Self-renewing Progenitors Research Articles

Thrombopoietin expands hematopoietic stem cells after transplantation.

Multiple lines of evidence indicate that thrombopoietin (TPO) contributes to the development of hematopoietic stem cells (HSC), supporting their survival and proliferation in vitro. To determine whether TPO supports the impressive expansion of HSC observed following transplantation, we transplanted normal marrow cells into lethally irradiated Tpo(-/-) and Tpo(+/+) mice and quantified HSC self-renewal and expansion and hematopoietic progenitor cell homing. Although essentially identical numbers of marrow-associated colony forming unit-culture (a surrogate measure of stem cell homing) were observed in each type of recipient 24 hours following transplantation, we found that a minimum of fourfold greater numbers of marrow cells were required to radioprotect Tpo-null mice than to radioprotect controls. To assess whether long-term repopulating (LTR) HSCs self-renew and expand in Tpo(-/-) recipients or controls, we performed limiting-dilution secondary transplants using donor cells from the Tpo(-/-) or Tpo(+/+) recipients 5-7.5 weeks following primary transplantation. We found that LTR HSCs expand to levels 10-20 times greater within this time period in normal recipients than in Tpo-null mice and that physiologically relevant amounts of TPO administered to the Tpo(-/-) recipients could substantially correct this defect. Our results establish that TPO greatly promotes the self-renewal and expansion of HSCs in vivo following marrow transplantation.

MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors.

The expression, activity and functions of mitogen-activated protein (MAP) kinases in primary human hematopoietic progenitors (HP) have not yet been fully clarified. To perform our experiments we used a stroma-free cell culture system in which the combination of FLT3 ligand (FL), stem cell factor (SCF) and thrombopoietin (TPO) induces massive expansion and proliferation of cord blood HP. The addition of IL-3 results in a rapid decrease of HP due to the prevalence of maturation and cell death. To detect extracellular regulated kinase (ERK) immunoenzymatic activity we recovered HP from FL, SCF and TPO stimulated long term cultures (LTC) after four weeks of culture. Some samples were recovered 16 h after addition of IL-3 to the LTC. We selectively immunoprecipitated p44/42 ERK kinase from 245 microg of cell lysates. We then analysed dual-phosphorylation of ERK-activating kinase-kinase (p45 MEK1/2) and of p44 ERK1 and p42 ERK2, and investigated MEK and ERK expression. ERK activity, MEK1, and p42 and p44 ERK dual-phosphorylation were undetectable in the expanding, greatly proliferating and self-renewing HP. However, after addition of IL-3 sustained (still detectable 16 h after the stimulus) and high levels of ERK activity and dual-phosphorylation of the kinases were seen. The levels of MEK and ERK expression were stable in the different phases. These findings add new information on the intracellular mechanisms of HP and help explain the very low levels of hematopoietic toxicity recently seen when treating cancer with down-modulators of ERK activity.

Changes of KCl sensitivity of proliferating neural progenitors during in vitro neurogenesis.

The effects of KCl-treatment on the survival and proliferation of NE-4C self-renewing neural progenitor cells were investigated during early phases of in vitro induced neurogenesis. NE-4C cells, derived from the anterior brain vesicles of embryonic mouse (E9), divided continuously under non-inducing conditions, but acquired neuronal features within 6 days, if induced by all-trans retinoic acid (RA). During the first 2 days of induction, the cells went on proliferating and did not show signs of morphological differentiation. In this stage, the resting membrane potential of RA-induced cells adopted more negative values in comparison to non-induced ones. Despite the increased membrane polarity and K+ conductance, addition of 20-50 mM KCl failed to elicit inward Na+ currents and did not induce an increase in the intracellular Ca+ level. Long-term treatment with 25 mM KCl, on the other hand, resulted in a selective loss of cells committed to neuronal fate by both decreasing the rate of cell proliferation and increasing the rate of cell death. The data indicate that the viability and proliferation of neural progenitors are influenced by extracellular K+-level in a differentiation stage-dependent manner.

MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors.

MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors.

Cell therapy and transplantation in Parkinson's disease.

Transplanted human fetal dopamine neurons can reinnervate the striatum in patients with Parkinson's disease (PD). Recent findings using positron emission tomography indicate that the grafts are functionally integrated and restore dopamine release in the patient's striatum. The grafts can exhibit long-term survival without immunological rejection and despite an ongoing disease process and continuous antiparkinsonian drug treatment. In the most successful cases, patients have been able to withdraw L-dopa treatment after transplantation and resume an independent life. About two-thirds of grafted patients have shown clinically useful, partial recovery of motor function. The major obstacle for the further development of this cell replacement strategy is that large amounts of human fetal mesencephalic tissue are needed for therapeutic effects. Stem cells hold promise as a virtually unlimited source of self-renewing progenitors for transplantation. The possibility to generate dopamine neurons from such cells is now being explored using different approaches. However, so far the generated neurons have survived poorly after transplantation in animals.

Conditional Clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells

The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of associated pulmonary neuroendocrine (PNE) cells. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for development of small cell lung carcinoma, mechanisms regulating expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. We have previously used a Clara cell-specific toxicant, naphthalene, to induce airway injury in mice and have demonstrated that naphthalene-resistant Clara cells, characterized by their expression of Clara cell secretory protein (CCSP), and PNE cells contribute to airway repair and associated hyperplasia of NEBs. This study was conducted to define the contribution of NEB-associated CCSP-expressing progenitor cells to PNE cell hyperplasia after Clara cell ablation. Transgenic (CCtk) mice were generated in which herpes simplex virus thymidine kinase was expressed within all CCSP-expressing cells of the conducting airway epithelium through the use of transcriptional regulatory elements from the mouse CCSP promoter. Chronic administration of ganciclovir (GCV) to CCtk transgenic mice resulted in selective ablation of CCSP-expressing cells within conducting airways. Proliferation and hyperplasia of PNE cells occurred in the absence of detectable proliferation among any other residual airway epithelial cell populations. These results demonstrate that PNE cells function as a self-renewing progenitor population and that NEB-associated Clara cells are not necessary for PNE cell hyperplasia.

Plasticity of mammary epithelia during normal development and neoplastic progression

The functional unit of the mammary gland is the epithelium. It consists of luminal epithelial cells and myoepithelial cells that are generated from self-renewing stem and progenitor cells. The latter two cell types are scattered throughout the mammary epithelium and are concentrated in specialized structures, the end buds. In transplantation studies the pluripotency of mammary stem cells has been confirmed by demonstrating that they can regenerate a complete mammary gland. The ability of mammary epithelial cells to produce an elaborate ductal system during puberty and to differentiate into milk-producing alveoli during pregnancy is not only influenced by their genetic make-up, but is also governed by local molecular signals. Recent studies suggest that the transdifferentiation of epithelial cells into tumor cells is under microenvironmental control, despite the prominence of genetic mutations in breast cancer. Consequently, disturbances of tissue homeostasis can alter mammary gland development or result in preneoplastic and neoplastic pathologies. The plasticity of mammary epithelia is not limited to the entry of cells into differentiation and transdifferentiation pathways, but extends to their ability to regain facets of their preceding stage of functionality. Deciphering the molecular cues that determine cell plasticity is prerequisite for establishing a unifying concept of mammary gland development and breast tumor progression.

Self-renewing canine oligodendroglial progenitor expanded as oligospheres

We have previously shown that oligodendroglial progenitors (OP) can be generated from multipotent rat neural precursor cells. We now report the generation of a homogeneous culture of canine OP from neural precursor cells. In non-adherent cultures, homogeneous OP cultures were obtained in 6-8 weeks of treatment with B104 cell conditioned medium (B104CM). In adherent cultures where astrocytes grew as a layer of substrate, colonies of OP invariably appeared at 10-14 days in vitro (DIV) and the colonies were expanded as free-floating spheres (oligospheres), in the presence of B104CM, suggesting that astrocytes facilitate the generation of canine OP. The oligosphere cells were characterized by self-renewal in the presence of B104CM and by terminal differentiation into oligodendrocytes after withdrawal of B104CM. Transplantation studies indicated that the extensively expanded oligosphere cells retained myelination capacity. The oligospheres thus provide a valuable source for experimental cell therapy studies.

The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors.

During development and in regenerating tissues such as the bone marrow, progenitor cells constantly need to make decisions between proliferation and differentiation. We have used a model system, normal erythroid progenitors of the chicken, to determine the molecular players involved in making this decision. The molecules identified comprised receptor tyrosine kinases (c-Kit and c-ErbB) and members of the nuclear hormone receptor superfamily (thyroid hormone receptor and estrogen receptor). Here we identify the glucocorticoid receptor (GR) as a key regulator of erythroid progenitor self-renewal (i.e. continuous proliferation in the absence of differentiation). In media lacking a GR ligand or containing a GR antagonist, erythroid progenitors failed to self-renew, even if c-Kit, c-ErbB and the estrogen receptor were activated simultaneously. To induce self-renewal, the GR required the continuous presence of an activated receptor tyrosine kinase and had to cooperate with the estrogen receptor for full activity. Mutant analysis showed that DNA binding and a functional AF-2 transactivation domain are required for proliferation stimulation and differentiation arrest. c-myb was identified as a potential target gene of the GR in erythroblasts. It could be demonstrated that delta c-Myb, an activated c-Myb protein, can functionally replace the GR.

Effect of stem cell factor on leukemic progenitor cell growth and sensitivity to cytosine-arabinoside

Recruitment of quiescent, clonogenic blasts from patients with acute myeloid leukemia (AMD by hematopoietic growth factors (HGFs) may improve the cytotoxic effects of cell-cycle-specific drugs like cytosine-arabinoside (Ara-C). Using the culture methods described by Nara and McCulloch and making a distinction between self-renewing and post-deterministic mitoses, we analyzed the effects of stem cell factor (SCF), a growth factor acting on early hematopoietic progenitor and stem cells. First, we demonstrated that SCF, used in combination with other HGFs included in fetal calf serum (FCS) and/or in 5637 cell line supernatant (5637-CM), stimulated both colony formation and self-renewal of blast progenitors from 10 patients, unlike SCF alone. We tested the effects of SCF on the recruitment of cells in the S-phase by using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM). We showed that SCF stimulated proliferation of AML cells significantly in 9 18 patients with AML. Second, we tested the influence of SCF on the sensitivity to Ara-C of self-renewing leukemic cells from 18 patients with AML. We showed that SCF was efficient in increasing the toxicity of Ara-C on the self-renewing blast progenitors, especially with high concentrations of Ara-C. However, a large patient-to-patient heterogeneity was found and the activity of SCF was not correlated with its effect on the cell cycle. These data indicate that SCF can enhance sensitivity to Ara-C of some leukemic cells with self-renewing capacity.

Acute avian leukemia viruses as tools to study hematopoietic cell differentiation.

Hematopoiesis is a multistep process during which an uncommitted, self-renewing progenitor divides and differentiates along one of eight distinct lineages, each with a unique function and each expressing a unique complement of proteins enabling them to carry out these functions. In adult vertebrates, multipotent hematopoietic precursors reside within the microenvironment of the bone marrow where they undergo a progressive maturation and commitment to one of several lymphoid and nonlymphoid cell lineages. Regulation of normal hematopoietic development requires an intricate set of cues from stromal cells involving signaling via soluble growth factors, cell-cell and cell-extracellular matrix interactions (Quesenberry 1992). Analysis of the cellular intermediates in these differentiation pathways has been greatly facilitated by the development of in vitro colony forming assays for multipotent and monopotent hematopoietic precursors and the identification of cytokines which regulate their growth and differentiation (Metcale 1984, 1988). Nevertheless, analysis of the molecular mechanisms governing differentiation and commitment to a given lineage has been technically difficult as bone marrow precursors are present in relatively low frequency and reagents allowing their isolation have only recently been developed. In addition, obtaining clonal precursor cells in sufficient numbers for biochemical analysis has been problematic since these precursors have a limited life span in vitro.

V-Myb DNA binding is required to block thrombocytic differentiation of Myb-Ets-transformed multipotent haematopoietic progenitors.

The E26 avian leukaemia virus encodes a fusion oncoprotein consisting of truncated versions of the c-Myb and c-Ets-1 transcription factors. When used to infect embryonic chicken haematopoietic cells two types of self-renewing progenitors are obtained, namely myeloblasts and 'MEPs' (Myb-Ets progenitors). In earlier work we have shown that myeloblasts transformed by the ts21 mutant of E26, which has a lesion in v-Myb, can be induced to differentiate into macrophages following shift to the non-permissive temperature. Here we show that the ts21 v-Myb is temperature sensitive for DNA binding in band shift experiments and that its inactivation in transformed MEPs induces their maturation into thrombocytes. The MEP transforming capacity of v-Myb is not confined to its fusion with v-Ets, as it is also seen with a virus that co-expresses tsMyb with v-ErbB. As with wild-type E26-transformed MEPs, ts21-transformed MEPs are multipotent, differentiating into eosinophils and myeloblasts following treatment with 12-O-tetradecanoylphorbol-13-acetate. In addition, ts21-transformed myeloblasts differentiate into macrophages when shifted to the non-permissive temperature. This shows that v-Myb blocks haematopoietic differentiation at two distinct stages. In contrast, v-Ets inactivation in MEPs transformed by a ts E26 mutant with a lesion in the corresponding oncoprotein leads to their differentiation into erythrocytes, myeloblasts and probably eosinophils. These data show that the two domains of Myb-Ets selectively affect decision making processes in different types and stages of haematopoietic cells.

Primary, self-renewing erythroid progenitors develop through activation of both tyrosine kinase and steroid hormone receptors.

Self renewal in the hematopoietic system is thought to be restricted to a class of pluripotent stem cells. The capacity of cells with the properties of committed progenitors to self renew in many leukemias is thought to be an abnormal property resulting from the mutations responsible for leukemic transformation. It is not known how cells that can self-renew differ from cells that cannot. The notion that only pluripotent stem cells self renew has recently been challenged: normal committed erythroid progenitors capable of sustained self renewal have been described. These cells, called SCF/TGF alpha progenitors, co-express the c-Kit receptor tyrosine kinase and c-ErbB, the avian receptor for epidermal growth factor and transforming growth factor (TGF) alpha, and they undergo continuous self renewal in response to TGF alpha and estradiol. In contrast, common erythroid progenitors (termed SCF progenitors) express only c-Kit and undergo a limited number of cell divisions in response to the c-Kit ligand, stem cell factor (SCF). Both types of progenitor faithfully reproduce terminal erythroid differentiation in vitro when exposed to differentiation factors. Here, we have investigated the developmental origin of these two classes of self-renewing erythroid progenitors. We show that SCF progenitors can develop into SCF/TGF alpha progenitors. This developmental conversion requires 10-14 days and is accompanied by a gradual up-regulation of bioactive TGF alpha receptor. Using sera depleted of endogenous growth factors, we demonstrate that the development of SCF progenitors into SCF/TGF alpha progenitors absolutely requires the simultaneous presence of SCF, TGF alpha and estradiol, and is strongly enhanced by an unknown activity in chicken serum. SCF progenitors can be induced to develop into self-renewing SCF/TGF alpha progenitors. The development of self renewal is triggered by specific combinations of growth factors and hormones. This has important implications for understanding leukemogenesis, as the self renewal of leukemic cells may reflect the normal potential of certain committed progenitor cells and not, as has been thought, a unique abnormal property of leukemic cells.

Radiobiological features of acute myeloblastic leukemia: comparison of self-renewal versus terminally differentiated populations

Purpose : To evaluate the radiosensitivity of self-renewing progenitor cells in acute myeloblastic leukemia (AML), we have compared the radiosensitivity of the cells grown either in methylcellulose alone for 7 days, or first in suspension culture for 7 days before being plated in methylcellulose. Methylcellulose selects for terminal-dividing cells and suspension cultures have been developed because they allow self-renewal to occur: The exponential growth of the progenitors of AML cultured in suspension is due to self-renewal. Methods and Materials : Cells were harvested from previously untreated leukemic human bone marrows. The myeloblastic lineage of the colonies was assessed by morphological, cytochemical, and immunophenotypic analysis, and by the use of growth factors that did not stimulate the growth of T-lymphocytes. The cell-cycle distribution of the blasts was analyzed by flow cytometry and was comparable for all samples. The irradiation was performed with γ-photons at a dose-rate of 0.05 Gy/min, similar to the clinical conditions used in our institution for total body irradiation (TBI). Results : The culture methods selected agressive leukemias. There were large variations of the individual radiosensitivity whatever culture method was used. The progenitor cells capable of self-renewal were more radiosensitive than terminal dividing cells. In two cases, a shoulder was found in the initial part of the cell-survival curves of cells capable of self-renewal. In these two cases, the best fit for the data was the linear quadratic model (survival = e −αD−βD2) with α/β values of 1.49 Gy and 3.12 Gy, respectively. Conclusion : The very low values of α/β suggest a reduced antileukemic effect in case of fractionated TBI, and may lead to more reliable screening methods to determine the most appropriate technique for radiation ablation of bone marrow prior to bone marrow transplantation (BMT).

PROGNOSTIC IMPLICATIONS OF BLAST SELF-RENEWAL CAPACITY IN ACUTE MYELOCYTIC-LEUKEMIA

The in vitro culture studies have revealed that the infinite proliferation of leukemic cells in acute myelocytic leukemia (AML) is supported by leukemic blast progenitors with self-renewal capacity. The self-renewal of blast progenitors is assessed by either secondary blast colony-formation in methylcellulose culture or clonogenic cell recovery in suspension culture. The patients whose leukemic blast progenitors had high self-renewal ability were more difficult in achieving complete remission and survived for shorter term than those with leukemic blast progenitors of low self-renewal capacity. Namely, the self-renewal capacity of leukemic blast progenitors was highly predictive of not only remission induction outcome but also survival duration of AML patients. Among several antileukemic agents tested, only a few drugs such as cytosine arabinoside were effective against blast self-renewal. To cure AML, a therapy that aims to eradicate self-renewing leukemic blast progenitors should be developed.

Dexamethasone recruitment of self-renewing osteoprogenitor cells in chick bone marrow stromal cell cultures

Bone marrow stromal cells are a mixed population that contribute to the formation of the hematopoietic microenvironment. The osteogenic lineage includes populations of cells that, in culture, form discrete nodules of mineralized tissue when grown in the presence of ascorbic acid and sodium beta-glycerophosphate. We have used nodule formation to assay for the self-renewal capacity of osteoprogenitor cells in chick bone marrow cultures. To examine the regulatory influence of dexamethasone (Dx), first subcultures were grown continuously or split 1:1 at repeated subculture. Cells in continuous culture exhibited less than two population doublings, while cellular proliferation and alkaline phosphatase area were inhibited by 10(-8) mol/L Dx. Cells in split (redistributed) cultures exhibited up to 14 population doublings and cellular proliferation was also inhibited by Dx. In contrast with continuous cultures, redistributed cultures treated with Dx had increased alkaline phosphatase area and 15-fold larger amounts of mineralized tissue formation than controls. Osteogenesis was sustained for up to four subcultures and the ratio of mineralized tissue area to alkaline phosphotase positive cell area was at most 0.55. These data indicate that the osteogenic lineage of bone marrow stromal cells contains self-renewing progenitors that are recruited by Dx in culture and that at a maximum, only 55% of the alkaline phosphatase-positive cell population contributes to osteogenesis.

Dexamethasone Recruitment of Self-Renewing Osteoprogenitor Cells in Chick Bone Marrow Stromal Cell Cultures

Bone marrow stromal cells are a mixed population that contribute to the formation of the hematopoietic microenvironment. The osteogenic lineage includes populations of cells that, in culture, form discrete nodules of mineralized tissue when grown in the presence of ascorbic acid and sodium beta-glycerophosphate. We have used nodule formation to assay for the self-renewal capacity of osteoprogenitor cells in chick bone marrow cultures. To examine the regulatory influence of dexamethasone (Dx), first subcultures were grown continuously or split 1:1 at repeated subculture. Cells in continuous culture exhibited less than two population doublings, while cellular proliferation and alkaline phosphatase area were inhibited by 10(-8) mol/L Dx. Cells in split (redistributed) cultures exhibited up to 14 population doublings and cellular proliferation was also inhibited by Dx. In contrast with continuous cultures, redistributed cultures treated with Dx had increased alkaline phosphatase area and 15-fold larger amounts of mineralized tissue formation than controls. Osteogenesis was sustained for up to four subcultures and the ratio of mineralized tissue area to alkaline phosphotase positive cell area was at most 0.55. These data indicate that the osteogenic lineage of bone marrow stromal cells contains self-renewing progenitors that are recruited by Dx in culture and that at a maximum, only 55% of the alkaline phosphatase-positive cell population contributes to osteogenesis.