Self-renewing Progenitors Research Articles

Myocyte deficiency as a target in the treatment of cardiomyopathy

Cardiomyopathy is a general term referring to any disorder of cardiac muscle function, and can result from a number of defined and unknown acquired and congenital disorders; the end result is generally reduced contractile function and heart failure. Heart failure may occur early or late in the course of cardiomyopathy depending on the etiology and the age of the patient. How cardiomyopathic disorders lead to heart failure is incompletely resolved, particularly for disorders associated with hypertrophy. A leading hypothesis is that heart failure is effectively a myocyte deficiency disorder, reflecting the view that cardiomyopathy leads to cell loss and thence to heart failure. Several approaches for addressing this deficiency have been studied, including gene therapy targeting specific molecular events, cell therapy using myocytes from various sources, and a combination of gene and cell therapy using gene-modified cell transplantation. The recent discovery of populations of self-renewing progenitor cells residing in the myocardium raises the possibility of enhancing endogenous repair and regeneration mechanisms, and suggests additional levels at which genetic disorders may lead to impairment of myocardial function. Here we will review the evidence for myocyte dropout as a functionally significant phenomenon in human myocardial disorders, briefly discuss mechanisms and effectors of myocyte death, and their potential as targets in the treatment of cardiomyopathy. Finally, we will consider ways in which cell replacement therapy may eventually provide a solution to myocyte deficiency disorders.

Enhanced Self-Renewal Capability in Hepatic Stem/Progenitor Cells Drives Cancer Initiation

Transformed hematopoietic stem/progenitor cells with an enhanced or acquired self-renewal capability function as leukemic stem cells. In a variety of solid cancers, stem/progenitor cells could be also targets of carcinogenesis. However, it remains unclear whether disruption of stem cell function directly contributes to cancer initiation. We sought to elucidate the mechanisms of self-renewal in hepatic stem/progenitor cells and the relation between stem cell function and hepatocarcinogenesis. Functional analyses of polycomb-group protein Bmi1 and Wnt/beta-catenin, the molecules that are responsible for the self-renewal capability of many types of stem cells, were conducted in c-Kit(-)CD29(+)CD49f(+/low)CD45(-)Ter-119(-) hepatic stem/progenitor cells using retrovirus- or lentivirus-mediated gene transfer. The tumorigenicity of these cells transduced with the indicated retroviruses was also assessed by transplantation into nonobese diabetic/severe combined immunodeficient mice. Forced expression of Bmi1 and constitutively active beta-catenin mutant similarly promoted the self-renewal of hepatic stem/progenitor cells. The transplantation of Bmi1- or beta-catenin-transduced cells clonally expanded from single hepatic stem/progenitor cells produced tumors, which exhibited the histologic features of combined hepatocellular and cholangiocarcinoma. These observations imply that the dysregulated self-renewal of hepatic stem/progenitor cells serves as an early event in hepatocarcinogenesis, and they highlight the important roles of Bmi1 and the Wnt/beta-catenin pathway in regulating the self-renewal of normal or cancer stem cells in liver.

Origin of dendritic cells in peripheral lymphoid organs of mice

Parabiosis experiments demonstrating that dendritic cells (DCs) do not equilibrate between mice even after prolonged joining by parabiosis have suggested that DCs are derived from self-renewing progenitors that divide in situ. However, here we found that unequal exchange of DCs between mice joined by parabiosis reflected uneven distribution of DC precursors in blood due to their short half-life in circulation. DCs underwent only a limited number of divisions in the spleen or lymph nodes over a 10- to 14-day period and were replenished from blood-borne precursors at a rate of nearly 4,300 cells per hour. Daughter DCs presented antigens captured by their progenitors, suggesting that DC division in peripheral lymphoid organs can prolong the duration of antigen presentation in vivo.

Lentivirus vector‐mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb

Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.

E6/E7 oncogenes increase the proportion of self-renewing neural progenitor cells

International Journal of Developmental NeuroscienceVolume 24, Issue 8 p. 530-531 Abstract [P82]: E6/E7 oncogenes increase the proportion of self-renewing neural progenitor cells K. Piltti, Corresponding Author K. Piltti n/[email protected] University of Helsinki, FinlandSearch for more papers by this authorL. Kerosuo, L. Kerosuo University of Helsinki, FinlandSearch for more papers by this authorJ. Hakanen, J. Hakanen University of Helsinki, FinlandSearch for more papers by this authorM. Eriksson, M. Eriksson University of Helsinki, FinlandSearch for more papers by this authorA. Angers-Loustau, A. Angers-Loustau University of Helsinki, FinlandSearch for more papers by this authorS. Leppä, S. Leppä University of Helsinki, FinlandSearch for more papers by this authorM. Salminen, M. Salminen University of Helsinki, FinlandSearch for more papers by this authorH. Sariola, H. Sariola University of Helsinki, FinlandSearch for more papers by this authorK. Wartiovaara, K. Wartiovaara University of Helsinki, FinlandSearch for more papers by this author K. Piltti, Corresponding Author K. Piltti n/[email protected] University of Helsinki, FinlandSearch for more papers by this authorL. Kerosuo, L. Kerosuo University of Helsinki, FinlandSearch for more papers by this authorJ. Hakanen, J. Hakanen University of Helsinki, FinlandSearch for more papers by this authorM. Eriksson, M. Eriksson University of Helsinki, FinlandSearch for more papers by this authorA. Angers-Loustau, A. Angers-Loustau University of Helsinki, FinlandSearch for more papers by this authorS. Leppä, S. Leppä University of Helsinki, FinlandSearch for more papers by this authorM. Salminen, M. Salminen University of Helsinki, FinlandSearch for more papers by this authorH. Sariola, H. Sariola University of Helsinki, FinlandSearch for more papers by this authorK. Wartiovaara, K. Wartiovaara University of Helsinki, FinlandSearch for more papers by this author First published: 16 November 2006 https://doi.org/10.1016/j.ijdevneu.2006.09.144Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume24, Issue8ABSTRACTS TO THE 16TH BIENNIAL MEETING OF THE INTERNATIONAL SOCIETY FOR DEVELOPMENTAL NEUROSCIENCE, 24‐28 AUGUST 2006, BANFF, CANADADecember 2006Pages 530-531 RelatedInformation

Identifying Direct Targets of the Hoxb4 Transcription Factor Involved in Hematopoietic Cell Self Renewal.

Hoxb4 is a member of the homeobox protein family which when overexpressed, promotes expansion of hematopoietic stem cells (HSCs) both in vivo and ex vivo. The molecular mechanisms underlying the capacity of Hoxb4 to induce self-renewal of HSCs are poorly understood. In order to identify the direct transcriptional targets of Hoxb4 in primary hematopoietic progenitor cells, we created a Hoxb4-ERT2 fusion gene to allow for temporally regulated Hoxb4 activity. This gene was introduced into mouse lineage negative bone marrow (Lin− BM) cells using retroviral-mediated gene transfer. When these cells were treated with Tamoxifen (TAM), translocation of the fusion protein into the nuclei was demonstrated using confocal microscopy. Transduced cells were treated with 300nM of TAM for varying time in suspension culture and then plated into semisolid medium for myeloid progenitor assays. After 7 days, colonies from these primary CFU-C cultures were dispersed and replated for secondary CFU-C as an assay for myeloid progenitor self renewal. Treatment with TAM for 12hr in Hoxb4-ERT2 introduced cells resulted in an increase in the number (5.5 fold) and size (10 fold) of secondary colonies comparing to mock vector transduced controls, verifying that TAM treatment resulted in self-renewal of myeloid progenitors. In order to identify the target genes for this response, RNA from 12hr and 24hr TAM treated cells was analyzed for differential gene expression using Affymetrix 430v2 chips. Analysis of three independent experiments showed good reproducibility and allowed identification of candidate genes. Local pooled error test was used to analyze the data and a false discovery rate threshold at <25% was applied to identify the probe sets significantly differing in samples from TAM-treated Hoxb4-ERT2 samples versus TAM-treated control vector samples. This analysis identified 78 probe sets from cells treated for 12 hours, 103 probe sets from 24hr treated samples, and 20 common probe sets from both time points. A computer analysis of 64 candidate promoters identified in our screen and available in the NCBI database found that 50 % of them contained more than one high or moderate affinity consensus Hoxb4 binding motif. Furthermore, 22 of our candidate genes contained more than two consensus Hoxb4 binding motifs in the promoter regions. In this selected candidate gene group, genes were identified that are involved in various cellular functions: four in signal transduction, two in cell cycle regulation/apoptosis, four in enzyme activity regulation, six in cellular metabolism, two in transcriptional regulation; and eight of them associated with signaling transduction pathways for hematopoietic cells proliferation/self renewal. Therefore, our current results have identified a relatively small number of candidate genes (20) with significant potential to be direct targets of the Hoxb4 transcription factor in primary, self-renewing hematopoietic progenitor cells. We are currently testing several of these genes for direct Hoxb4 binding activity and for functional activity in hematopoietic cells.

Glioma Vaccines Generated from Electrofusion of Dendritic Cells and Brain Tumor Stem Cells.

Malignant gliomas and medulloblastomas contain a subset of self-renewing tumor progenitor cells designated brain tumor stem cells (BTSCs) that are an attractive target for T cell mediated immunotherapy. BTSCs are able to resist current therapy because theycan reside in the G0 state then reenter the cell cycle,can migrate away from the tumor and infiltrate surrounding normal brain parenchyma, andexpress transporter molecules, which efflux chemotherapeutic agents.To develop mouse models of BTSC immunotherapy, we have generated five murine medulloblastoma tumor lines (MM) derived from cerebellar tumors removed from Ptc+/−p53−/− transgenic mice. Mouse DCs were generated by injecting Flt3L (5 mcg) for 8 days, followed by MACS positive selection of CD11c+ splenocytes and overnight culture in medium supplemented with GM-CSF and IL-4. Electrofusion of DC and irradiated mouse medulloblastoma cells was performed by mixing DC and tumor cells at a 1:1 ratio (2 × 107 cells/ml) in electrofusion buffer [5% dextrose, 0.5 mM Mg (CH3COO)2, 0.1 mM Ca(CH3COO)2 pH 7.2]. Cells were subjected to dielectrophoresis using an ac current of 220V/cm × 9s to align the cells in a chain-like configuration between the electrodes, then pulsed with a 1200 V/cm × 25 μs to induce transient membrane breakdown. The following day, non-adherent DC were decanted and adherent cells, consisting of multinucleated heterokaryons and unfused tumor cells were harvested with a yield of 8% to 22% of cells expressing both DC and tumor markers. In previous therapy experiments DC/GL261 or DC/fibrosarcoma electrofusion vaccines were injected intrasplenically into mice bearing 7-day established intracranial tumors in combination with 5Gy local brain irradiation, and adjuvant anti-CD134 mAb. Therapy induced complete regression of established tumors, which was immunologically specific for the tumor cell component of the vaccine. Cured survivors resisted subsequent intracranial challenge. To investigate whether human glioma vaccines could be prepared, we established BTSC lines. The BTSCs were derived from tumor specimens using serum-free medium supplemented with EGF and FGF2 and they grew as non-attached neurospheres. BTSC lines were confirmed to contain genetic changes concordant with the original tumor and several lines expressed CD133 and nestin. Human DCs were prepared from normal donor PBMC cultured in GM-CSF, IL-4 and matured with PGE2 and TNF-α. Single cell suspensions of DCs and irradiated, fluorescently labeled BTSCs were mixed at a 2:1 ratio in electrofusion buffer and were subjected to ac current of 220 V/cm × 9s then dc pulse of 1200 V/cm × 99 μs. Multinucleated heterokaryons were readily apparent on cytospin preparations and FACS analysis demonstrated double positive cells containing fluorescently labeled tumor proteins and DC markers, such as HLA-DR, CD11c, CD80, and CD86. Four independent BTSC lines yielded heterokaryons incorporating between 25% and 33% of the total tumor cells. These experiments demonstrate that BTSC lines can be derived from patient samples and undergo efficient heterokaryon formation with DCs. Heterokaryons contain the entire complement of tumor antigens as well as the antigen-processing and co-stimulatory molecules found on antigen-presenting cells. Future experiments will explore whether a common set of tumor antigens are shared between BTSCs derived from GBM samples to establish whether it is feasible to prepare brain tumor vaccines.

A revised model for spermatogonial expansion in man: lessons from non-human primates

We have recently described a revised scheme for spermatogonial expansion in non-human primates. We proposed that A(pale)-spermatogonia act as self-renewing progenitors and premeiotic germ cells are organized and divide as small clones. Here, we are revisiting the model described for man and propose a modified scheme for spermatogonial expansion. Our revised model shows high similarity to the scheme proposed for non-human primates and is in accordance with all previous and present data.

Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon

Our understanding of the cellular and molecular mechanisms underlying the adult neural stem cell state remains fragmentary. To provide new models on this issue, we searched for stem cells in the adult brain of the zebrafish. Using BrdU tracing and immunodetection of cell-type-specific markers, we demonstrate that the adult zebrafish telencephalon contains self-renewing progenitors, which show features of adult mammalian neural stem cells but distribute along the entire dorso-ventral extent of the telencephalic ventricular zone. These progenitors give rise to newborn neurons settling close to the ventricular zone within the telencephalon proper. They have no equivalent in mammals and therefore constitute a new model of adult telencephalic neural stem cells. In addition, progenitors from the ventral subpallium generate rapidly dividing progenitors and neuroblasts that reach the olfactory bulb (OB) via a rostral migratory stream and differentiate into GABAergic and TH-positive neurons. These ventral progenitors are comparable to the mammalian neural stem cells of the subependymal zone. Interestingly, dorsal and ventral progenitors in the adult telencephalon express a different combination of transcription factors than their embryonic counterparts. In the case of neurogenin1, this is due to the usage of different enhancer elements. Together, our results highlight the conserved and unique phylogenic and ontogenic features of adult neurogenesis in the zebrafish telencephalon and open the way to the identification of adult neural stem cell characters in cross-species comparative studies.

E6/E7 oncogenes increase and tumor suppressors decrease the proportion of self-renewing neural progenitor cells

Many if not most tissues need a controlled number of stem cells to maintain normal function. Cancer can be seen as a process of disturbed tissue homeostasis, in which too many cells have or acquire too primitive identity. Here we measured how oncogenes and tumour suppressors affect the differentiation capacity, proportion and characteristics of progenitor cells in a model tissue. Neural progenitor cells (NPCs) were exposed to human papilloma virus E6, E7 or E6/E7 oncogenes, which degrade tumour suppressors p53 and pRb family members, respectively. E6/E7-expressing or p53-/- NPCs were able to differentiate, but simultaneously retained high capacity for self-renewal, proliferation, ability to remain multipotent in conditions promoting differentiation and showed delayed cell cycle exit. These functions were mediated through p53 and pRb family, and involved MEK-ERK signalling. Decreased amount of p53 increased self-renewal and proliferation, whereas pRb affected only proliferation. Our results suggest that the oncogenes increase whereas p53 and pRb family tumour suppressors decrease the number and proportion of progenitor cells. These findings provide one explanation how oncogenes and tumour suppressors control tissue homeostasis and highlight their importance in stem cell self- renewal, linked both to cancer and life-long tissue turnover.

Neurogenic peripheral blood‐derived adult stem cells

We have isolated several clonal lines of angiogenic self-renewing progenitor cells from the blood of adult green fluorescent protein transgenic swine. The cells are designated peripheral blood der...

Angiogenic peripheral blood‐derived adult stem cells

We have isolated several clonal lines of angiogenic self-renewing progenitor cells from the blood of adult green fluorescent protein transgenic swine. The cells are designated “peripheral blood derived multipotent adult progenitor cells” (PBD-MAPCs). These cells grow as spheroids and readily proliferate under conditions chosen to promote self-renewal without differentiation. We have isolated 5 independent lines of these cells from 2 different animals. When plated under angiogenic differentiation conditions PBD-MAPCs rapidly differentiate into cells that biochemically, immunologically and morphologically resemble either smooth muscle cells or endothelial cells, depending upon the differentiation conditions. When grown on collagen and fed a media that is typically used to grow vascular smooth muscle (VSM) cells, PBD-MAPCs differentiate into cells that express α-actin and are morphologically indistinguishable from bona fide VSM cells. Alternatively, cells differentiated on fibronectin and fed endothelial-specific media appear cobblestone in appearance and do not express α-actin but are positive for von Willebrand factor (vWF) and diI-Ac-LDL uptake, indicative of an endothelial phenotype. Finally, PBD-MAPCs seeded on MatrigelTM and fed angiogenic media form three-dimensional tube structures that are lined with vWF+ cells. We conclude PBD-MAPCs can differentiate into angiogenic cells that may prove useful in cell therapies directed at vascular disease. Supported by NIH PO1HL52490.

TA-p63-γ regulates expression of ΔN-p63 in a manner that is sensitive to p53

Genetic analysis indicates that TP63 is required for establishment and preservation of self-renewing progenitors within the basal layer of several epithelial structures, however, the specific contributions of transactivating (TA-p63) and dominant-negative (DeltaN-p63) isoforms remain largely undefined. Recent studies have suggested a model in which TA-p63 plays an important role in the establishment of progenitor populations in which expression of DeltaN-p63 contributes to the preservation of self-renewing capacity. Our previous studies indicate that DeltaN-p63 is a transcriptional target of p53, however, the absence of overt epithelial deficiencies in p53-/- mice and reports of increased expression of DeltaN-p63 in p53-/- mice suggest p53-independent mechanisms also contribute to expression of DeltaN-p63. Here, we present data indicating that, prolonged loss of p53 leads to the activation of a p53-independent mechanism for transcriptional regulation of DeltaN-p63. This p53-independent mechanism is sensitive to ectopic p53 but not to a p53 mutant that lacks the transactivation domain. We further show that in cells in which p53 is expressed TA-p63-gamma protein is destabilized in a manner that is p53 dependent and sensitive to pharmacologic inhibition of the 26S proteosome. Consistent with this observation, we demonstrate that loss of p53 leads to the stabilization of TA-p63-gamma that is reversible by ectopic p53. Finally, we present evidence that disruption of TA-p63-gamma expression leads to decreased expression of DeltaN-p63 and that overexpression of TA-p63-gamma was sufficient to enhance the activity of the DeltaN-p63 promoter. Taken together, our studies indicate that TA-p63-gamma is capable of activating expression of DeltaN-p63 and that this mechanism may account for p53-independent expression of DeltaN-p63.

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)–positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.

Graft-versus-Leukemia Target Antigens in Chronic Myelogenous Leukemia Are Expressed on Myeloid Progenitor Cells

Donor lymphocyte infusion (DLI) reliably induces durable remission in 75% to 80% of patients with relapsed chronic myelogenous leukemia (CML) following allogeneic bone marrow transplantation. We previously reported the identification of a high titer-specific immunoglobulin G response against two novel leukemia-associated antigens, CML28 and CML66, which correlated with immune-induced remission. The present studies characterize expression of CML28 and CML66 in primary hematopoietic tissues. Specific monoclonal antibodies to CML28 and CML66 were developed and used to detect antigen expression in leukemia cell lines and primary leukemia tissue on Western blot and immunohistochemistry. Expression patterns were confirmed by antigen-specific real-time PCR. Both CML28 and CML66 were highly expressed in leukemic blasts from patients with acute myelogenous leukemia and CML blast crisis but barely detectable in normal bone marrow, normal peripheral blood, or leukemic cells from patients with stable-phase CML. In contrast, purified CD34+ progenitors from normal individuals and patients with stable-phase CML expressed high levels of CML28 and CML66 transcript and protein. Immunohistochemical staining for CML66 confirmed rare staining of myeloid precursors in normal marrow and diffuse staining of myeloblastic cells in acute myelogenous leukemia and blast crisis CML marrows. The expression patterns of CML28 and CML66 are strikingly similar and suggest that antigen expression may play a role in shaping the post-DLI antibody repertoire. The CD34+ restricted pattern of expression of CML28 and CML66 is particularly relevant in light of the notion that DLI likely exerts its curative effect by targeting antigens present in self-renewing malignant progenitor populations in CML.

Hnf6 and Tcf2 (MODY5) are linked in a gene network operating in a precursor cell domain of the embryonic pancreas.

During pancreatic organogenesis endocrine cells arise from non self-renewing progenitors that express Ngn3. The precursors that give rise to Ngn3+ cells are presumably located within duct-like structures. However, the nature of such precursors is poorly understood. We show that, at E13-E18, the embryonic stage during which the major burst of beta-cell neogenesis takes place, pancreatic duct cells express Hnf1beta, the product of the maturity-onset diabetes of the young type 5 (MODY5) gene. Ngn3+ cells at this stage invariably cluster with mitotically competent Hnf1beta+ cells, and are often intercalated with these cells in the epithelium that lines the lumen of primitive ducts. We present several observations that collectively indicate that Hnf1beta+ cells are the immediate precursors of Ngn3+ cells. We furthermore show that Hnf1beta expression is markedly reduced in early pancreatic epithelial cells of Hnf6-deficient mice, in which formation of Ngn3+ cells is defective. These findings define a precursor cellular stage of the embryonic pancreas and place Hnf1beta in a genetic hierarchy that regulates the generation of pancreatic endocrine cells.

Pulling the Soxs off proliferation

A paper in this issue discloses the secret of eternal youth—at least for neural progenitors. During neurogenesis, self-renewing progenitor cells that reside in the ventricular zone of the developing CNS exit the cell cycle, migrate toward the marginal zone, and differentiate into neurons. Previous studies have identified transcription factors that promote the formation of new neurons by directing stem cells to stop dividing. However, the mechanisms responsible for countering differentiation to maintain a pool of undifferentiated neural progenitor cells have been less clear.

Stem cells at the dawn of the 21st century.

It is rare that a field of scientific research can both have an enormous potential impact on human health and quality of life and be a fount of new basic research discovery. Stem cell biology is surely one such field, offering hope for curing scourges like diabetes, Parkinson's disease, neurological degeneration, and congenital heart disease, as well as bringing together many disciplines of cell and molecular biology. Five years ago, stem cell biology was an exciting but rather restricted area of science with growing basic science and clinical implications. Currently, the existence of stem cells is a matter of public discussion, with religious, ethical, political, and economic implications. A week does not go by without some new revelation, about either the politics or biology of stem cells in the general press. What has changed? Clearly we know more about the biology of these cells, but the public interest has been driven by their potential in the treatment of disease on the one hand and concerns for the ethical implications of their use on the other. Some of the arguments are semantic and can be resolved by making sure that everyone is using the same terms to discuss the topic. Other concerns are theoretical and religious, such as defining when human life begins, and reflect beliefs and philosophies rather than the facts and data that scientists are restricted to when formulating coherent models. Science relies on facts, and many of the extraordinary claims made about stem cells in the scientific and public domain need to pass the important test of independent verification. Stem cells are loosely defined as self-renewing progenitor cells that can generate one or more specialized cell type. In vertebrates, stem cells have been traditionally subdivided into …

Dynamics of dendritic cell development from precursors maintained in stroma-dependent long-term cultures.

Two distinct subsets of dendritic cells are produced within the non-adherent cell population of the stroma-dependent long-term culture system. These are the small subset containing dendritic cell precursors and their progeny, large long-term culture-dendritic cells, which resemble immature CD11c+CD11b+MHCIIloCD8alpha- dendritic cells. The replicative and developmental potential of cells produced in long-term culture were investigated as a model for production of dendritic cells from progenitors. Cell proliferation and apoptosis were examined by labelling with bromodeoxyuridine and Annexin-V, respectively. The developmental potential of cells was analysed following transfer on to stromal monolayers or into in vitro colony and transwell assays. Results demonstrate that small long-term culture-dendritic cells are stromal cell-dependent. In the absence of stroma, they become apoptotic and die. Furthermore, direct contact with stromal cells is necessary for the differentiation and proliferation of small precursor cells. The small cell subset contains no long-term self-renewing cells, but instead appears to contain cells committed to developing into large long-term culture dendritic cells. The large long-term culture dendritic cell subset also contains dividing cells. Survival of large long-term culture-dendritic cells is dependent on soluble stroma-derived factor(s) and not direct contact with the stromal layer. All data suggest that the long-term culture system supports dendritic cell development from a self-renewing progenitor population resident within the stroma that gives rise to committed dendritic cell precursors and immature dendritic cells.

Mammary stem cell repertoire: new insights in aging epithelial populations

The proliferative lifespan of mammary stem cells was examined in serially transplanted clonal-dominant epithelial populations. Five successive transplant generations were done. The epithelial cell number in each outgrowth expands ∼500-fold in nulliparous hosts and ∼10 000-fold in impregnated hosts. Despite this, all resulting mammary outgrowths showed lineal identity with the original. Growth senescence was observed in some implants beginning at the third generation in impregnated recipients. The ability of an individual implant to support ductal morphogenesis and also secretory lobule development decayed at independent rates. Individual implants from a single clonal-dominant outgrowth occasionally gave rise to markedly different ductal development within the same host indicating an epithelial cell autonomous mechanism in ductal patterning. Both premalignant and malignant populations appeared focally within the aging transplants. These populations were also lineally related to the original outgrowth supporting the conclusion that the primary growth was derived clonally from one or a few lineally related antecedents. The premalignant and malignant descendant populations no longer exhibit growth senescence suggesting that they are supported by a perpetually self-renewing progenitor. Our evidence indicates that a single mammary cell may have the capacity to self-renew through five transplant generations. Even some sixth generation implants show vigorous growth.