scholarly journals Effects of Caspase Inhibitors on Hematopoietic Engraftment after Short-Term Culture

2002 ◽  
Vol 11 (4) ◽  
pp. 351-358 ◽  
Author(s):  
Anne Wiesmann ◽  
A. Elena Searles ◽  
L. Jeanne Pierce ◽  
Gerald J. Spangrude
Keyword(s):  
Mouse Bone Marrow ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Caspase Inhibitors ◽  
Stem And Progenitor Cells ◽  
Short And Long Term ◽  
Cells Cultured

The induction of apoptosis during cytokine-induced proliferation of hematopoietic stem and progenitor cells (HSPC) may result in the loss of hematopoietic function. We tested the ability of several caspase inhibitors to maintain transplantation potential of mouse HSPC during in vitro culture. HSPC were isolated from mouse bone marrow by cell sorting and cultured in the presence of steel factor (STL) with or without various caspase inhibitors. After incubation, cells were harvested and tested for in vitro colony-forming cell (CFC) potential and transplantation activity in both short- and long-term in vivo assays. HSPC required STL to retain CFC activity during a 24-h culture at 37°C, and none of three caspase inhibitors could substitute for STL in this respect. In transplant assays, a twofold higher frequency of animals showed donor-derived blood cells 12 weeks after competitive transplantation of 50 HSPC cultured for 4 h in the presence of STL plus n-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (ac-YVAD) compared with 50 cells cultured in STL alone. To evaluate the effect of ac-YVAD on short-term engraftment, 500 cultured HSPC were transplanted into lethally irradiated mice. Animals transplanted with cells cultured in the presence of ac-YVAD showed a higher survival rate and a faster recovery of platelets and hematocrit compared with animals transplanted with cells cultured in STL alone. We conclude that both the short-term and the long-term engraftment potentials of HSPC cultured in the presence of STL + ac-YVAD were superior to that obtained from cells cultured in STL alone.

PROLACTIN SECRETION AND SYNTHESIS IN SHORT- AND LONG-TERM ORGAN CULTURE OF PITUITARY TUMOURS FROM ACROMEGALIC PATIENTS

1978 ◽  
Vol 87 (4) ◽  
pp. 701-715 ◽  
Author(s):  
F. Peillon ◽  
F. Cesselin ◽  
P. E. Garnier ◽  
A. M. Brandi ◽  
M. Donnadieu ◽  
...  
Keyword(s):  
Organ Culture ◽  
Prolactin Secretion ◽  
Pituitary Tumours ◽  
Short Term ◽  
Ultrastructural Studies ◽  
Gh Cells ◽  
Short And Long Term

ABSTRACT To evaluate the in vitro PRL secretion and synthesis by pituitary tumours from acromegalic patients, six somatotrophic and two somatomammotrophic adenomas were collected after surgery and divided into fragments 1 mm3 in size. Fragments were cultured in short-term (1–2–3–4 h) and in long-term organ culture (7 and 14 days). Media were collected for GH and PRL radioimmunoassay and fragments studied by electron microscopy. In two experiments, fragments from a somatotrophic adenoma and from a somatomammotrophic adenoma were cultured for 1 to 4 h and 9 to 16 days with [3H]leucine. In addition the effect of somatostatin (2.5 nmole/ml) upon PRL secretion was studied in short-term incubation. In long-term organ culture PRL concentrations increased in 8 out of 16 media samples collected from the five tumours and one normal pituitary cultured for 14 days, while GH concentrations decreased during the same time in all the experiments. The [3H]PRL/[3H] proteins ratios were 9.7 % on the 9th day of culture and 20.8 % on the 16th day while for GH, the ratios were respectively 40 and 34.7 %. Ultrastructural studies showed, besides GH cells, the presence of PRL secreting cells in the different tumours, after 7 or 14 days of culture. In short-term incubation, PRL concentrations in media increased in most of the samples collected from the three tumours incubated for 1 to 4 h and the [3H]PRL/[3H]proteins ratio was 10.6 % after 1 h and 21 % after 2 h of incubation. A decrease of PRL content (< 50 %) was observed in two of the three tumours incubated with somatostatin. These data indicate that 1) somatotrophic adenomas, as well as somatomammotrophic ones, are able to secrete and synthesize PRL in vitro, 2) PRL synthesis from these adenomas increases during culture, as from normal pituitary, presumably because of the release of PRL-secreting cells from the PRL inhibitory hypothalamic control (PIF), 3) somatostatin may reduce PRL release in vitro from somatotrophic adenomas as reported in some cases in vivo.

scholarly journals E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction

Blood ◽  
2011 ◽  
Vol 117 (13) ◽  
pp. 3529-3538 ◽  
Author(s):  
Qi Yang ◽  
Brandt Esplin ◽  
Lisa Borghesi
Keyword(s):  
Energy Metabolism ◽  
Myeloid Differentiation ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Altered Expression ◽  
Helix Loop Helix ◽  
Multipotent Progenitors

Abstract The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix transcription factors, contribute to HSC and MPP activity, but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However, long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover, E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism, and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together, these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism, and demonstrate that E47 is not required for short-term myeloid differentiation.

A Novel Road Map for Blood Lineage Commitment and Development: Identification of a Lympho-Myeloid Stem/Progenitor Cell Lacking Erythro-Megakaryocytic Potential.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2790-2790
Author(s):  
Liping Yang ◽  
Jörgen Aolfsson ◽  
Robert Månsson ◽  
David Bryder ◽  
Ole-Johan Borge ◽  
...  
Keyword(s):  
T Cell ◽  
Lineage Commitment ◽  
Mouse Bone Marrow ◽  
Erythroid Progenitors ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Developmental Potential ◽  
Road Map

Abstract The recent identification of common myeloid and lymphoid progenitors (CMPs and CLPs, respectively) lends support to the classical and currently prevailing model for hematopoietic commitment and blood lineage development, suggesting that the first and decisive lineage commitment step of adult hematopoietic stem cells (HSCs) results in an immediate and complete separation of myelopoiesis and lymphopoiesis. Virtually all of multipotent (lympho-myeloid) stem and progenitor cells in adult mouse bone marrow (BM) reside in the small Lin−Sca1+kithi (LSK) compartment. We now present data demonstrating that the LSK compartment in adult BM, can be separated into three phenotypically and functionally distinct HSC subsets, based on differential expression of CD34 and flt3. Long-term HSCs (with extensive self-renewing potential) reside in the LSKCD34−flt3− fraction. The LSKCD34+ short-term HSC compartment consists of LSKCD34+flt3− cells fulfilling all criteria of short-term HSCs, being highly enriched in CFU-S activity, and capable of rapidly reconstituting myelo-erythropoiesis and thereby rescuing myeloablated mice. The short-term LSKCD34+flt3− HSCs give upon transplantation rise to LSKCD34+flt3+ cells, that although sustaining a combined lymphoid (B and T cell) and myeloid (granulocyte and monocyte) developmental potential at the single cell level, loose their ability to adapt megakaryocytic and erythroid fates in vitro as well as in vivo. These evidence for the existence of LSKCD34+flt3+ cells with granulocyte, monocyte, B and T cell, but not megakaryocyte and erythroid development potentials, are not compatible with the first lineage commitment step of HSCs resulting in strict separation of common lymphoid and common myeloid differentiation pathways. Based on the present findings we rather propose an alternative road map for blood lineage development in which LSKCD34+flt3− short-term HSCs genereate megakaryocyte-erythroid progenitors and LSKCD34+flt3+ cells upon asymmetric cell divisions.

scholarly journals Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization

Blood ◽  
1995 ◽  
Vol 85 (6) ◽  
pp. 1472-1479 ◽  
Author(s):  
CL Li ◽  
GR Johnson
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Stem And Progenitor Cells

Murine bone marrow cells were fractionated by fluorescence-activated cell sorting into Rh123lo Lin- c-kit+ Ly6A+, Rh123hi Lin-c-kit+ Ly6A+, and Lin- c-kit+ Ly6A- populations within which most, if not all, of the hematopoietic activities of the marrow resided. The Rh123lo Lin- c- kit+Ly6A+ cells, which consist exclusively of small- or medium-sized lymphocyte-like cells, are highly enriched for long-term hematopoietic in vivo repopulating cells. The enrichment factor for these cells from the marrow was estimated as 2,000-fold. The Rh123hi Lin- c-kit+ Ly6A+ cells, although also highly enriched for day-12 spleen colony-forming units, were relatively depleted of long-term in vivo repopulation capacity. Most, if not all Lin- c-kit+ Ly6A- cells were Rb123hi. In contrast to both Rh123lo and Rh123hi Lin- c-kit+ Ly6A+ stem cell populations, the Lin- c-kit+ Ly6A- cells can be stimulated to proliferate in vitro in the presence of single cytokines, which is a characteristic of committed progenitor cells. No marked synergistic interactions between individual cytokines were observed with this cell population. Both Rh123hi Lin- c-kit+ Ly6A+ mature stem cell and Lin- c- kit+ Ly6A- progenitor cell populations displayed in vivo repopulation kinetics resembling those of the putative short-term hematopoietic repopulating cells.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brianna J. Klein ◽  
Anagha Deshpande ◽  
Khan L. Cox ◽  
Fan Xuan ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Critical Role ◽  
Cellular Transformation ◽  
Acute Leukemias ◽  
Hematopoietic Stem ◽  
Nucleosome Core ◽  
Stem And Progenitor Cells ◽  
Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

2021 ◽  
Author(s):  
Zixian Liu ◽  
Jinhong Wang ◽  
Miner Xie ◽  
Peng Wu ◽  
Yao Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Pcr Analysis ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Colony Assay ◽  
Megakaryocyte Progenitors ◽  
Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

Lithium Restores Abnormal Platelet 5-HT Transport in Patients with Affective Disorders

1980 ◽  
Vol 136 (3) ◽  
pp. 235-238 ◽  
Author(s):  
Alec Coppen ◽  
Cynthia Swade ◽  
Keith Wood
Keyword(s):  
Blood Platelets ◽  
Lithium Treatment ◽  
Before And After ◽  
Short And Long Term ◽  
5 Ht Uptake ◽  
Depressive Patients ◽  
Rate Of Accumulation

SummaryKinetic analysis of the transport of 5-HT into the blood platelets of depressed patients and recovered depressive patients has shown that the rate of accumulation of 5-hydroxytryptamine (5-HT) is significantly decreased both before and after recovery from the illness. This abnormality is corrected by both short and long-term lithium treatment. As a corollary to these studies, the effect of lithium in vitro on 5-HT uptake has been studied and the results are opposite to those reported in vivo. These findings suggest that lithium acts indirectly, and possible mechanisms of its action are discussed.

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