Progenitor Cell Biology Research Articles

Neural stem cells: From basic biology to tissue repair

The isolation and characterization of neural stem cells in the adult brain offers considerable hope for the treatment of CNS injury and disease. I will discuss the fundamental biology of neural stem and progenitor cells in the adult brain and our work to date using small molecules to activate these endogenous neural precursor cells to effectively enhance “self‐repair” of the stroke injured brain.

In vitro biology of human hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells from human umbilical cord blood have been the focus of both basic and clinical research during the last 20 years. It has been clearly demonstrated that such sells possess higher proliferation and expansion potentials, as compared to their adult counterparts, and their capacity to reconstitute the hematopoietic system of mammals has also been shown. Different in vitro systems have been used to characterize the biology of these hematopoietic cells and some culture methods are being currently used to expand the numbers of such cells for clinical purposes.

Wound Healing Angiogenesis: Innovations and Challenges in Acute and Chronic Wound Healing

Formation of new blood vessels, by either angiogenesis or vasculogenesis, is critical for normal wound healing. Major processes in neovascularization include (i) growth-promoting or survival factors, (ii) proteolytic enzymes, (iii) activators of multiple differentiated and progenitor cell types, and (iv) permissible microenvironments. A central aim of wound healing research is to "convert" chronic, disease-impaired wounds into those that will heal. Reduced ability to re-establish a blood supply to the injury site can ultimately lead to wound chronicity. (1) Human fetal endothelial progenitor cells can stimulate wound revascularization and repair following injury, as demonstrated in a novel mouse model of diabetic ischemic healing. (2) Advances in bioengineering reveal exciting alternatives by which wound repair may be facilitated via the creation of vascularized microfluidic networks within organ constructs created ex vivo for wound implantation. (3) A "personalized" approach to regenerative medicine may be enabled by the identification of protein components present within individual wound beds, both chronic and acute. Despite the development of numerous therapies, impaired angiogenesis and wound chronicity remain significant healthcare problems. As such, innovations in enhancing wound revascularization would lead to significant advances in wound healing therapeutics and patient care. Insights into endothelial progenitor cell biology together with developments in the field of tissue engineering and molecular diagnostics should not only further advance our understanding of the molecular mechanisms regulating wound repair but also offer innovative solutions to promote the healing of chronic and acute wounds in vivo.

Helicobacter pylori infection induced gastric cancer; advance in gastric stem cell research and the remaining challenges

Helicobacter pylori infection is the major cause of gastric cancer, which remains an important health care challenge. Recent investigation in gastric stem cell or progenitor cell biology has uncovered valuable information in understanding the gastric gland renewal and maintenance of homeostasis, they also provide clues for further defining the mechanisms by which gastric cancer may originate and progress. Lgr5, Villin-promoter, TFF2-mRNA and Mist have recently been identified as gastric stem/progenitor cell markers; their identification enriched our understanding on the gastric stem cell pathobiology during chronic inflammation and metaplasia. In addition, advance in gastric cancer stem cell markers such as CD44, CD90, CD133, Musashi-1 reveal novel information on tumor cell behavior and disease progression implicated for therapeutics. However, two critical questions remain to be of considerable challenges for future exploration; one is how H. pylori or chronic inflammation affects gastric stem cell or their progenitors, which give rise to mucus-, acid-, pepsinogen-, and hormone-secreting cell lineages. Another one is how bacterial infection or inflammation induces oncogenic transformation and propagates into tumors. Focus on the interactions of H. pylori with gastric stem/progenitor cells and their microenvironment will be instrumental to decipher the initiation and origin of gastric cancer. Future studies in these areas will be critical to uncover molecular mechanisms of chronic inflammation-mediated oncogenic transformation and provide options for cancer prevention and intervention. We review recent progress and discuss future research directions in these important research fields.

P2-01-13: Impact of Erlotinib on MSC-Mediated TIC Expansion and EMT.

Abstract Background: Recently, we have demonstrated that mesenchymal stem cells (MSC) and MSC secreted factors (MSC-CM) have a profound effect on tumor initiating cells (TIC) enriched mammosphere formation and latency of tumor xenografts formation from breast cancer cell lines. Furthermore these interactions increased the expression of epithelial mesenchymal transition (EMT)-associated proteins which are associated with tumor cell invasion and metastasis as well as the TIC phenotype. (Klopp, A. H. et al., 2010, PLoS One. 5, e12180). Our data suggest that the presence of MSC in the tumor microenvironment may increase metastases by conferring stem progenitor cell biology on more differentiated non-metastatic cells. In addition, preliminary data suggested MSC-CM upregulated EGFR signaling in breast cancer cells. Therefore, we hypothesized that inhibiting EGFR signaling with erlotinib (tyrosin kinase inhibitor) can suppress MSC-mediated TIC expansion and EMT. Methods & Results: In order to demonstrate that erlotinib inhibits MSC-CM promoted expansion of TIC, we cultured breast cancer cells lines (SUM149, SUM159, SUM190, MDA-IBC3 and MCF-7) in anchorage independent conditions with MSC-CM and treated them with increasing concentrations of erlotinib. The efficiency of mammosphere formation was examined after 5 days. We found that erlotinib inhibited MSC mediated increase in mammosphere formation in triple negative cell lines SUM149 and SUM159, and HER2−positive cell lines SUM190 and MDA-IBC3, but not in ER-positive, erlotinib resistant MCF-7 cells. Furthermore, we evaluated the impact of erlotinib on cell cultures grown with breast cancer patient-derived fluids, such as seroma and malignant pleural effusions. We observed that the effect of erlotinib on mammospheres formation was attenuated by both types of patient fluids. Discussion: Patients with triple negative breast cancer have the highest rates of metastases and no available targeted therapies for treatment. EGFR is expressed in a significant proportion of triple negative breast cancers, and recent clinical and preclinical studies suggest that EGFR may contribute to the metastasis or aggressiveness of triple negative breast cancer. Here we demonstrate that host and environmentally-derived factors are critical for determining resistance to erlotinib. In vivo studies regarding the ability of erlotininb to prevent MSC-enhanced TIC survival and metastases are underway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-13.

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay. To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 μL of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin βB, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony. In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 μL of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin βB, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

Abstract 16907: Modification of Hemeoxygenase-1 Expression Enhances the Therapeutic Potency of CD34+ Cells in Ischemic Tissue Repair

The potency of cell-based therapy in ischemic tissue repair is limited by low viability and retention of transplanted cells. The hemeoxygenase system catabolizes heme cleavage and has been shown to exert cytoprotection via its anti-oxidative, -apoptotic, -inflammatory properties. HO-1, inducible in various pathologic states, has been shown to influence progenitor cell biology. We hypothesized that HO-1 over-expression enhances the therapeutic potency of CD34+ cells for ischemic tissue repair. CD34+ from healthy donors were enriched from the peripheral blood mononuclear cell fraction of healthy donors after G-CSF mobilization (Isolex 300i). Adenoviral transduction of HO-1/GFP or GFP led to a substantial increase of HO-1 expression (HO-1/18s RNA expression: 62-fold increase, protein expression: 20-fold increase; n=3). After allowing 48 hrs for target gene expression, CD34+ cells were FACS-sorted for GFP followed by overnight recovery in-vitro. A low-therapeutic dose (10^5 cells/kg) of HO-1+/GFP+ (HO-1) or GFP+ (GFP) CD34+ cells was locally injected into the border zone after complete ligation of the left coronary artery in 8-week-old, male nude mice (saline control: 2x 15 μl). At day 28, LV ejection fraction of mice treated with HO-1 over-expressing CD34+ cells was significantly improved (fractional shortening: saline 19±2%, HO-1 30±1%, GFP 24±1%; n=6-8/group, P<0.01). This functional improvement can be explained by a preservation of the border-zone: the number of lectin+ capillaries was increased (lectin staining: saline 73±5/hpf, HO-1 99±4/hpf, GFP 79±7/hpf; n=7-8/group, P<0.01) with the use of HO-1 modified CD34+ cells while lectin+/alpha-smooth-muscle actin+ structures indicative for resistance vessels remained unchanged (P=ns). Apoptosis of resident myocardial cells was attenuated (TUNEL staining: saline 69±5/hpf, HO-1 31±3, GFP 54±9, P<0.05). As a result, the infarct size in mice treated with HO-1 modified cells was reduced (Masson-trichrome staining: saline 39±1%, HO-1 36±2%, GFP 42±1%; n=6-8/group, P<0.05). Ongoing studies are addressing the underlying mechanism(s) on cellular basis as well as the clinical relevance. Conclusion: HO-1 gene modification enhances the therapeutic potency of CD34+ in ischemic tissue repair.

Endothelial Progenitor Cells in Cardiovascular Disease and Chronic Inflammation: from Biomarker to Therapeutic Agent

The discovery of endothelial progenitor cells in the 1990s challenged the paradigm of angiogenesis by showing that cells derived from hematopoietic stem cells are capable of forming new blood vessels even in the absence of a pre-existing vessel network, a process termed vasculogenesis. Since then, the majority of studies in the field have found a strong association between circulating endothelial progenitor cells and cardiovascular risk. Several studies have also reported that inflammation influences the mobilization and differentiation of endothelial progenitor cells. In this review, we discuss the emerging role of endothelial progenitor cells as biomarkers of cardiovascular disease as well as the interplay between inflammation and endothelial progenitor cell biology. We will also review the challenges in the field of endothelial progenitor cell-based therapy.

Common Recurrent DKC1 Mutation A353V Does Not Support Telomere Maintenance in Induced Pluripotent Stem Cells

Abstract 50Dyskeratosis Congenita (DC) is a rare bone marrow failure syndrome showing considerable genetic and clinical heterogeneity. The most common form is the X-linked form due to mutations in the DKC1 gene encoding dyskerin, a protein important in telomere maintenance and ribosomal RNA biogenesis. Six other genes, all of whose products are involved in telomere maintenance, have been shown to be mutated in DC, together the seven genes accounting for about half of the known cases. The X-linked form can cause severe disease for which therapeutic options are limited. It is known that mutant dyskerin destabilizes telomerase RNA leading to rapidly shortening telomeres, accelerated stem cell aging and bone marrow failure. However the precise mechanism by which this occurs is not known. So far studies of the cell biology of DC stem and progenitor cells have been hampered by their scarcity in patients and their short life span and attempts to create mouse models have suffered from differences in telomere biology between mouse and human. An alternative approach that has recently become feasible is the production of induced pluripotent stem cells (iPSC) from patient fibroblasts that can then be used to investigate disease pathogenesis. Accordingly we generated iPSC from skin fibroblast from X-linked DC patients carrying DKC1 mutations Q31E, δ37A and 353V, and by using the classical OCT4, KLF4, SOX2 and cMYC 4-transcription factor system. Of particular interest is the A353V mutation since this is a recurrent mutation and accounts for about 40% of DKC1 mutations. In total, we obtained two Q31E clones, three δ37 clones and eight A353V clones. We found that all these DKC1 mutant iPS cells express decreased levels of dyskerin, in agreement with our mouse studies that show mutant proteins are relatively unstable. Mutant iPSC have very low levels of TERC (only 20–30% of the levels in WT iPSC) while TERT expression is the same as in WT cells. By using the TRAP assay, we found that both A353V and δ37 iPSC showed dramatically decreased telomerase activity; only 10–20 % compared to WT iPSC. After measuring the telomere length of both patient skin fibroblast cells and DKC1 mutant iPSC, we found A353V and δ37 iPSC lost the ability to elongate the telomere end during iPSC reprogramming while WT iPSC showed significantly increased telomere length compared to WT skin fibroblast cells. These results indicated that DKC1 iPSC are defective in telomere maintenance. In terms of ribosome biogenesis, we found that some snoRNA expression was slightly decreased including H/ACA snoRNAs E2, E3, U69, ACA10 and scaRNAs U90 and U93 while all C/D snoRNA we investigated were unchanged compared with WT iPS cells. We also found that DKC1 mutant iPS cells did not show significantly changes in ribosomal profiles or in the kinetics of rRNA processing. Together these results suggest that the iPSC faithfully reproduce the molecular features of the human disease and will prove to be a useful tool in investigations of the pathogenesis and treatment of DC. Disclosures:Bessler:Alexion Phamaceutical: Membership on an entity's Board of Directors or advisory committees; National Organization for Rare Dieases: Speakers Bureau.

Mitochondria Determine the Differentiation Potential of Cardiac Mesoangioblasts

An understanding of cardiac progenitor cell biology would facilitate their therapeutic potential for cardiomyocyte restoration and functional heart repair. Our previous studies identified cardiac mesoangioblasts as precommitted progenitor cells from the postnatal heart, which can be expanded in vitro and efficiently differentiated in vitro and in vivo to contribute new myocardium after injury.Based on their proliferation potential in culture, we show here that two populations of mesoangioblasts can be isolated from explant cultures of mouse and human heart.Although both populations express similar surface markers, together with a panel of instructive cardiac transcription factors, they differ significantly in their cellular content of mitochondria. Slow dividing (SD) cells, containing many mitochondria, can be efficiently differentiated with 5-azacytidine (5-aza) to generate cardiomyocytes expressing mature structural markers. In contrast, fast dividing (FD) mesoangioblasts, which contain decreased quantities of mitochondria, do not respond to 5-aza treatment.Notably, increasing mitochondrial numbers using pharmacological nitric oxide (NO) donors reverses the differentiation block in FD mesoangioblasts and leads to a progressive maturation to cardiomyocytes; conversely decreasing mitochondrial content, using respiratory chain inhibitors and chloramphenicol, perturbs cardiomyocyte differentiation in SD populations. Furthermore, isolated cardiac mesoangioblasts from aged mouse and human hearts are found to be almost exclusively mitochondria low FD populations, which are permissive for cardiomyocyte differentiation only after NO treatment. Taken together,this study illustrates a key role for mitochondria in cardiac mesoangioblast differentiation and raises the interesting possibility that treatments, which increase cellular mitochondrial content, may have utility for cardiac stem cell therapy.

Endothelial progenitor cell biology in disease and tissue regeneration

Endothelial progenitor cells are increasingly being studied in various diseases ranging from ischemia, diabetic retinopathy, and in cancer. The discovery that these cells can be mobilized from their bone marrow niche to sites of inflammation and tumor to induce neovasculogenesis has afforded a novel opportunity to understand the tissue microenvironment and specific cell-cell interactive pathways. This review provides a comprehensive up-to-date understanding of the physiological function and therapeutic utility of these cells. The emphasis is on the systemic factors that modulate their differentiation/mobilization and survival and presents the challenges of its potential therapeutic clinical utility as a diagnostic and prognostic reagent.

Selection of Brain Metastasis-Initiating Breast Cancer Cells Determined by Growth on Hard Agar

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44(+) and CD133(+) and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice.

Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo (153.5)

Abstract Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are broadly used to study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells is suboptimal in these hosts. We hypothesized that this defect was due to reduced cross-reactivity of mouse cytokines on the human receptor. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. We generated RAG2-/-γc-/- mice in which we replaced the gene encoding mouse TPO by its human homologue, and we transplanted human CD34+ cells in these recipients. Homozygous humanization of TPO (TPOh/h) led to increased levels (higher than 80%) and longer maintenance (more than 6 months) of human cell engraftment in the bone marrow of the hosts. Multilineage differentiation of hematopoietic cells was also improved, with an increased ratio of myelomonocytic verus lymphoid lineages. Moreover, maintenance of human stem and progenitor cells was improved : we observed a significant increase in the fraction of human Lin-CD34+CD38-CD90+CD45RA- cells in TPOh/h compared to control recipients; and CD34+ cells purified from TPOh/h primary recipient had a significantly increased capacity to efficiently engraft secondary recipients. Therefore, RAG2-/-γc-/- TPO-humanized mice represent a novel model to study human hematopoiesis in vivo.

Human neural progenitors from different foetal forebrain regions remyelinate the adult mouse spinal cord

Improving oligodendroglial differentiation from human foetal neural progenitor cells remains a primordial issue to accomplish successful cell-based therapies in myelin diseases. Here, we combined in situ, in vitro and in vivo approaches to assess the oligodendrogenic potential of different human foetal forebrain regions during the first trimester of gestation. We show for the first time that the initial wave of oligodendrocyte progenitor emergence in the ventral telencephalon onsets as early as 7.5 weeks into gestation. Interestingly, in vitro, isolation of ganglionic eminences yielded oligodendrocyte progenitor-enriched cultures, as compared with cortex and thalamus. Most importantly, single injection of human neural progenitors into rodent models of focal gliotoxic demyelination revealed the great capacity of these cells to survive, extensively migrate and successfully remyelinate the spinal cord, irrespective of their origin. Thus, our study brings novel insights into the biology of early human foetal neural progenitor cells and offers new support for the development of cellular therapeutics for myelin disorders.

Vascular Smooth Muscle Progenitor Cells

Molecular pathways that control the specification, migration, and number of available smooth muscle progenitor cells play key roles in determining blood vessel size and structure, capacity for tissue repair, and progression of age-related disorders. Defects in these pathways produce malformations of developing blood vessels, depletion of smooth muscle progenitor cell pools for vessel wall maintenance and repair, and aberrant activation of alternative differentiation pathways in vascular disease. A better understanding of the molecular mechanisms that uniquely specify and maintain vascular smooth muscle cell precursors is essential if we are to use advances in stem and progenitor cell biology and somatic cell reprogramming for applications directed to the vessel wall.

Developmental and Regenerative Biology of Multipotent Cardiovascular Progenitor Cells

Our limited ability to improve the survival of patients with heart failure is attributable, in part, to the inability of the mammalian heart to meaningfully regenerate itself. The recent identification of distinct families of multipotent cardiovascular progenitor cells from endogenous, as well as exogenous, sources, such as embryonic and induced pluripotent stem cells, has raised much hope that therapeutic manipulation of these cells may lead to regression of many forms of cardiovascular disease. Although the exact source and cell type remains to be clarified, our greater understanding of the scientific underpinning behind developmental cardiovascular progenitor cell biology has helped to clarify the origin and properties of diverse cells with putative cardiogenic potential. In this review, we highlight recent advances in the understanding of cardiovascular progenitor cell biology from embryogenesis to adulthood and their implications for therapeutic cardiac regeneration. We believe that a detailed understanding of cardiogenesis will inform future applications of cardiovascular progenitor cells in heart failure therapy and regenerative medicine.

Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo

Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are broadly used to study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells are suboptimal in these hosts. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. We generated RAG2(-/-)γ(c)(-/-) mice in which we replaced the gene encoding mouse TPO by its human homolog. Homozygous humanization of TPO led to increased levels of human engraftment in the bone marrow of the hosts, and multilineage differentiation of hematopoietic cells was improved, with an increased ratio of myelomonocytic verus lymphoid lineages. Moreover, maintenance of human stem and progenitor cells was improved, as demonstrated by serial transplantation. Therefore, RAG2(-/-)γ(c)(-/-) TPO-humanized mice represent a useful model to study human hematopoiesis in vivo.

Salivary gland progenitor cell biology provides a rationale for therapeutic salivary gland regeneration

An irreversible loss of salivary gland function often occurs in humans after removal of salivary tumors, after therapeutic radiation of head and neck tumors, as a result of Sjögren's syndrome and in genetic syndromes affecting gland development. The permanent loss of gland function impairs the oral health of these patients and broadly affects their quality of life. The regeneration of functional salivary gland tissue is thus an important therapeutic goal for the field of regenerative medicine and will likely involve stem/progenitor cell biology and/or tissue engineering approaches. Recent reports demonstrate how both innervation of the salivary gland epithelium and certain growth factors influence progenitor cell growth during mouse salivary gland development. These advances in our understanding suggest that developmental mechanisms of mouse salivary gland development may provide a paradigm for postnatal regeneration of both mice and human salivary glands. Herein, we will discuss the developmental mechanisms that influence progenitor cell biology and the implications for salivary gland regeneration.

P73 deficiency results in impaired self renewal and premature neuronal differentiation of mouse neural progenitors independently of p53

The question of how neural progenitor cells maintain its self-renewal throughout life is a fundamental problem in cell biology with implications in cancer, aging and neurodegenerative diseases. In this work, we have analyzed the p73 function in embryonic neural progenitor cell biology using the neurosphere (NS)-assay and showed that p73-loss has a significant role in the maintenance of neurosphere-forming cells in the embryonic brain. A comparative study of NS from Trp73−/−, p53KO, p53KO;Trp73−/− and their wild-type counterparts demonstrated that p73 deficiency results in two independent, but related, phenotypes: a smaller NS size (related to the proliferation and survival of the neural-progenitors) and a decreased capacity to form NS (self-renewal). The former seems to be the result of p53 compensatory activity, whereas the latter is p53 independent. We also demonstrate that p73 deficiency increases the population of neuronal progenitors ready to differentiate into neurons at the expense of depleting the pool of undifferentiated neurosphere-forming cells. Analysis of the neurogenic niches demonstrated that p73-loss depletes the number of neural-progenitor cells, rendering deficient niches in the adult mice. Altogether, our study identifies TP73 as a positive regulator of self-renewal with a role in the maintenance of the neurogenic capacity. Thus, proposing p73 as an important player in the development of neurodegenerative diseases and a potential therapeutic target.