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Biomedical subjects

M Kletzel

Publications and source records attributed to M Kletzel.

At least 19 recordsLinked to original sources
PubMed2006

Weight loss and reduced body mass index: a critical issue in children with multiorgan chronic graft-versus-host disease.

Weight loss and malnutrition are major problems in patients with chronic graft-versus-host disease (GVHD). In adults, low body mass index (BMI) is a predictor for mortality; however, weight loss and BMI have not been studied in pediatric chronic GVHD. A retrospective study on 18 children with extensive chronic GVHD was completed. Median age at SCT was 12.3 (range 0.6-23) years; age at chronic GVHD diagnosis was 12.5 (1-23) years. Patients with multiorgan involvement had a mean maximal decrease in BMI of 20.9% and most dropped below 10th percentile in expected weight-for-age. This change in BMI not only indicates a significant decrease in weight but often a plateau in stature. In contrast, patients with one organ system involved had a mean maximal decrease in BMI of 5% and did not fall below 10th percentile. All patients with multiorgan involvement required salvage therapy beyond steroids and CSA. Three patients died due to complications of chronic GVHD. Weight loss and malnutrition (as reflected by a decrease in BMI) are clinically significant issues in children with multisystem chronic GVHD. Weight loss is likely another systemic manifestation of the disease and may contribute, along with other factors such as increased immunosuppression and infection, to increased mortality in this group.

Adolescent↗
PubMed2005

Monosomy 7 associated with pediatric acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS): successful management by allogeneic hematopoietic stem cell transplant (HSCT).

Pediatric acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) with monosomy 7 is associated with poor disease-free survival when treated by conventional chemotherapy, immunosuppression or supportive measures. Hematopoietic stem cell transplant (HSCT) may improve outcomes; however, data to support this are limited. To better understand the curative potential of HSCT in these patients, all cases of AML and MDS with monosomy 7 treated by two transplant programs (1992 to present) were reviewed. A total of 16 patients were treated, all by allogeneic HSCT. Primary diagnoses were MDS (N = 5), therapy-related MDS (N = 3), AML (N = 5) and therapy-related AML (N = 3). In all, 11 patients (69%) survive event-free at 2 years with median follow-up of 986 days (range 330-2011 days). Toxicity caused deaths of the five nonsurviving patients, four of whom were transplanted with active leukemia. Allogeneic HSCT is effective therapy for childhood AML and MDS associated with monosomy 7, particularly for patients with AML in complete remission and MDS.

Acute Disease↗
PubMed2005

Wilms' tumor 1 (WT1) gene in hematopoiesis: a surrogate marker of cell proliferation as a possible mechanism of action?

BACKGROUND: Wilms' tumor 1 (WT1) gene expression is seen in a significant number of cases of human neoplasia; however, the mechanism of action remains to be clarified. We hypothesized that WT1 gene is a surrogate marker of proliferation in normal hematopoietic cells and leukemias. While we and others have recognized its value as a tool for the detection of minimal residual disease (MRD), the objective of this study was to confirm our hypothesis regarding normal. METHODS: Samples from healthy donors (n=16) and UC blood (n=9) were cultured in Methocult for 21 days. Colonies were analyzed on days 7, 14 and 21 by RT-PCR for WT1 gene expression. Our positive controls were samples from patients with leukemia (n=91). Negative controls were from normal volunteers without stimulation (n=26). RESULTS: Results showed a statistically significant difference (P<0.0001) between cultured groups, with the highest level of WT1 gene expression in the positive controls and on day 14, when cells are at their maximal proliferation. DISCUSSION: In conclusion, WT1 gene expression in the proliferating colonies was highest on day 14, although less than in leukemia samples, confirming our hypothesis that WT1 gene is a surrogate marker of proliferation, not only in leukemogenesis but also, to a lesser degree, in normal cell proliferation.

Cell Proliferation↗
PubMed2005

Comparison of Tc-99 measurement of glomerular filtration rate vs. calculated creatinine clearance to assess renal function pretransplant in pediatric patients undergoing hematopoietic stem cell transplantation.

We compared the results of Tc-99 evaluation of glomerular filtration rate (GFR) vs. the calculation of the creatinine clearance (CCrC) as a predictor for the development of renal insufficiency in pediatric patients following hematopoietic stem cell transplantation (HSCT). We reviewed 95 consecutive patients receiving autologous (n = 37) or allogeneic (n = 58) HSCT at Children's Memorial Hospital between January, 1995 and February, 1998. Diagnoses included leukemia (n = 43), solid tumor (n = 27), bone marrow failure syndrome (n = 12), non-malignant disease (n = 8), CNS tumors (n = 5) and immunodeficiency (n = 3). Tc-99 GFR was compared with a calculated creatinine clearance derived from the Schwartz formula (CCrC) prior to HSCT. These measures of renal function were compared with the patient's subsequent clinical course to determine if patients who developed renal insufficiency of sufficient magnitude as to require continuous veno-venous hemofiltration (CVVH) or dialysis, could have been identified. Overall comparison of the two methods of evaluation of renal function showed low correlation with values obtained by CCrC, which were consistently higher in most patients (r-value 0.01 in the regression analysis and a p = 0.08 95% CI -24.15 to 1.48). When stratified for age, correlation between the two methods was excellent only in children younger than 5 yr of age p = 0.02 95%, CI 0.032-0.49). Eleven patients required therapy with CVVH or dialysis but neither CCrC nor Tc-99 GFR prior to transplant predicted this event. Patients who received TBI were statistically more prone to develop renal insufficiency than those without TBI (p < 0.0001, 95% CI 0.25-0.008). Neither the Tc-99 GFR nor the CCrC was predictive of the development of renal insufficiency in HSCT patients as the majority of patients who required dialysis had normal Tc-99 GFR prior to transplant. The characteristics found in the patients who developed renal insufficiency and required dialysis include: the use of total body irradiation as part of the transplant-conditioning regimen (p < 0.0001) and the use of continuous infusion CSA (p = 0.04).

Adolescent↗
PubMed2004

Identification of two pseudogenes with sequence homology to human and gorilla MHC class IA genes: ancestral haplotype in the Filipino population.

While characterizing exons 2 and 3 of the class I human leukocyte antigen (HLA)-A locus in human lymphocytes, two similar but unexpected PCR products were detected in six samples of Filipino ethnicity. A nucleotide sequence analysis of the two amplicons, tentatively named HLA-COQ and HLA-DEL, rendered them as two novel and seemingly related sequences, both with homology to the gorilla and human major histocompatibility complex (MHC) A locus. Exon 2 is similar to the published human pseudogenes HLA-BEL, HLA-Y, and to primate MHC Gogo-A*0501, differing by 2 bp from HLA-BEL, and HLA-Y, and by 4 bp from Gogo-A*0501. Exon 3 is most similar to HLA-A*2902 and A*310102, differing by 7 bp from A*2902, and by 8 bp from A*31012. Genomic sequence comparison of exons 1 to 8 indicates that their closest published match is to the Gogo-A*0501. Complete typing at the HLA-A, -B, -C, DRB1, and DRB5 loci for the six samples yielded the reoccurring types: HLA-A*3401, -B*1521/1525, -Cw*0403, -DRB1*150201, and DRB5*010101. Thus far, HLA-COQ and HLA-DEL have been detected only in Filipino samples containing these HLA types. The HLA-COQ gene is nonfunctional based on a stop codon located in exon 4. HLA-DEL is also a nonfunctional gene because of the dual cytosine insertion in exon 4, with a reading frame shift generating a stop codon downstream. Parsimony analysis of the two pseudogenes with 31 other primate A locus coding regions resulted in a phylogenetic tree that segregated the two pseudogenes with the Gogo-A*0501, suggesting that HLA-COQ, HLA-DEL, and Gogo-A*0501 evolved from a common ancestral allele.

Alleles↗
PubMed2004

Outcomes of unrelated cord blood transplants and allogeneic-related hematopoietic stem cell transplants in children with high-risk acute lymphocytic leukemia.

Acute lymphocytic leukemia (ALL) is a common indication for hematopoietic stem cell transplantation (HSCT) in children. Use of unrelated cord blood (UCB) has become increasingly popular as a stem cell source, given the rapid availability and decreased GVHD potential. Publications describing outcomes of children with leukemia who underwent UCB transplants have compared them to those having received unrelated donor marrow transplants. Results are similar. We compared our outcomes using UCB vs allogeneic-related hematopoietic stem cells in pediatric ALL patients since 1992. A total of 49 patients were analyzed. All patients were either in CR1 with high-risk features (n=21) or in CR2 (n=28) with initial remission less than 36 months. Patients received myeloablation with fractionated total body irradiation, cyclophosphamide, and etoposide and GVHD prophylaxis with cyclosporine and methotrexate. Antithymocyte globulin was added for UCB recipients to address the HLA differences. In all, 23 patients underwent allogeneic -related HSCT and 26 underwent UCB transplantation. Other than increased time to engraftment for UCB recipients, results are equivalent. The 3-year overall survival is 64% and 3-year event-free survival is 60% for both groups. Rates of GVHD and transplant-related mortality are also equivalent. UCB is a reasonable option for children with ALL who are referred for HSCT.

Adolescent↗
PubMed2003

Rapid immune reconstitution following autologous hematopoietic stem cell transplantation in children: a single institution experience.

In this retrospective study, we review the immune reconstitution of children undergoing autologous hematopoietic stem cell transplantation. A total of 125 patients underwent autologous transplantation between 1992 and 2000. The report includes data on 58 patients. Data were not available on the remaining patients who either died before testing or data were not obtained. The parameters evaluated include: (a) immunophenotype by flow cytometry to quantify lymphocyte subpopulations (b) mitogen stimulation assays, and (c) quantitative immunoglobulins. The analysis reveals that CD3+ cells did not reach the normal range during the first year post-transplant. The median percentage of CD4+ cells was below normal up to 6 months post-transplant, while the absolute number remain low throughout the first year. The CD8+ percentage and absolute numbers remain normal at all times post-transplant. The CD19+ cells were also normal post-transplantation. The mitogen lymphocyte stimulation was normal in 27 out of 31 patients tested after 6 months post-transplant. Our analysis of immune reconstitution shows a similar pattern to previous studies with a faster recovery of the CD4/CD8 ratio, especially in those patients who did not receive TBI. In conclusion, the observed deficiencies are transient and have very little clinical significance because, historically, the rate of serious infections is low despite prolonged immune suppression. The recovery post-autologous transplant is fast.

Antigens, CD↗
PubMed2003

Platelet chimerism by polymerase chain reaction (PCR) utilizing variable number of tandem repeats (VNTR) in allogeneic stem cell transplant in children: a new novel approach to full chimerism analysis.

Evaluation of chimerism following allogeneic transplantation has been performed traditionally focusing on two cellular compartments, namely lymphoid and myeloid. However, none has been described so far to evaluate platelet chimerism. In order to achieve full chimerism in all three cellular compartments, we prospectively obtained 138 samples of peripheral blood in 55 patients at different post transplant periods following allogeneic hematopoietic transplantation. Evaluation of chimerism was performed utilizing tests of variable number of tandem repeat (VNTR) and sex determination by quantitative polymerase chain reaction (PCR). Tests for platelet chimerism using platelet-rich plasma were simultaneously analyzed with samples for T-cell lymphoid and myeloid compartments. Complete donor chimerism was noted in 49 of 55 patients (89%), while the remaining six have split chimerism ranging from 34 to 98%. There is significant difference (P=0.0004) between the percentages of donor DNA in all three cellular compartments comparing the means+/-s.e.m. (myeloid 95.60+/-0.9, T-cell lymphocytes 87.6+/-1.9, and the platelets 90.8+/-1.5); however, comparison between the medians is not statistically significant. This study represents an additional step towards achieving full chimerism and the observation may help reduce the number of unnecessary platelet transfusions once chimerism is noted in that cellular compartment.

Adolescent↗
PubMed2002

Evaluation of mixed hematopoietic chimerism in pediatric patients with leukemia after allogeneic stem cell transplantation by quantitative PCR analysis of variable number of tandem repeat and testis determination gene.

In order to monitor the clinical outcome of pediatric patients with leukemia following allogeneic hematopoietic transplantation, tests of variable number of tandem repeat (VNTR) and sex determination by quantitative polymerase chain reaction (PCR) were performed. PCR results combined with the blast counts from 21 leukemia patients were analyzed. Complete chimerism (100% donor cells) was found in 15 cases with remission, and incomplete chimerism in six cases with relapse. In the majority of cases, complete chimerism was always associated with no detectable blasts, while blasts were often detected in association with incomplete chimerism. There is significant correlation (P<0.0001) between the percentage of donor DNA and blast percentage in these patients. Early detection of incomplete chimerism may therefore predict a poor prognosis. In one patient (case 15), a differing percentage of donor DNA was observed between samples of bone marrow and peripheral blood collected on the same day. This may be due to the fact that allogeneic stem cells proliferate at different rates depending on their environment (bone marrow or peripheral blood). In addition, 100% donor cells found in the peripheral blood may not reflect the number of cells in the bone marrow. In case 17, asynchronous engraftment of donor cells was present between the white and red blood cell lineages, indicating that the degree of chimerism may not be the same in all cell lineages. At the time of this report, the significance of this observation is unknown and needs further investigation.

Adolescent↗
PubMed2001

Treatment with nephrectomy only for small, stage I/favorable histology Wilms' tumor: a report from the National Wilms' Tumor Study Group.

PURPOSE: Children younger than 24 months with small (< 550 g), favorable histology (FH) Wilms tumors (WTs) were shown in a pilot study to have an excellent prognosis when treated with nephrectomy only. PATIENTS AND METHODS: A study of nephrectomy only for the treatment of selected children with FH WT was undertaken. Stringent stopping rules were designed to insure closure of the study if the true 2-year relapse-free survival rate was 90% or lower. RESULTS: Seventy-five previously untreated children younger than 24 months with stage I/FH WTs for which the surgical specimen weighed less than 550 g were treated with nephrectomy only. Three patients developed metachronous, contralateral WT 1.1, 1.4, and 2.3 years after nephrectomy, and eight patients relapsed 0.3 to 1.05 years after diagnosis (median, 0.4 years; mean, 0.51 years). The sites of relapse were lung (n = 5) and operative bed (n = 3). The 2-year disease-free (relapse and metachronous contralateral WT) survival rate was 86.5%. The 2-year survival rate is 100% with a median follow-up of 2.84 years. The 2-year disease-free survival rate (excluding metachronous contralateral WT) was 89.2%, and the 2-year cumulative risk of metachronous contralateral WT was 3.1%. CONCLUSION: Children younger than 24 months treated with nephrectomy only for a stage I/FH WT that weighed less than 550 g had a risk of relapse, including the development of metachronous contralateral WT, of 13.5% 2 years after diagnosis. All patients who experienced relapse on this trial are alive at this time. This approach will be re-evaluated in a clinical trial using a less conservative stopping rule.

Disease-Free Survival↗
PubMed2001

Developing a pediatric outpatient transplantation program. The Children's Memorial Hospital experience.

We describe the development of a pediatric outpatient transplant program and our initial experience with autologous and allogeneic transplants performed partially or completely in the outpatient setting. Forty-eight autologous and seven allogeneic transplants have been performed in our institution in the outpatient setting between June 1994 and July 2000. The ablation used for the autologous outpatient transplants was VP-16 1000 mg/m2/ day as a continuous infusion for 2 days and Carboplatinum 667mg/m2/day for 2 days. The autologous inpatient transplants received Thio-tepa 300-mg/ m2per day x 3 days and cyclophosphamide 60 mg/kg/day for 4 days. For those patients who received an immune-ablative allogeneic outpatient transplant, the regimen consisted of Fludarabine 30 mg/m2/day for 6 days, followed by busulfan for children less than five years of age 1 mg/kg/dose x 8 doses and for those five years and older 0.8 mg/kg/dose x 8 doses, followed by ATG 40mg/kg/day x 4 days. Engraftment was complete in all transplants achieving an ANC >500 for the outpatient transplant in 15 days (10-35) vs. the inpatient in 15 days (14-58). This was not statistically significant. They achieved un-sustained platelets >20.0 by day 19 (14-58) for the outpatients and day 32 10-64) for the inpatient. The allogeneic immune ablative transplants were considered engrafted when their VNTRs were greater that 30% which was achieved at a median of 13 days (10-27). The economic data showed a statistically significant decrease in charges and direct costs between the outpatient (median charges $30 775, direct costs $8 389) and the inpatient (median charges $99 838, direct costs $42 757) transplants (p0.001). There was no difference in morbidity and mortality between the two groups but the use of empiric amphotericin B was markedly decreased in the outpatient transplants. In conclusion it is feasible and less costly to perform autologous hematopoietic stem cell transplants in the outpatient setting with no increase in morbidity and mortality. For the allogeneic transplants there is not yet enough data to establish a similar conclusion.

Ambulatory Surgical Procedures↗
PubMed2001

Natural killer cell lymphoma: report of two pediatric cases, therapeutic options, and review of the literature.

BACKGROUND: Natural killer (NK) cell lymphomas are rapidly fatal malignancies that to the authors' knowledge are rare in children. In the current study, the authors report the cases of two boys with NK cell lymphomas with refractory disease who both were salvaged with high dose chemotherapy and stem cell transplantation and compare these patients with those in the published experience. METHODS: A comprehensive literature review was performed to identify other cases of pediatric patients with NK cell lymphomas, their treatment, and outcome. RESULTS: One of the patients in the current study developed two recurrences and the other patient experienced early disease progression during front-line treatment. Both then were treated with high dose chemotherapy followed by stem cell rescue. At last follow-up, the patients remained free of disease at 15 months and 16 months, respectively, after transplantation (48 months and 22 months, respectively, from the time of diagnosis). In addition to the 2 patients in the current study, the authors found 13 pediatric patients reported in the literature to date. Of the 7 patients with localized (Stage I-II) disease, 5 patients (71%) were reported to be alive 1-107 months after diagnosis. Of the 6 patients with Stage IV disease, only the 2 patients who received high dose chemotherapy and stem cell rescue (33%) were alive at the time of last follow-up (at 30 months and 12 months, respectively). Including the patients reported in the current study, 9 of 15 children with NK cell lymphoma (all stages) (60%) were reported to be alive at the time of last follow-up. CONCLUSIONS: Although pediatric NK cell lymphomas rapidly can become fatal, it appears that high dose chemotherapy followed by stem cell transplantation is effective therapy, especially in patients with advanced or resistant disease. Further follow-up is needed to determine whether this treatment approach will be curative.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

Treatment of congenital erythropoietic porphyria in children by allogeneic stem cell transplantation: a case report and review of the literature.

Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder of porphyrin metabolism in which the genetic defect is the deficiency of uroporphyrinogen III cosynthase (UIIIC). Deficiency of this enzyme results in an accumulation of high amounts of uroporphyrin I in all tissues leading to hemolytic anemia, splenomegaly, erythrodontia, bone fragility, exquisite photosensitivity and mutilating skin lesions. We describe the case of a 23-month-old boy who was cured of his CEP by a matched-sibling allogeneic bone marrow transplant, and review the published clinical experience regarding transplantation in this disease. He is alive and disease-free 15 months post transplant. All of his disease manifestations except for the erythrodontia have resolved. His UIIIC level and stool and erythrocyte porphyrin metabolites have almost completely corrected. He is the sixth child reported to be cured of this disease by stem cell transplantation, five cases being long-term survivors. If patients with this disease have an HLA-matched sibling, then stem cell transplantation should be strongly considered because this is currently the only known curative therapy.

Blood Donors↗
PubMed2001

Detection of EBV DNA in the cord blood donor for a patient developing Epstein-Barr virus-associated lymphoproliferative disorder following mismatched unrelated umbilical cord blood transplantation.

Epstein-Barr virus-associated post-transplant lymphoproliferative disorder (PTLD) has been well described as a complication following allogeneic stem cell transplantation but has only recently been reported following umbilical cord blood (UCB) transplant. We report the case of a child transplanted with unrelated mismatched UCB for juvenile chronic myelogenous leukemia (JCML) who developed EBV-associated PTLD, which was confirmed pathologically, 139 days following stem cell infusion. There was no clinical response to reduction of immune suppression, high-dose acyclovir, or alpha interferon. The patient died 160 days after transplantation. EBV was detected by polymerase chain reaction in the cord blood unit used for transplantation. This case demonstrates that EBV-associated PTLD can occur following mismatched unrelated UCB transplant and may be related to transmission of EBV infection by donor lymphocytes.

B-Lymphocytes↗
PubMed2001

Unrelated cord blood transplant experience by the pediatric blood and marrow transplant consortium.

Cord blood (CB) has emerged as a potential source of hematopoietic stem cells for patients who are in need of hematopoietic stem cell transplant (HSCT). The authors analyzed the Pediatric Blood and Marrow Transplant Consortium's (PBMTC) data of consecutive unrelated CB transplants performed during the initial 2 years of using placental blood grafts. From January 1995 to December 1996 PBMTC performed a total of 44 unrelated CB transplant for a variety of diseases consisting of acute leukemias (n = 29), congenital conditions (n = 9), and bone marrow failure (n = 6). There were 15 females and 29 males with median age of 5 years (range 0.4-20.6 years) and median weight of 18.2 kg (range 6.3-70 kg). The median volume of CB units was 80 mL (range 44.5-215 mL) and the median cell dose given was 4.3 x 10(7)/kg of recipient weight (range 1.1-23 x 10(7)/kg). Techniques used for human leukocyte antigen (HLA) matching were serologic typing for class I HLA antigens and high-resolution molecular typing for HLA-DRB1 alleles. HLA disparities were as follows: 4 were 6/6 matches, 21 were 5/6, 15 were 4/6, and 4 were 3/6. Twenty-nine (66%) of CB units were DRB1 matched with recipients. Conditioning regimens consisted of either total body irradiation containing (n = 31) or chemotherapy only (n = 11) regimens. All but 3 patients receive cyclosporine as part of graft vs. host disease (GvHD) prophylaxis in combination with either methotrexate (MTX) or methylprednisolone (Pred). The other 3 patients had FK506 and MTX for GvHD prophylaxis. Myeloid engraftment (absolute neutrophil count > or = 500) occurred at a median of 21 days (range 10-43 days) and platelet > or = 50,000/mm3 was noted at a median of 44 days (range 16-102 days). Eight patients died too early (< day + 28) for evaluation of engraftment (5 for infection, 2 for multiorgan failure, 1 for toxic epidermolysis). The probability of having grade II-IV acute GvHD for all patients was 44 +/- 0.7%. The incidence of a GvHD is similar for 4/6 and 5/6 antigen when DRB1 matched, at 47 and 52%, respectively. Chronic GvHD was noted in 28% of patients surviving > 90 days. The Kaplan-Meier estimate of 4-year event-free survival was 43%. A Cox model for analysis of factors associated with survival was DRB1 matching, p = .001; cell dose, p = .009; and younger age, p = .03. In conclusion, CB transplant offers a good alternative to bone marrow transplant Although GvHD occurs, it is usually of low severity despite the high frequency of multiple HLA antigen mismatches. It also appears that a 4/6 is as good as a 5/6 matched antigen CB unit when DRB1 matched especially in the pediatric setting.

Adolescent↗
PubMed2001

A flow cytometric technique using thiazole orange to detect platelet engraftment following pediatric stem-cell transplants.

BACKGROUND: Thiazole orange (TO) is a nucleic-acid-specific dye that enters cells without pretreatment. When it binds to either RNA or DNA, there is an increase in fluorescence emission. This property has been utilized to measure the amount of newly released platelets using flow cytometry. These newly released platelets differ from more mature platelets because they still contain residual amounts of RNA, and have become known as reticulated platelets. METHODS: Peripheral blood samples were collected at least 48 h following platelet infusion. For validation, manual reticulocyte counts obtained in the laboratory were compared with results obtained using TO and flow cytometry. Following validation, experiments using platelet-rich plasma were performed to evaluate the presence of reticulated platelets in the sample. RESULTS: Validation experiments comparing the manual and flow cytometric reticulocyte counts gave a strong relationship between the two values (r(2) = 0.92). Reticulated platelet studies performed on platelet-rich plasma samples yielded the following results. Patients who did not engraft within 4 days were significantly different from patients who did engraft within 4 days, idiopathic thrombocytoperic purpura (ITP) patients, and donor platelet segments (all P < 0.0008). Patients who engrafted within 4 days, ITP patients, and donor platelet segments were all statistically similar (all P > 0.08). DISCUSSION: The statistical difference between patients who did engraft within 4 days and those who did not suggests that this method could have an important clinical impact in determining those patients who are still in need of platelet support. However, great care must be taken when performing and analyzing the results.

Adolescent↗
PubMed2001

Expansion of megakaryocyte precursors and stem cells from umbilical cord blood CD34+ cells in collagen and liquid culture media.

Umbilical cord blood (UCB) is now commonly used as a source of stem cells for hematopoietic reconstitution following myeloablative therapy in patients with a variety of diseases. Although UCB is a rich source of stem cells, platelet engraftment occurs at a median of 71 days which is significantly prolonged compared to allogeneic bone marrow. The number of megakaryocyte (MK) precursors in stem cell harvests appears to correlate inversely with the time to platelet engraftment. In an effort to increase the number of platelet precursors, we cultured CD34-selected cord blood mononuclear cells (MNC) in serum-free collagen medium with numerous cytokine combinations. The cells were cultured with four cytokines: interleukin-3 (IL-3), thrombopoietin (TPO), stem cell factor (SCF), and Flt-3); five cytokines, IL-3, TPO, SCF, Flt-3 plus granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo); or all six cytokines in combination. After 16 days, significant expansion of MK precursors (CD41(+)) and stem cells (CD34(+) and AC133(+) cells) were seen in cells cultured in IL-3, TPO, SCF, and Flt-3 with or without GM-CSF compared to the combinations that contained Epo (p < 0.05). Similar studies were performed using liquid culture medium, and after 14 days the number of MNCs, CD34(+), AC133(+), CD41(+), and CD61(+) cells were higher in the UCB cells cultured in IL-3, TPO, SCF, and Flt-3 compared to those cultured with those four cytokines plus GM-CSF. These results demonstrate that UCB stem cells can be effectively expanded ex vivo and enriched with platelet precursors using TPO, SCF, Flt-3, and IL-3, whereas the addition of Epo and GM-CSF is unnecessary.

AC133 Antigen↗