Capacity Of Hematopoietic Stem Cells Research Articles

Stem cell leukemia protein directs hematopoietic stem cell fate

AbstractStem cell leukemia (SCL) protein has been shown to be an essential transcription factor during hematopoietic development in the embryo. In adult hematopoiesis, however, the role for SCL has remained largely unknown, whereas it is expressed in bone marrow hematopoietic stem cells (HSCs). In this study, we performed HSC transplantation and an in vitro HSC differentiation assay using retrovirally transduced HSCs with wild-type (WT) and dominant-negative (DN) SCL. The transplantation experiments showed that SCL does not affect the long-term repopulating capacity of HSCs but that WT SCL and DN SCL increase the short-term contribution of the transduced HSCs in myeloid and lymphoid lineages, respectively. An in vitro single-cell assay using a fetal thymus organ culture system further demonstrated that WT SCL facilitates HSCs to differentiate into the myeloid lineage but that DN SCL facilitates HSCs to differentiate into the lymphoid lineage. We conclude that the up-regulation or down-regulation of SCL directs HSCs toward myeloid or lymphoid lineage, respectively, although SCL does not affect their long-term repopulating capacity. (Blood. 2004;103: 3336-3341)

How hematopoietic stem cells know and act in cardiac microenvironment for stem cell plasticity? Impact of local renin–angiotensin systems

Bone marrow-derived hematopoietic stem cells (HSC) can exhibit tremendous differentiation activity in numerous non-hematopoietic organs. This enigmatic process is called as `stem cell plasticity' (SCP). HSC may promote structural and functional repair in several organs such as heart, liver, brain, and skeletal muscle via the SCP. The differentiation capacity of HSC is dependent on the specific signals present in the local tissue microenvironment. Those specific molecular signals required for the interactions of HSC and host tissues are currently unknown. The aim of this report is to propose a hypothesis on how HSC reach, recognize, and function in cardiac tissues in the context of SCP. Locally signaling cardiac microenvironment is essential for the seeding, expansion, and `cardiomyocyte differentiation' of the HSC in the heart. Our hypothesis is that the receptors, ligands, and signaling pathways of the tissue renin–angiotensin system (RAS) serve as the link between HSC and local cardiac microenvironment in SCP. The RAS is considered as a `tissue-based system' exhibiting paracrine functions within many organs. The presence of local hematopoietic bone marrow RAS and local cardiac RAS have been suggested. Both local tissue RASs share similar angiotensin peptide-signaling pathways such as JAK-STAT and mitogen-activated protein kinases. HSC have angiotensin type I (AT1a) receptors for the binding of angiotensin II, the active component of the RAS. Binding of angiotensin II to AT1a can increase hematopoietic progenitor cell proliferation. Local cardiac RAS has critical (patho)biological functions in the cardiomyocyte survival, renewal, and growth, as well as in cardiac remodeling. Therefore, the components of the local cardiac RAS and hematopoietic RAS could interact with each other during the SCP through myocardial tissue repair. Activation of the local myocardial RAS after injury may be related to homing and engraftment of the HSC to the cardiac tissue. Regenerating myocardial tissue may exert regulatory functions on circulating or resident HSC via the locally active RAS. Understanding the exact molecular basis of SCP in relation to local tissue RAS could offer new frontiers in the better management of ischemic cardiac diseases.

Cord blood stem cell banking and transplantation.

Stem cells have the ability to divide for indefinite periods in culture and to give rise to specialized cells. Cord blood as a source of hematopoietic stem cells (HSC) has several advantages as it is easily available, involves non-invasive collection procedure and is better tolerated across the HLA barrier. Since the first cord blood transplant in 1988, over 2500 cord blood HSC transplants have been done world wide. Since then, the advantages of cord blood as a source of hematopietic stem cells for transplantation have become clear. Firstly, the proliferative capacity of HSC in cord blood is superior to that of cells in bone marrow or blood from adults. A 100 ml unit of cord blood contains 1/10th the number of nucleated cells and progenitor cells (CD34+ cells) present in 1000 ml of bone marrow, but because they proliferate rapidly, the stem cell in a single unit of cord blood can reconstitute the entire haematopoietic system. Secondly, the use of cord blood reduces the risk of graft vs host disease. Cord Blood Stem Cell banks have been established in Europe and United States to supply HSC for related and unrelated donors. Currently, more than 65,000 units are available and more than 2500 patients have received transplants of cord blood. Results in children have clearly shown that the number of nucleated cells in the infused cord blood influences the speed of recovery of neutrophils and platelets after myeloablative chemotherapy. The optimal dose is about 2 x 10(7) nucleated cells/kg of body weight. The present study was carried out for collection, separation, enumeration and cryopreservation of cord blood HSC and establishing a Cord Blood HSC Bank. 172 samples of cord blood HSC were collected after delivery of infant prior to expulsion of placenta. The average cord blood volume collected was 101.20 ml. Mononuclear cell count ranged from 7.36 to 25.6 x 10(7)/ml. Viability count of mononuclear cells was 98.1%. After 1 year of cryopreservation, the viability count on revival was over 82.1%. Related cord blood stem cell transplantation was carried out in three cases at Army Hospital (R&R), Delhi Cantt.

Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation

AbstractTelomere shortening ultimately limits the replicative life span of cultured human somatic cells. Telomeres also shorten during replicative aging in vivo in hematopoietic cells, including early hematopoietic progenitors and hematopoietic stem cells (HSCs), from humans and mice, despite readily detectable levels of telomerase in these cells. To assess the relevance of telomerase to the long-term replicative capacity of HSCs in vivo, we serially transplanted HSCs from wild-type and telomerase-deficient mice until exhaustion and monitored telomere length in HSCs during this process. Telomerase-deficient HSCs could be serially transplanted for only 2 rounds, whereas wild-type HSCs could be serially transplanted for at least 4 rounds. Furthermore, the rate of telomere shortening was increased approximately 2-fold during serial transplantation of telomerase-deficient HSCs. These findings suggest that one role for telomerase in the HSC is to partially counter the rate of telomere shortening during division of HSCs, thereby preventing premature loss of telomere function and providing added replicative capacity.

Correlation between telomere length and proliferative capacity of hematopoietic stem cells

Correlation between telomere length and proliferative capacity of hematopoietic stem cells

Cryopreserved human haematopoietic stem cells retain engraftment potential after extended (5–14 years) cryostorage

Harvesting of stem cells during the early phases of treatment with no immediate intention to perform a stem cell transplant is becoming an increasingly common practice. Such “insurance” harvests are often stored for many years before being needed for transplant in a subsequent relapse. The effect of long-term cryostorage (5–14 years) on the viability and functional capacity of haematopoietic stem cells (HSCs) was investigated in 40 bone marrow and peripheral blood harvests using standard in vitro methods, the colony forming unit-granulocyte/macrophage (CFU-GM) assay and a single platform viable CD34 + cell absolute count by flow cytometry. Forty percent of harvests had CD34 + HSC counts of at least 0.7×10 6/kg bodyweight and 85% had CFU-GM counts of at least 1.0×10 5/kg bodyweight, these values representing our institutional minimum requirements for safe transplantation. Based on these results, it appears that HSC collections can remain adequate for safe transplantation after up to 14 years of cryostorage. However, as deterioration of HSC quality and viability may occur, some precautions may be warranted, namely harvesting higher than normal numbers of HSCs in collections intended for long-term storage and repeating in vitro assays on harvests after long-term storage prior to transplantation.

Replicative senescence of hematopoietic stem cells during serial transplantation: does telomere shortening play a role?

Hematopoietic stem cells (HSC) have a finite proliferative lifespan, based upon the limited number of times they can be serially transplanted in mice. Telomeres have been shown to shorten during the division of many normal somatic cells in humans, and the attrition of telomeres has been shown to ultimately cause replicative senescence in vitro for a number of different human cell strains. Whereas most human cell types have little to no detectable levels of telomerase activity, hematopoietic cells, including HSC, express low to moderate levels of telomerase, and yet telomeres shorten considerably during replicative aging of these cells. Here we consider the role telomerase may play in the hematopoietic system as well as the effect that over-expression of telomerase reverse transcriptase may have on the replicative capacity of hematopoietic stem cells during transplantation.

The aging of lympho-hematopoietic stem cells

The extensive self-renewal capacity of hematopoietic stem cells (HSCs) implies that this cell population may not age and thus may provide undiminished replenishment of blood cells throughout the lifespan of an organism. In contrast, accumulating experimental evidence supports the premise that HSCs show signs of aging and may have a limited functional lifespan. We summarize here the evidence for HSC aging, discuss the possible molecular mechanisms that may be involved and show evidence of a genetic connection between the effects of age on blood-forming cells and the longevity of mice. We speculate that age-related functional decline in adult tissue HSCs limits longevity in mammals.

Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells.

Reactivation of telomerase and maintenance of telomere length can lead to the prevention of replicative senescence in some human somatic cells grown in vitro. To investigate whether telomere shortening might also play a role in the limitation of hematopoietic stem cell (HSC) division capacity in vivo, we analyzed telomere length during serial transplantation of murine HSCs. Southern blot analysis of telomere length in donor bone marrow cells revealed extensive shortening ( approximately 7 kb) after just two rounds of HSC transplantation. The number of cycling HSCs increased after transplantation and remained elevated for at least 4 mo, while the frequency of HSCs in the bone marrow was completely regenerated by 2 mo after transplantation. Direct analysis of telomeres in HSCs by fluorescent in situ hybridization during serial transplantation also revealed a reduction in telomere size. Together, these data show that telomeres shorten during division of HSCs in vivo, and are consistent with the hypothesis that telomere shortening may limit the replicative capacity of HSCs.

Globin Gene Expression Is Reprogrammed in Chimeras Generated by Injecting Adult Hematopoietic Stem Cells into Mouse Blastocysts

To elucidate whether the differentiation capacity of hematopoietic stem cells (HSCs) is influenced by specific microenvironments, adult mouse bone marrow–derived HSCs were injected into mouse blastocysts. Embryos developing from injected blastocysts contained donor-derived cells at various developmental stages, and progeny of the stem cells were detected in hematopoietic tissues. Thus, HSCs derived from an adult animal survive after injection into blastocysts and are able to participate in hematopoietic development. We further find that the erythroid progeny of transplanted adult HSCs express embryonic/fetal-type globin genes and, conversely, that embryonic and fetal progenitor cells transplanted into adult recipients transcribe the adult-type globin gene. Thus, the developmental potential of adult HSCs is evidently more plastic than previously thought, and the developmental stage of the hematopoietic microenvironment controls the developmental fate of transplanted progenitor cells.

Hematopoietic reconstitution by transplanted grafts in lethally irradiated recipients

Persistence of multilineage microchimerism has been shown in recipients of successful solid organ transplantation.1,2 Continuous existence for decades of a trace number of donor cells implicates long-lasting engraftment of donor hematopoietic progenitor cells originating from the transplanted organs. We have recently showed that syngeneic organ transplantation was able to protect animals from lethal irradiation.3 This study further describes multilineage differentiation and self-renewal capacity of hematopoietic stem cells existing in solid organs.

Targeted disruption of the DNA repair methyltransferase gene renders mice hypersensitive to alkylating agent.

Alkylation of DNA at the O(6)-position of guanine is one of the most critical events leading to induction of mutation as well as to cancer. The enzyme O(6)-methylguanine-DNA methyltransferase repairs this and related lesions in DNA. By means of gene targeting, we established mouse lines deficient in the methyltransferase gene and tissues from these mice contained no methyltransferase activity. Administration of methylnitrosourea to these gene-targeted mice led to early death, and normal mice treated in the same manner showed no untoward effects. In mice given methylnitrosourea treatment, the bone marrow became hypocellular and there was a drastic decrease in the number of leukocytes and platelets, thereby indicating an impaired reproductive capacity of hematopoietic stem cells. Methyltransferase apparently protected these mice from the pancytopenia caused by the alkylating agent.

Do the preclinical effects of thrombopoietin correlate with its in vitro properties?

In the short time since its cloning, much has been learned of the in vitro properties of thrombopoietin (TPO). In addition to effects on the differentiation of megakaryocytes, TPO has also been shown to stimulate the proliferation of megakaryocytic progenitor cells, colony-forming units-megakaryocytes (CFU-MK), to act in synergy with interleukin 3 or c-kit ligand and erythropoietin (Epo) to stimulate the development of early and the generation of late erythroid progenitor cells, and to affect the rate of entry into the cell cycle and proliferative capacity of hematopoietic stem cells. An important question posed by these observations, for both TPO and for hematopoietic research in general, is whether the in vitro effects of a cytokine are mirrored by its preclinical and clinical biology. The results of recent studies in mice and nonhuman primates will be presented which have attempted to address this issue. In normal animals, TPO increases the numbers of marrow and spleen CFU-granulocyte/erythroid/macrophage/megakaryocyte, CFU-MK, CFU-GM and BFU-E, but its effects in the peripheral blood are limited to marked increases in the platelet count. The reason for widespread progenitor cell effects, yet stable leukocyte and erythrocyte blood counts, is likely the predominant regulatory effects of G-CSF and Epo; in the absence of elevated levels of the lineage-dominant regulator of each of these cell types, expanded progenitor cell numbers are not translated into increased peripheral blood counts. However, in states of increased blood cell demand such as follows myelosuppressive therapy, elevated levels of Epo and G-CSF allow the effects of TPO on erythropoiesis and myelopoiesis to become manifest. The administration of TPO to myelosuppressed animals is associated with not only greatly expanded hematopoietic progenitor cell recovery, but also improvement in platelet, red cell and leukocyte nadir levels and greatly accelerated recovery of all three cell lineages. These results indicate that the panhematopoietic properties of TPO identified by in vitro culture techniques correlate well with its effects in animals. The results of ongoing clinical trials should soon establish whether these conclusions can be extended to patient care.

Limited proliferation of stem cells surviving alkylating agents

HAYFLICK observed that when human foetal tissue was cultivated in vitro the cells underwent 50±10 population doublings before death1. Whether this phenomenon is a property of all diploid cells in culture and is pertinent to cell renewal systems in vivo has been the subject of much discussion. Haematopoietic failure is rarely the cause of death in animal or man, presumably because this cell renewal system has a proliferative capacity which is either unlimited or, if limited, exceeds the life span of a normal animal. The proliferative capacity of the haematopoietic system, if restricted, might become exhausted when stressed by the prolonged use of cytotoxic agents. In these circumstances, significant stem cell division would be necessary to return to the steady state, thereby using much of the stem cell proliferative capacity. We have carried out experiments in mice attempting to simulate the clinical use of chemotherapeutic agents that might result in exhausting the proliferative capacity of haematopoietic stem cells. Two alkylating agents, Busulfan and L-phenylalanine (L-Pam) were investigated. Mice treated with Busulfan and to a lesser extent L-Pam demonstrate permanent damage to the proliferative capacity of surviving stem cells as measured by serial transplantation. Such data may have relevance to the use of these agents in the clinic where there is great interest in such agents to destroy presumed subclinical micrometastases in patients expected to have extended survival.

The repopulating potential and differentiation capacity of hematopoietic stem cells from the blood and bone marrow of normal mice.

AbstractUsing the hematopoietic colony technique, we have investigated the repopulating potential of bone marrow cells and leukocytes of blood from normal mice and have demonstrated that the frequency of hematopoietic stem cells in bone marrow is 50 to 150 times that of stem cells in the circulating blood. The differentiation capacity of these stem cells has also been examined. Results of comparative studies of the serial sections of hematopoietic colonies formed from marrow and blood leukocytes indicate that the differentiation capacity of stem cells from marrow and blood is similar, and that at least 80% of these cells differentiate along a single cell line. Thus, peripheral blood stem cells can effect a complete hematopoietic graft, establishing in the host, donor red cells, granulocytes, and platelets.The possibility that blood leukocytes may serve as a potential source of stem cells for hematopoietic transplants has been considered. Although blood contains stem cells, their frequency is so low as to make it unlikely that they would become a useful source of precursor cells for transplantation purposes.