONS: Processing of volume-reduced and thawed UCB transplants within a closed-bag system before immunomagnetic CD34+ cell selection allows for the preparation of CD34+ cells of significant purity at technically useful cell recoveries. However, these experiments indicate a potential impairment of engraftment capacity for the CD34+ cell-enriched fraction.

Animals↗

PubMed2002

Prenatal HLA genotyping of uncultured amniotic fluid samples contaminated with maternal blood.

OBJECTIVE: Allogenic transplantation of umbilical cord blood (UCB) from HLA-identical siblings is a therapeutic concept of increasing importance. Prenatal HLA typing results can provide important information as to whether the UCB should be collected. Therefore, we tested the suitability of two methods based on polymerase chain reaction (PCR) for low-resolution HLA genotyping of uncultured amniocytes contaminated with maternal blood. STUDY DESIGN: Amniotic fluid samples (~10 mL, n = 4) were collected and divided into 5 equal sized portions. Subsequently, maternal blood was added to produce a series of varying degrees of artificial contamination ranging from 0% to 5% (vol/vol). Corresponding maternal blood and cord blood samples were collected and served as reference material. After DNA preparation, all samples were subjected to both PCR-SSP (sequence-specific primers) and PCR-SSO (sequence-specific oligonucleotides) typing procedures, designed for low-resolution typing of the highly polymorphic HLA-B locus. RESULTS: Both PCR tests were able to accurately predict the fetal HLA-B type when pure amniotic fluid was used in all cases. The PCR-SSP method could still determine the fetal HLA type at 0.1% contamination but not at levels above this; in contrast, PCR-SSO failed if any degree of contamination was present. CONCLUSION: Fetal cells from amniotic fluid represent a reasonable source for prenatal testing of the fetal HLA genotype with either the PCR-SSP or the PCR-SSO method. Low levels of contamination with maternal blood in the amniotic fluid are tolerated by the more robust PCR-SSP method. In contrast, the PCR-SSO procedure is more sensitive to any degree of contamination, but it is the method of choice if the amount of DNA is limited because of the very low volume of specimens of amniotic fluid available.

Amniotic Fluid↗