WWW.DISSERTATION.XLIBX.INFO
FREE ELECTRONIC LIBRARY - Dissertations, online materials
 
<< HOME
CONTACTS



Pages:     | 1 |   ...   | 5 | 6 || 8 | 9 |   ...   | 14 |

«3. Medizinische Klinik und Poliklinik – Hämatologische Forschung Cks1 is a critical regulator of hematopoietic stem cell cycling, quiescence and ...»

-- [ Page 7 ] --

RESULTS Mice LSK cells expressing high levels of the SLAM family receptor CD150 are known to possess high self-renewal activity and one of the most immature HSC populations is defined as CD34-CD150+ LSK [46, 48, 173]. Flow cytometry analysis of the WT control and Cks1-/- mice demonstrated that the CD150high LSK cells are part of the CD34- LSK fraction (Fig. 8C). Based on that finding, CD150+ LSK in this study are referred as LT-HSC.

In order to determine how the different subsets are represented in a steady state mice lacking Cks1, the absolute numbers and the percentages of HSC/HPC from Cks1-/- mice was compared to those of WT animals. Consistent with the decreased total cell numbers in Cks1-/- BM (Fig. 7E), the absolute MPP, CMP and GMP numbers were significantly decreased (Fig. 9A), whereas the frequencies of these subpopulations were unaffected in Cks1-/- BM (Fig.

9A). No significant changes were observed in the LSK subsets of Cks1-deficient mice compared to WT (Fig. 9B). However, analysis of the very early hematopoietic stem cell subsets of CD34- LSK and CD34-CD150+ LSK revealed a significant increase in their frequency in the BM of Cks1-deficient mice (Fig. 9B). This observation led to the assumption that the early stage of hematopoiesis is disturbed at steady state in the absence of Cks1.

RESULTS

Figure 9: Decreased absolute HPC numbers and increased relative HSC numbers in Cks1-/- mice.

(A) Total cell number and percentage of total cell number of progenitor populations (n=20 for each genotype, 6 independent experiments). (B) Total cell number and percentage of total cell number of LSK; CD34-LSK (n=20 for each genotype, 6 independent experiments) and CD34CD150+ LSK (n=15 for each genotype, 5 independent experiments).

RESULTS

4.2.3. Loss of Cks1 results in decreased hematopoietic colony formation A well-established method to test the clonogenetic capacity of hematopoietic cells is the methylcellulose assay [168].To investigate whether the quality of colony forming hematopoietic cells was affected by the loss of Cks1, methyl cellulose assays with freshly isolated FL, BM and Lin- BM cells were performed and the number of colonies for the respective genotype was determined. A significantly reduced ability of Cks1-/- BM and FL cells to form colonies was observed (Fig. 10), indicating reduced clonogenetic capacity in the absence of Cks1. Since an equal number of cells were used for both genotypes, and there were no significant differences in the percentage of colony forming progenitors (Fig. 9A) and even an increase in the percentage of stem cells (Fig. 9B), it was assumed that HPC/HSC lacking Cks1 are impaired in their differentiation capacity, proliferation or survival.

Figure 10: Loss of Cks1 results in decreased colony formation.

Colony count from methylcellulose assays with fetal liver cells (Cks1+/+ n=10; Cks1-/- n=9), bone marrow (Cks1+/+ n=12; Cks1-/- n=11) and lineage-depleted cells (Cks1+/+ n=20; Cks1-/n=18).

4.2.4. Decreased colony formation and HPC number after cultivation of Lin- Cks1-/- cells on stromal cells The stromal cell line EL08-1D2 is known to maintain hematopoietic stem and progenitor cells in vitro [163]. Co-cultures of Cks1-/- and WT cells were established to further observe the behavior of the colony forming cells. Therefor the EL08-1D2 cell line was used to support 5000 lineage-depleted WT or Cks1BM cells. Freshly isolated cells were incubated with irradiated (30 Gy, in order to restrain overgrow) stromal cells for 14 days (Fig. 11A). Afterwards, the bone marrow cells were harvested, half well was seeded in methyl cellulose medium, and the rest was used for FACS analysis. Consistent with the results at steady RESULTS state (Fig. 10), significantly fewer colonies were formed from Lin- Cks1-/- cells after co-culture than from the WT cells (Fig. 11B). Furthermore, FACS analysis revealed that there was a correspondingly significant decrease in the remaining Cks1-/- MPP cells after the 14 days of co-culture (Fig. 11C). Interestingly, similar to the steady state condition (Fig. 9B), there was no drop in the LSK subset, but a slight increase compared to the WT LSK (Fig. 11C), proving that Cks1-deficient hematopoietic cells tend to accumulate in the early hematopoietic subsets.

Figure 11: Decreased colony formation and progenitor cell number after cultivation of Lin- Cks1-/- cells on stromal cells.

(A) Scheme of the experiment: 5000 Lin- BM cells from WT and Cks1-/- mice were cultivated for 14 days on irradiated stromal cells. (B) CFU Assay after cultivation on stromal cells. (C) FACS analysis representing the remaining LSK and MPP cells after 2 weeks of cultivation on stromal cells (Cks1+/+ n=6; Cks1-/- n=6; 3 independent experiments).

4.2.5. Cks1-/- hematopoietic cells proliferate slower in culture Since Cks1-/- cells formed fewer colonies at steady state condition and after cocultivating them with stromal cells, and the Cks1-/- MPP pool was decreased in vivo, it was hypothesized, that the cells lacking Cks1 are impaired in their proliferation or in their survival. To test this, ex vivo experiments were RESULTS performed (Fig. 12A, 13A). First, the cell cycle in cultivated bone marrow cells was analyzed. Bone marrow cells from WT and Cks1-/- mice were isolated and depleted of lineage-committed cells using micro beads. The lineage negative cells were then cultivated in the presence of cytokines, which have been shown to maintain early hematopoietic cells in culture [174-176]. To analyze cell cycle progression an assay with the synthetic nucleoside BrdU was applied. FACS analysis of the cells after 5 days of cultivation showed a significantly reduced S phase fraction in Cks1-/- Lin- cells as compared to WT Lin- cells (Fig. 12B, C), indicating that Cks1 is important for entry into S phase. A similar effect can be observed in WT vs. Cks1-/- MEFs [177].





Figure 12: Cks1-/- cells proliferate slower in culture.

(A) Experimental procedure: Lin- BM WT and Cks1-/- cells were cultivated for 5 days with supplemented growth factors. (B) Representative dot blots from cell cycle analysis with BrdU.

(C) Frequency of Lin- Cks1+/+ and Cks1-/- cells in the different cell phases (n=2 for each genotype).

RESULTS

4.2.6. Cks1 regulates survival of progenitor cells in vitro The lower percentage of cells in the S phase (Fig. 12C) implied that the decrease in MPP numbers in vivo (Fig. 9A) and in vitro (Fig. 11C) and the decline in Cks1-/- colonies of BM or FL cells (Fig. 10) or after co-culture (Fig.

11B) are due to decelerated proliferation in the cells lacking Cks1. Consistent with reduced S Phase progression, significantly lower cell numbers were recovered after 5 days of cytokine stimulated culture of lineage depleted Cks1-/deficient cells compared to WT cells (Fig. 13A, B). In particular, the number of Cks1-/- MPP and LSK was significantly reduced (Fig. 13B). An alternative or accessory explanation for the observed differences in HSC/HPC generated in culture is that Cks1-/- cells could be more susceptible to the induction of apoptosis. To find out whether this was the case, an apoptosis analysis was performed (Fig. 13A). To induce apoptosis the stimulated Cks1-/- or WT cell cultures were deprived of cytokines for 24 hours and subsequently stained with Annexin V and PI for FACS analysis. The cytokine depletion induced a significantly increased apoptotic rate in the Cks1-/- Lin- and MPP fraction but not in the Cks1-deficient LSK (Fig. 13C).

Taken together, loss of Cks1 resulted in accumulation of LT-HSC at steady state, reduced proliferation of BM cells and sensitization of MPP towards apoptosis upon growth factor withdrawal. This finding suggests that Cks1 function is required for optimal growth and survival of hematopoietic cells.

RESULTS

Figure 13: Cks1 regulates survival of progenitor cells in vitro.

(A) Experimental procedure: Lin- BM WT and Cks1-/- cells were cultivated for 5 days with supplemented growth factors and subsequently remaining cells were counted and analyzed by FACS for LSK and MPP populations. In the second part of the experiment, cells were washed from the growth factors and analyzed for apoptosis 12 hours later. (B) Cell number and remaining LSK and MPP cells after 5 days cultivation with growth factors (n=7 for each genotype; 3 independent experiments). (C) On the left: percentage of apoptotic cells after

growth factor withdrawal (n=8 for each genotype, three independent experiments); on the right:

representative FACS dot blots (Lin- fraction) of the Annexin V/PI apoptosis analysis.

RESULTS

4.2.7. Increased B-Lymphocytes- and decreased granulocytes frequency after cytotoxic stress in Cks1-knockout mice Cell culture is an in vitro model for cellular stress and the results in this study so far demonstrate that Cks1 affects the response to hematopoietic stress in vitro.

To further analyze whether Cks1 is also involved in the response towards hematopoietic stress in vivo, Cks1-/- and WT control mice were injected with the cytotoxic agent 5-Fluorouracil (5-FU). 5-FU kills actively cycling cells, thus ablating the progenitor pool and all dividing hematopoietic cells but sparing the pool of quiescent, non-dividing HSC [171, 178]. Such chemo-ablative stress has been shown to efficiently activate the proliferation of the dormant HSC in order to replenish the blood system [7, 179, 180]. Using cell counting and flow cytometry the regeneration of the mature blood cells and of the HSC/HPC pool was determined on day 6 after 5-FU injection. To facilitate the detection of dividing cells, the mice were injected with BrdU 12 hours before sacrifice (Fig.

14A). No differences were observed in the regeneration of the white blood cells (WBC) and the lymphoid and myeloid blood populations in WT and Cks1deficient mice 6 days after 5-FU injection (Fig. 14B, C). Though, detailed FACS analysis revealed significantly decreased granulocytes and a significantly increased B cell fraction (Fig. 14D, E) in the blood of Cks1-/- mice, indicating a disturbed distribution of the different mature populations.

RESULTS

Figure 14: Increased B cells- and decreased granulocytes frequency after cytotoxic stress in Cks1-/- mice.

(A) Schematic representation of the experiment: WT and Cks1-/- mice were treated i.p. with 5FU (150 µg/g body weight); after 6 days BrdU was injected and 12 hours later the peripheral blood, MPP and LSK subsets were analyzed by FACS. (B) WBC counts 6 days after 5-FU injection. (C) Fractions of recovered myeloid (B220-CD4/8a-) and lymphoid (Gr1-CD11B-) cells.

(D) Detailed FACS analysis of the B cells (B220+), T cells (CD4+ CD8a+), granulocytes (Gr1++CD11B+) and monocytes (Gr1+CD11B+) 6 days after 5-FU treatment. (E) Representative FACS dot blots from the blood analysis in (D) (Cks1+/+ n=14 Cks1-/- n=15, 3 independent experiments).

RESULTS

4.2.8. Cks1 controls stress hematopoiesis in LSK cells: impaired regeneration after cytotoxic stress Consistent with the steady state results (Fig. 8), the CFU potential was significantly reduced in the BM of 5-FU-treated Cks1-/- mice as compared to the WT controls (Fig. 15A). Also, the absolute BM and specifically MPP and LSK cell numbers were reduced in 5-FU treated Cks1-/- animals and the reduction was significant in the LSK population (Fig. 15B). To further estimate the recovery of HSC/HPC after cytotoxic stress, the ratio of absolute numbers of BM cells, MPP, and LSK (determined using flow cytometry) on day 6 to the absolute number of a matched control group of the respective genotype that was left untreated was calculated. The ratio of BM cells in the Cks1-/- mice was comparable to that in the control mice and the ratio of Cks1-/- MPP was decreased but not significantly (Fig. 15C). However, there was a striking decay in the ratio of the LSK (Fig. 15C). These results confirm that lack of Cks1 predominantly affects the immature hematopoietic compartment. Most importantly, there was a significant decay in the incorporation of BrdU in Cks1-/LSK (Fig. 15D) strongly suggesting that the reduced numbers of LSK cells was the result of reduced cell cycling. Collectively, these results point to Cks1 being a crucial regulator of the regenerative response of LSK and colony-forming cells after cytotoxic stress.

RESULTS

Figure 15: Cks1 controls stress hematopoiesis in LSK cells: impaired LSK regeneration after cytotoxic stress.

(A) Colony counts after methyl cellulose assays with 5-FU treated WT and Cks1-/- bone marrow cells 6 days after 5-FU injection (n=8 for each genotype). (B) Total BM, MPP and LSK cell number 6 days after BrdU treatment (Cks1+/+ n=14 Cks1-/- n=15, 3 independent experiments).

(C) Ratio (day 6/day 0) of absolute cell numbers. (D) On the left: representative dot blots from the analysis of LSK cells after 5-FU and subsequent BrdU treatment; on the right: BrdU incorporation in the LSK of wild type and Cks1-/- mice (Cks1+/+ n=14 Cks1-/- n=15, 3 independent experiments).

RESULTS

4.2.9. Bone marrow transplantations: Cks1-/- bone marrow cells are able to reconstitute recipient mice 5-FU treatment experiments address the initial stages of hematopoietic regeneration after challenge. The most articulate parameter for enduring hematopoietic recovery is, however, long-term engraftment of HSC. The gold standard for analyzing engraftment and self-renewal capability of HSC is the transplantation of BM cells into lethally irradiated hosts [31, 33]. The irradiation of the recipients leads to cell death of most of the hematopoietic cells, therefore the transplanted BM cells are initiated to divide and replenish the hematopoietic system of the host. Serial transplantations of BM cells deliver information about the self-renewal potential of HSC since only the early (LT-HSC) are capable of long-term engraftment [33].

To study engraftment and self-renewal capacity of Cks1-deficient hematopoietic cells, serial BM transplantations were performed. In order to track donor,

recipient and helper hematopoietic cells the CD45 congenic system was used:



Pages:     | 1 |   ...   | 5 | 6 || 8 | 9 |   ...   | 14 |


Similar works:

«Presentation to House Committee on Public Health Thomas Suehs Executive Commissioner Billy Millwee Deputy Executive Commissioner for Health Services Operations January 24, 2012 House Bill 300 • The bill delineates responsibilities and prohibitions associated with handling protected health information and establishes enhanced penalties for existing acts or offenses associated with misuse of information when those acts or offenses include medical information • Several sections of the bill...»

«THE ROLE OF TAF PROTEINS DURING EARLY ZEBRAFISH DEVELOPMENT by ANDREAS ZAUCKER A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY College of Medicine & Dentistry The University of Birmingham April 2011 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The...»

«Assuring Best Practice in First Year First Session Assessment: Report on Tier One and Tier Two Course Reports Report prepared for Senate Education Committee September, 2013 Report prepared by: Dr Betty Gill Associate PVC Education, Health & Science, Leader, UWS STaRS Project With contributions from: Dr Lynn Burnett Dr Caroline Henderson-Brookes Ms Erika Matruglio Dr Evelyn Hibbert Ms Lauren Ross Assoc Professor Bronwyn Cole Assoc Professor Craig Ellis Dr Thuy Vu Assuring Best Practice in First...»

«ANNEX I SUMMARY OF PRODUCT CHARACTERISTICS 1 1. NAME OF THE MEDICINAL PRODUCT Zometa 4 mg powder and solvent for solution for infusion 2. QUALITATIVE AND QUANTITATIVE COMPOSITION One vial contains 4 mg zoledronic acid, corresponding to 4.264 mg zoledronic acid monohydrate. For the full list of excipients, see section 6.1. 3. PHARMACEUTICAL FORM Powder and solvent for solution for infusion White to off-white powder and clear, colourless solvent. 4. CLINICAL PARTICULARS 4.1 Therapeutic...»

«Colenbrander Basic Low Vision Exam THE BASIC LOW VISION EXAM August Colenbrander, MD, San Francisco This chapter is based (with some modifications) on a chapter that appeared in the (1994) Vision Rehabilitation issue of the Ophthalmology Clinics of North America, 7-2: 151-162 INTRODUCTION Before we embark on any intervention we should know our starting points. This holds true for Low Vision Rehabilitation as it holds for traditional medical interventions. When patients arrive for a Low Vision...»

«Drugs and Alcohol Policy Cowtown Bus Charters, Inc. is dedicated to the health and safety of our drivers. Drug and/or alcohol use may pose a serious threat to driver health and safety. Therefore, it is the policy of Cowtown Bus Charters, Inc. to prevent the use of drugs and abuse of alcohol from having an adverse effect on our drivers. The serious impact of drug use and alcohol abuse has been recognized by the federal government. The Federal Highway Administration (FHWA) has issued regulations...»

«BYLAWS OF VILLAGE AT MILL POND II HOMEOWNERS ASSOCIATION, INC. A Nonprofit Corporation BYLAWS OF VILLAGE AT MILL POND II HOMEOWNERS ASSOCIATION, INC. A Nonprofit Corporation ARTICLE I Name and Principal Office Section 1.1. The name of the Corporation is “Village at Mill Pond II Homeowners Association, Inc.” Its principal office shall be at P.O. Box 907 in Williston, Vermont, or at such other place as may be determined by the Board of Directors from time to time. ARTICLE II Purposes Section...»

«PLANTAS MEDICINALES DE USO POPULAR EN LA AMAZONIA PERUANA 3 KEMBER MEJIA * ELSA RENGIFO Plantas Medicinales de Uso Popular en la Amazonía Peruana La Dra. Cáliz Vila B. ha preparado el capítulo «Enfermedades frecuentes y su tratamiento fitoterapéutico» ILUSTRACIONES DE Edgardo Canayo Arirama 4 Mejía, Kember; Rengifo, Eisa Plantas Medicinales de Uso Popular en la Amazonía Peruana Lima, Agencia Española de Cooperación Internacional. Primera edición: 1995 Segunda edición corregida y...»

«Curriculum Vitae Cam Escoffery, PhD, MPH, CHES Associate Professor Department of Behavioral Sciences and Health Education (BSHE) Rollins School of Public Health 1518 Clifton Road, N.E., 5th Floor Atlanta, Georgia 30322 Telephone: (404) 727-4701 Fax: (404) 727-1369 Email: cescoff@emory.edu EDUCATION 2002 University of Georgia, Athens, Georgia Ph.D., Health Promotion and Behavior 1995 Rollins School of Public Health, Emory University, Atlanta, Georgia M.P.H., Health Promotion and Education 1992...»

«Copyright by Yeojin Lee The Dissertation Committee for Yeojin Lee certifies that this is the approved version of the following dissertation: THE EFFECT OF EMPOWERMENT ON BURNOUT OF FEMALE EMPLOYEES IN HEALTH AND HUMAN SERVICE ORGANIZATIONS IN THE STATE OF TEXAS Committee: Michael L. Lauderdale, Supervisor Shelley W. Dumas Noel G. Landuyt Dennis L. Poole Calvin L. Streeter THE EFFECT OF EMPOWERMENT ON BURNOUT OF FEMALE EMPLOYEES IN HEALTH AND HUMAN SERVICE ORGANIZATIONS IN THE STATE OF TEXAS by...»

«Package leaflet: Information for the user Sulfasalazine 250mg/5ml Oral Suspension Read all of this leaflet carefully before you start taking this medicine because it contains important information for you. ● Keep this leaflet. You may need to read it again. ● If you have any further questions, ask your doctor or your pharmacist. ● This medicine has been prescribed only for you. Do not pass it on to others. It may harm them, even if their signs of illness are the same as yours. ● If you...»

«PACKAGE LEAFLET: INFORMATION FOR THE USER Zanipress 20 mg/20 mg film-coated tablets Enalapril maleate/Lercanidipine hydrochloride Read all of this leaflet carefully before you start taking this medicine because it contains important information for you.Keep this leaflet. You may need to read it again.If you have any further questions, ask your doctor or pharmacist.This medicine has been prescribed for you only. Do not pass it on to others. It may harm them, even if their signs of illness are...»





 
<<  HOME   |    CONTACTS
2016 www.dissertation.xlibx.info - Dissertations, online materials

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.