Total Number Of Stem Cells Research Articles

Prior Daratumumab Exposure and It's Correlation to Parameters Related to Apheresis Prior to Hematopoietic Stem Cell Transplantation in Multiple Myeloma

A number of recent studies have brought to light the potential detrimental effect the biologic monoclonal antibody daratumumab, directed towards CD38, may have on the collection of stem cell collection for hematopoietic stem cell transplantation (HSCT). In our evaluation of this claim, we retrospectively reviewed multiple myeloma patients who underwent autologous HSCT at a major tertiary transplant center between 2017 and 2022. A total of 240 patients were identified which included 62 patients who received daratumumab prior to stem cell collection and 178 patients who had no prior daratumumab exposure. Basic demographic data was collected as well as radiation, chemotherapy, and smoking history. No significant differences were identified between the groups based on demographics, smoking, or radiation exposure. Parameters related to peripheral stem cell collection were identified and recorded. We found that in patients exposed to daratumumab prior to apheresis, the mean number of days of apheresis required to reach the target collection goal was statistically increased compared to control (1.42 days ± 0.07 vs 1.26 ± 0.03; p<0.05). Furthermore, medications used to increase peripheral stem cell collection yield were also increased. It was demonstrated that the mean number of doses of plerixafor and G-CSF analogs were increased in the daratumumab group vs the control (1.08 ± 0.12 vs 0.6 ± 0.05; p<0.001 and 5.98 ± 0.31 vs 5.17 ± 0.06; p <0.001 respectively). Total number of stem cells collected both as an absolute number and controlled for the number of days of apheresis were lower in the daratumumab group compared to the control (5.31x10^6 ± 0.31 vs 6.68x10^6 ± 0.20; p<0.05 and 4.16x10^6 ± 0.32 vs 5.84x10^6 ± 0.21; p < 0.001 respectively). These results suggest that pre-apheresis exposure to daratumumab has a detrimental effect on peripheral stem cell collection. These results have a significant implications for both long term patient care and healthcare system resource utilization. The clinical outcome implications of daratumumab are currently not well known and further study is needed to determine if alterations in stem cell collection protocols would result in improved stem cell collection yields for patients with pre-collection daratumumab exposure. All data is expressed as mean ± SEM

Bone Marrow Grafts From Pediatric Donors May Contain A Considerable Number of Hematogones.

During CD34 + stem cell count to determine the number of stem cells in the allografts from pediatric donors, we noticed a considerable amount of early hematogones (eHGs) within the stem cell gate in flow cytometry. Since the number of hematogones causes a decrease in the total number of stem cells counted within the graft, we planned a retrospective study to analyze the effect of eHGs on transplant outcomes. We also wanted to show how allografts containing high amounts of early HGs affect transplant outcomes. Quantification of CD34 numbers and the number of eHGs were determined by flow cytometry. Patients were divided into 2 groups according to the number of CD 34+ cells calculated after subtracting the number of hematogones within the allograft. Those who received < 2 × 106/kg CD34+ cells and ≥ 2 × 106/kg were defined as group 1 and 2, respectively. Twenty-six patients and their 26 donors were included in the study. The median age of patients was 6.5years and 5.4years in Group 1 and 2, respectively. The median donor age was 9years in Group 1 and 7years in Group 2. The ages and genders were similar in the two groups (p > 0.05). The number of nucleated cells given to both groups was not different. The number of early hematogones given to both groups was similar (p = 0.93). The mean times to myeloid and platelet engraftments were also similar in the two groups. In this study, we provided trilineage engraftment to all patients in two groups. We could not find a considerable effect of these eHGs in myeloid and platelet engraftments. However, the number of patients included in our study is low, therefore we suggest a study including a large number of donors in order to confirm our findings.

Impact of Induction Treatment on Stem Cell Collection: A COVID Related Study

Introduction: Standard induction therapy for multiple myeloma consists of 3-6 cycles of bortezomib, lenalidomide, and dexamethasone (VRd) or carfilzomib, lenalidomide and dexamethasone (KRd). Receiving greater than 6 cycles of a lenalidomide containing regimen is thought to negatively impact the ability to collect sufficient CD34+ stem cells for autologous stem cell transplant (Kumar, Dispenzieri et al. 2007, Bhutani, Zonder et al. 2013). Due to the COVID-19 pandemic, at least 20 patients at University of Maryland Greenebaum Comprehensive Cancer Center (UMGCC) had transplant postponed, potentially resulting in prolonged exposure to lenalidomide containing induction regimens. Here, in the context of modern stem cell mobilization methods, we describe a retrospective study that suggests prolonged induction does not inhibit adequate stem cell collection for transplant.Methods: By chart review, we identified 56 patients with multiple myeloma who received induction with VRd or KRd and underwent apheresis or stem cell transplant at UMGCC between 10/1/19 and 10/1/20. Patients were excluded if they received more than 2 cycles of a different induction regimen, had a past medical history of an inborn hematological disorder, or participated in a clinical trial of novel stem cell mobilization therapy. We defined 1 cycle of VRd or KRd as 1 cycle of “lenalidomide containing regimen”. In accordance with routine clinical practice, we defined standard induction as having received 3-6 cycles of lenalidomide containing regimen and prolonged induction as having received 7 or more cycles.Results: 29 patients received standard induction (Standard induction cohort) and 27 received prolonged induction (Prolonged induction cohort) with lenalidomide containing regimens. The median number of cycles received by the Standard cohort was 6 (range 4-6), and the median number of cycles received by the Prolonged cohort was 8 (range 7-13). The frequency of KRd use was similar between patients who received standard induction and prolonged induction (27.58% vs. 25.93%, respectively). Standard induction and Prolonged induction cohorts were similar with respect to clinical characteristics (Fig 1), as well as the mobilization regimen used for stem cell collection (p = 0.6829).55/56 patients collected sufficient stem cells for 1 transplant (≥ 4 x 10 6 CD34 cells/kg), and 40/56 patients collected sufficient cells for 2 transplants (≥ 8 x 10 6 CD34 cells/kg). There was no significant difference in the total CD34+ stem cells collected at completion of apheresis between standard and prolonged induction (10.41 and 10.45 x 10 6 CD34 cells/kg, respectively, p = 0.968, Fig 2). Furthermore, there was no significant correlation between the number of cycles of lenalidomide containing regimen a patient received and total CD34+ cells collected (R 2 = 0.0073, p = 0.5324).Although prolonged induction did not affect final stem yield, prolonged induction could increase the apheresis time required for adequate collection or result in more frequent need for plerixafor rescue. There was no significant difference in the total number of stem cells collected after day 1 of apheresis between patients who received standard or prolonged induction (8.72 vs. 7.96 x 10 6 cells/kg, respectively, p = 0.557). However, patients who received prolonged induction were more likely to require 2 days of apheresis (44% vs. 25%, p = 0.1625) and there was a trend toward significance in which patients who received prolonged induction underwent apheresis longer than patients who received standard induction (468 vs 382 minutes, respectively, p = 0.0928, Fig 3). In addition, longer apheresis time was associated with more cycles of lenalidomide containing regimen, which neared statistical significance (R 2 = 0.0624, p = 0.0658, Fig 4). There was no significant difference between standard and prolonged induction with respect to the frequency of plerixafor rescue.Conclusions: Prolonged induction with lenalidomide containing regimens does not impair adequate stem cell collection for autologous transplant. Prolonged induction may increase the apheresis time required to collect sufficient stem cells for transplant, but ultimately clinicians should be re-assured that extending induction when necessary is not likely to increase the risk of collection failure. [Display omitted] DisclosuresBadros: Janssen: Research Funding; J&J: Research Funding; BMS: Research Funding; GlaxoSmithKline: Research Funding.

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation

ABSTRACTTo investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.

Abstract LB-147: Colonic crypt neuroendocrine cells regulate colonic stem cells during colorectal cancer development

Abstract Our studies on tissues from colon cancer patients show that in the development of CRC, stem cells (SC) overpopulation underlies tumor initiation and progression. Specifically, we have involved studying patients with Familial Adenomatous Polyposis (FAP) during CRC development, which carry cancer-predisposing germline mutations in the adenomatous polyposis coli (APC) gene. These results indicate that SC overpopulation caused by APC mutations is the driver mechanism. However, how the APC mutations cause SC overpopulation needs clarification. Similar to SCs, Neuroendocrine cells (NECs) comprise ∼1% of the total cells in the colon and both reside in the crypt stem cell niche in the colonic epithelium. NECs are involved in autocrine and paracrine signaling to help regulate the function of surrounding cells. This leads to an overall goal of this project, to study the relationship between NECs and SCs in young-onset CRC, especially those individuals with a genetic predisposition to CRC. Our overall hypothesis is that NECs play a key role in maintaining the SC population in the normal colon and, aberrant signaling from NECs contributes to tumor initiation and increased stemness. To begin to investigate underlying mechanisms, we studied human colon tissues with varying APC genotypes: (i) normal crypts (homozygous-wildtype APC); (ii) normal-appearing FAP crypts (heterozygous-mutant APC); (iii) adenomatous FAP crypts (homozygous-mutant APC); (iv) CRCs (homozygous-mutant APC&amp; other mutations). Preliminary data from our laboratory has shown that in colon cancer progression: (i) there is a decrease in the number of cells that co-express both SC and NEC markers (ii) there is a decrease in the total number of NECs, and (iii) there is an increase in the total number of SCs. Furthermore, we have established an in vitro co-culture model system to study regulation of SCs by NECs. Results from our model show that SSTR1 and GLP-2R NEC signaling plays a role in modulating colonic SC populations. This in vitro model system can help analyze the interaction between SCs and NECs during CRC progression. Taken together, our findings indicate that: (1) NE factors contribute to colonic SC regulation via an autocrine or paracrine mechanism in normal colon, and (2) dysregulation of these mechanisms contributes to colon cancer development. Using our established in vitro model system, we can track the changes in the SC and NEC population size, based on different conditions of anchorage independent growth and presence of potential therapeutic agents. Citation Format: Shirin R. Modarai, Lynn M. Opdenaker, Bruce M. Boman. Colonic crypt neuroendocrine cells regulate colonic stem cells during colorectal cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-147. doi:10.1158/1538-7445.AM2015-LB-147

Effect Of Cumulative Adverse Clinical Risk Factors On Patients With Multiple Myeloma Undergoing First GCSF Apheresis

With the new treatments for multiple myeloma (MM), increasing numbers of patients fail to mobilize sufficient peripheral blood stem cells (PBSC) for autologous stem cell transplant (ASCT). Multiple studies have identified clinical and laboratory factors, such as age, number of lines of chemotherapy, radiation exposure, bone marrow involvement, and low PLT count as risks for poor mobilization. However, there are fewer studies that analyze only the effect of multiple clinical risk factors on mobilization outcomes. We retrospectively analyzed 259 MM patients who underwent first apheresis after GCSF mobilization between December 2000 and December 2012. Clinical risk factors analyzed include age, number of lines of chemotherapy, number of cycles of chemotherapy, number of doses of cyclophosphamide, number of doses of lenalidomide, and prior external beam radiation. The standard dose of GCSF was 10 mcg/kg/day, however the exact dose for a significant number of patients was not known. Patients were assessed as to whether optimal (≥8x106 CD34+ cells/kg) or minimal (≥4x106 CD34+ cells/kg) number of stem cells for two ASCTs were collected. The median age of the entire cohort was 59.7 years (27.9-76.0). Overall 10.8% and 32.6% failed to collect the minimal and optimal number of stem cells after one round of apheresis. Of the twenty patients who underwent a second round of apheresis, 9 (45%) collected minimal and 7 (35%) collected optimal total number of stem cells, with an overall failure rate of 4.6% and 29.8%, respectively. The effect of the number of clinical risk factors on the mobilization failure during first apheresis is summarized in Table 1. For each additional clinical risk factor, the likelihood of collecting the minimal and optimal number of CD34+ cells is reduced by 34% (CI=0.484-0.893, p=0.0072) and 32% (CI=0.538–0.860, p=0.0013) respectively. On univariate analysis, all risk factors were analyzed as continuous variables, except for prior radiation which was analyzed as a categorical variable. Prior lenalidomide exposure (odds ratio=0.502, CI=0.297–0.845, p=0.0096), and prior radiation therapy (odds ratio=0.502, CI=0.293–0.861, p=0.0123) had the greatest negative predictive value. Of the 38 patients who were exposed to lenalomide (median 4 cycles; range 1-24 cycles), 13% and 42% failed to collect minimal and optimal number of stem cells in the first apheresis cycle, respectively. An association was seen between number of days required to collect target number of stem cells and number of risk factors (p≤0.001). Median number of days required to collect target number of stem cells for 0, 1 or 2+ clinical risk factors was 2, 2, and 4 days, respectively. When the effect of clinical risk factors were analyzed according to number of CD34+ cells/kg collected on each day of apheresis, statistically significant differences in collection efficiency were seen on the first 3 days of apheresis (Figure 1). In summary, clinical characteristics of patients with MM can potentially be used to predict mobilization failure. The presence of 2 or greater clinical risk factors adversely affect the ability to successfully collect the target stem cell dose. These risk factors may help in identifying high-risk MM patients who may benefit from alternative mobilization regimens that can be tested in prospective clinical trials.Number of Clinical Risk Factors012+N1128561% Patients not collecting optimal # of cells on 1st apheresis cycle23.231.850.895% CI15.8-32.122.1-42.833.7-63.9p-value0.19710.0003% Patients not collecting minimal # of cells on 1st apheresis cycle5.411.818.095% CI2.0-11.35.8-20.69.4-30.0p-value0.11950.0096Median number of days of collection224Range1-81-91-10p-value0.4233&lt;0.0001 Disclosures: No relevant conflicts of interest to declare.

Fractionated Stem Cell Infusions for Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplant

AbstractAbstract 4550 Background:High dose therapy with autologous stem cell transplant (SCT) is an established therapeutic modality for multiple myeloma (MM). Although effective, it is associated with significant symptom burden due to the obligate period of neutropenia in addition to an increased risk of infectious complications. In animal models, infusing stem cells over a period of days enhances immune reconstitution (Felfly H, et al. Br J Haematol. 2009; 148: 646–658). We hypothesized that multiple stem cell infusions could reduce the period of neutropenia and enhance immune recovery resulting in a better tolerated procedure. Methods:We are conducting a phase II trial of multiple fractionated autologous stem cell infusions after high-dose Melphalan. We compared initial engraftment kinetics of the first 14 patients to receive multiple infusions (Days 0, +2, +4, +6) after high-dose Melphalan (study group) to 23 patients receiving high-dose Melphalan followed by the standard single stem cell infusion (Day 0) (standard group). Pegfilgrastim was given to all patients on Day +1. All patients underwent SCT between October 2011 and July 2012. Results:Results are shown in Table 1. Similar numbers of stem cells were collected in the study and standard groups. The total number of stem cells infused was 9.7 × 10∧6 CD34+ cells/kg in the study patients compared to 5.0 × 10∧6 CD34+ cells/kg in the standard patients. To date, the median duration of neutropenia (days of ANC < 500) and lymphopenia (days of ALC < 500) as well as the days to neutrophil, lymphocyte and platelet engraftment are similar. There are no significant differences in the number of red cell or platelet transfusions, days of fever, diarrhea, empiric antibiotics or documented infections. Despite receiving multiple infusions, none of the patients in the study group experienced an infusion reaction. Engraftment syndrome occurred in 3/14 (21%) of the study patients. The median duration of hospitalization was 15 days in the study cohort versus 17 days in the concurrent control group (P = 0.01). Conclusion:Multiple stem cell infusions following high dose Melphalan in patients with MM were not associated with a higher incidence of adverse side effects from DMSO nor unexpected frequency of engraftment syndrome. Fractionating stem cells over a period of days may result in less antibiotic use, reduced frequency of infection and was associated with shorter hospitalizations. We continue to collect patient reported symptom scores in order to determine if we can improve patient experience during SCT. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Prognosis of Low- or Intermediate-1-Risk MDS with Del(5q) Chromosome Abnormality During Lenalidomide Therapy Depends on the Capacity of the Neoplastic Stem Cell to Differentiate Into Maturing Erythroid Committed Stem Cells,

Abstract 3819Transfusion-dependent anemia is a characteristic complication of myelodysplastic syndromes (MDS) with del(5q) chromosome abnormality. Background appears to be a haplo-insufficiency of the RPS-14 gene located within the CDR. Lenalidomide has improved the treatment of MDS with 5q deletion inducing a hematologic (erythroid) and cytogenetic response in the majority of patients. The exact mechanisms of action of lenalidomide explaining its effects on erythropoiesis and their possible prognostic relevance are not clear so far. PATIENTS:We present the first data on in-vivo changes at the level of hematopoietic stem cells (SC), erythroid committed SC (SC-e), maturation of SC-e to erythroblastic islands and normoblasts observed during lenalidomide therapy within the bone marrow from a total of 39 transfusion-dependent patients with low- or intermediate-1-risk MDS with del(5q) chromosome aberration (MDS.5q-) who were recruited and treated with lenalidomide (10 mg / d) at the European site of the MDS-003 study and whose bone marrow samples (aspirates and biopsies) were taken prospectively at 6-month intervals after start of treatment. Mature erythroid precursors were marked by anti-hemoglobin-A-antibody (HbA); immature, HbA- erythroid precursors by anti-glycophorin-C-antibody (GPC), and the SC compartment by anti-CD34 antibody. Due to their low number, the numerical densities of SC-e were estimated indirectly by the numerical density of erythroblastic islands within bone marrow whereas the total number of SC was estimated by a statistical approach assuming that SC give rise to clusters of CD34+ precursors. The results were correlated with lenalidomide dose, RPS-14, SPARC, and glycophorin A (GPA) gene expression, degree of anemia, transfusion dependence, cytogenetic and molecular (FISH) response, clonal evolution, AML-free and overall survival of patients. RESULTS:Lenalidomide therapy resulted in a marked increase of SC-e, differentiation of SC-e to GPC+HbA- erythroid precursors, pronounced production of HbA+ precursors and erythrocytes resulting in transfusion independence in 54 % of patients. Lenalidomide-induced increase of SC-e did not correlate with a rise in the total number of SC. It was more pronounced in patients with a cytogenetic response, but it occurred also in patients without a cytogenetic remission. 17 / 39 patients with a minimal therapy effect on erythropoiesis achieving the 1% percentile of normal healthy marrow and continuing for > 12 months showed an excellent prognosis with a probability of survival > 90 % 60 months after start of treatment and 120 months after diagnosis of disease irrespective of their hematologic response whereas the median survival time of the patients without minimal effect on erythroid precursors (n = 22) was shorter than 20 months after start of treatment (P < 0.0002). In multivariate survival analysis, changes of erythropoiesis occurring during lenalidomide treatment, clonal evolution of disease, and age of patients were the only variables providing independent prognostic information (P < 0.00005). Classification of disease (FAB, WHO, IPSS, WPSS), changes of peripheral blood cell counts, transfusion independence, cytogenetic and molecular response, RPS-13, SPARC, or GPA gene expression did not provide additional independent prognostic information (P > 0.05). CONCLUSIONS:Lenalidomide promotes erythroid differentiation at stem cell level as well as at more mature stages of erythropoiesis affecting both, the neoplastic clone as well as non-neoplastic hematopoiesis. The anti-neoplastic effect of lenalidomide does not seem to be relevant for the prognosis of the patients; (1) genetic instability of neoplastic SC / precursor resulting in clonal evolution on the one and (2) the capacity of neoplastic SC / precursors to respond to lenalidomide therapy with the production of SC-e and more mature erythroid precursors on the other hand appear to be significant. By combining both variables, three groups can be discriminated allowing for a therapy-specific prognostic scoring: one group with excellent prognosis (100 % survival 5 years after start of therapy) comprising ∼ 33 % of patients, another with poor prognosis (0 % survival 5 years after start of therapy) comprising ∼ 28 % of patients, and a third group with intermediate prognosis (53 % dying during this period of observation) comprising ∼ 39 % of patients (P < 0.000001). Disclosures:Buesche:Celgene Corp.: reference pathologist of the MDS-004 and the MDS-Le-Mon-5 studies. Giagounidis:Celgene Corp: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Göhring:Celgene Corp.: reference cytogeneticist of the MDS-004 study. Schlegelberger:Celgene Corp.: reference cytogeneticist of the MDS-004 study. Knight:Celgene Corp.: Employment.

Changing phenotypes of hematopoietic stem cells.

Identification of the surface phenotypes of hematopoietic stem cells is an important subject in both clinical stem cell transplantation and basic research in hematopoiesis. Transplantation of pure populations of stem cells should eliminate the occurrence of graft-vs-host disease (GVHD) in allogeneic transplantation and may reduce the recurrence rate of malignancies in autologous transplantation. Availability of highly enriched populations of stem cells should facilitate investigations in a number of areas such as in vitro expansion, cryogenic storage, and gene therapy using stem cells. An ultimate goal is to develop a method to quantitate the number of living stem cells based on their surface characteristics. During the last two decades, investigators identified a number of surface molecules, which, in combinations, were thought to provide exact definition of murine and human stem cells. Recent studies of murine stem cells, however, clearly demonstrated that expression of the surface antigens of stem cells is under the influence of developmental stages and the kinetic state of the stem cells. This review summarizes the concept of the changing phenotypes of hematopoietic stem cells.

The Kinetics of Murine Hematopoietic Stem Cells In Vivo in Response to Prolonged Increased Mature Blood Cell Production Induced by Granulocyte Colony-Stimulating Factor

Because of the complexity of appropriate stem cell assays, little information on the in vivo regulation of murine stem cell biology or stemmatopoiesis is available. It is unknown whether and how in vivo the primitive hematopoietic stem cell compartment is affected during a continued increased production of mature blood cells. In this study, we present data showing that prolonged (3 weeks) administration of granulocyte colony-stimulating factor (G-CSF), which is a major regulator of mature granulocyte production, has a substantial impact on both the size and the location of various stem cell subset pools in mice. We have used the novel cobblestone area forming cell (CAFC) assay to assess the effects of G-CSF on the stem cell compartment (CAFC days 7, 14, 21, and 28). In marrow, in which normally 99% of the total number of stem cells can be found, G-CSF induced a severe depletion of particularly the most primitive stem cells to 5% to 10% of normal values. The response after 7 days of G-CSF treatment was an increased amplification between CAFC day 14 and 7. However, this response occurred at the expense of the number of CAFC day 14. It is likely that the resulting gap of CAFC day 14 cell numbers was subsequently replenished from the more primitive CAFC day 21 and 28 compartments, because these cell numbers remained low during the entire treatment period. In the spleen, the number of stem cells increased, likely caused by a migration from the marrow via the blood, leading to an accumulation in the spleen. The increased number of stem cells in the spleen overcompensated for the loss in the marrow. When total body (marrow and spleen) stem cell numbers were calculated, it appeared that a continued increased production of mature granulocytes resulted in the establishment of a higher, new steady state of the stem cell compartment; most committed stem cells (CAFC day 7) were increased threefold, CAFC day 14 were increased 2.3-fold, CAFC-day 21 were increased 1.8-fold, and the most primitive stem cells evaluated, CAFC day 28, were not different from normal, although now 95% of these cells were located in the spleen. Four weeks after discontinuation of the G-CSF treatment, the stem cell reserve in the spleen had returned to a normal level, whereas stem cell numbers in marrow had recovered to values above normal. This study shows that the primitive stem cell compartment is seriously perturbed during an increased stimulation of the production of mature blood cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The kinetics of murine hematopoietic stem cells in vivo in response to prolonged increased mature blood cell production induced by granulocyte colony-stimulating factor

Because of the complexity of appropriate stem cell assays, little information on the in vivo regulation of murine stem cell biology or stemmatopoiesis is available. It is unknown whether and how in vivo the primitive hematopoietic stem cell compartment is affected during a continued increased production of mature blood cells. In this study, we present data showing that prolonged (3 weeks) administration of granulocyte colony-stimulating factor (G-CSF), which is a major regulator of mature granulocyte production, has a substantial impact on both the size and the location of various stem cell subset pools in mice. We have used the novel cobblestone area forming cell (CAFC) assay to assess the effects of G-CSF on the stem cell compartment (CAFC days 7, 14, 21, and 28). In marrow, in which normally 99% of the total number of stem cells can be found, G-CSF induced a severe depletion of particularly the most primitive stem cells to 5% to 10% of normal values. The response after 7 days of G-CSF treatment was an increased amplification between CAFC day 14 and 7. However, this response occurred at the expense of the number of CAFC day 14. It is likely that the resulting gap of CAFC day 14 cell numbers was subsequently replenished from the more primitive CAFC day 21 and 28 compartments, because these cell numbers remained low during the entire treatment period. In the spleen, the number of stem cells increased, likely caused by a migration from the marrow via the blood, leading to an accumulation in the spleen. The increased number of stem cells in the spleen overcompensated for the loss in the marrow. When total body (marrow and spleen) stem cell numbers were calculated, it appeared that a continued increased production of mature granulocytes resulted in the establishment of a higher, new steady state of the stem cell compartment; most committed stem cells (CAFC day 7) were increased threefold, CAFC day 14 were increased 2.3-fold, CAFC-day 21 were increased 1.8-fold, and the most primitive stem cells evaluated, CAFC day 28, were not different from normal, although now 95% of these cells were located in the spleen. Four weeks after discontinuation of the G-CSF treatment, the stem cell reserve in the spleen had returned to a normal level, whereas stem cell numbers in marrow had recovered to values above normal. This study shows that the primitive stem cell compartment is seriously perturbed during an increased stimulation of the production of mature blood cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Steel factor influences the distribution and activity of murine hematopoietic stem cells in vivo.

To determine the effects of steel factor (SIF) on the number and distribution of phenotypically defined hematopoietic stem cells in vivo, mice were treated with continuous s.c. infusions of SIF for up to 7 days. The bone marrow demonstrated a transient 5-fold increase in the number of c-kit-positive lineage-negative/low cells with no change in cellularity. The radioprotective capacity of bone marrow cells was significantly reduced, and a 30% decrease in Thylo Lin-/lo Sca-1+ stem cells (Sca+ cells) was observed. In marked contrast, in the spleen a 2-fold increase in cellularity was accompanied by a 24-fold increase in c-kit-positive lineage-negative/low cells. SIF-treated spleen cells provided increased radioprotection and a corresponding 4-fold increase in the number of Sca+ cells. In the peripheral blood, an increase in both neutrophils and lymphocytes resulted; however, the number of c-kit-positive lineage-negative/low cells remained < 1%. SIF produced a 25-fold increase in radioprotection capacity and a 20-fold increase in the number of Sca+ cells in the peripheral blood. The increased radioprotection capacity of both the spleen cells and peripheral blood cells was associated with donor-derived, long-term multilineage reconstitution of recipient mice. The total number of Sca+ cells isolated per mouse after SIF treatment was not significantly increased. These results show that exogenous SIF treatment causes a redistribution of Sca+ cells and stem cell activity while having little effect on the total number of stem cells in the mouse.

Hemopoietic Stem Cells as a Function of Erythropoiesis

hematocrit values and red cell volume per 100 g of body weight. Influences on the results of the stem cell assays of factors related to red cell mass other than the level of erythropoietic activity were thereby equalized for the two groups. The 30-day survival rate, the number of endogenous spleen colonies, and the rate of erythropoietic recovery following x-irradiation were substantially increased in mice with increased erythropoietic activity as compared to mice in which erythropoiesis had ceased at the time of the stem cell assay. It was concluded that an increase in erythropoietic activity is associated with an increase in the total number of hemopoietic stem cells in an animal. The concentration of stem cells in femoral bone marrow was identical for the two experimental groups. It was suggested that an increase in the total number of stem cells is accomplished by an increase in the number of stem cells of the spleen.