Form Of Stem Cell Factor Research Articles

Key Discoveries in Melanocyte Development

Melanocytes are melanin pigment-producing cells. Mammalian melanocytes are categorized as ‘‘cutaneous’’ (follicular and epidermal) and ‘‘extracutaneous’’ (e.g., choroidal, cochlear). Epidermal melanocytes contribute to photoprotection and thermoregulation by packaging melanin pigment into melanosomes and delivering them to neighboring keratinocytes. Melanocytes are derived from the neural crest that is a migratory multipotent population that gives rise to multiple cell lineages, including neurons, glial cells, medullary secretory cells, smooth muscle cells, and bone and cartilage cells. Coat color mutants in different species have been useful for identifying genes involved in melanocyte development. Embryonic transplantation experiments played a significant role in early efforts to investigate melanocyte development. Rawles (1947) conducted a series of elegant transplantation experiments, which revealed that melanocytes originate from the neural crest. The investigator transplanted various axial levels of the embryonic central nervous system, adjacent tissues of the somite and lateral plate, and limb-bud regions, separately and in combination, from various developmental stages of black mouse embryos, to the coelom of White Leghorn chick embryos. Only tissues containing the neural crest or cells migrating from the neural crest were found to produce melanophores. In addition, Rawles found that the portions of embryo that produce pigment cells vary at different developmental stages of donor embryos. The quail nucleus exhibits condensed heterochromatin, which could serve as a marker to distinguish quail cells from chick cells. Teillet and Le Douarin (1970) studied melanoblast migration from the neural crest using a quail-chick xenograft transplantation model. The investigators transplanted different axial levels of quail neural tube and neural crest to White Leghorn chick embryos. They found that (1) at embryonic days E4 and E5, the transplanted quail cells localize mainly in the mesenchyme; (2) at E6 when the dermis and the epidermis are formed, quail cells (melanoblasts) begin to migrate into the epidermis; (3) at E9 quail cells (melanoblasts) increase their cytoplasmic volume and start producing melanin pigments; (4) at E10 and E11 quail cells (melanoblasts) localize in the basal layer of epidermis and become dendritic. Cell type-specific markers are useful tools to study the development of certain cell types. However, it is challenging to identify these markers. Steel et al. (1992) found Tyrp-2/Dct to be a specific marker of melanoblasts, the precursors of melanocytes. Using in situ hybridization, the investigators found that Tyrp-2 expression was detectable in melanoblasts as early as 10 days post coitum. The finding of a marker for early melanoblasts enabled scientists to look for the mechanisms of coat color mutations. Steel and W mutants exhibited a white spotting color pattern. W and Steel encode, respectively, a receptor tyrosine kinase, Kit, and its ligand, which is known by several different names: steel factor, stem cell factor, mast cell growth factor, and Kit ligand. The mutation Steel-dickie (Sl) is a deletion of its transmembrane and cytoplasmic domains so that only a secreted form of stem cell factor is produced. Steel et al. (1992) found that the number of melanoblasts in (Sl/ Sl) mutants began to decrease at around 11 days post coitum. They also found that the melanoblasts caudal of the optic vesicle failed to migrate toward the vesicle. These results suggested that the cell surface form of stem cell factor is important for both the survival and migration of melanoblasts. Wehrle-Haller and Weston (1995) performed in situ hybridization with Kit, Tyrp-2, and stem cell factor probes in Sl (null) and Sl mutants to examine the early dispersal and fate of melanoblasts in order to elucidate the function of stem cell factor in more detail. They concluded that soluble stem cell factor is sufficient for responsive melanoblast precursors to initiate their dispersal onto the lateral migration pathway, and that cell-bound stem cell factor was necessary for the survival of melanoblasts in the newly formed dermal mesenchyme. Dorsky et al. (1998) found that cranial neural crest cells destined to encode pigment cells were located adjacent to the Wnt-1 and Wnt-3a expression domain, whereas neurons were far from the Wnt-expressing domain in zebrafish. Most lateral cells, which become neurons when forcibly overexpressing an activated form of b-catenin, adopted a pigment-cell fate. Conversely, when the investigators overexpressed a mutant form of Tcf-3 or a dominant-negative Wnt-1 to inhibit Wnt signaling in medial neural crest cells, the number of pigment cells decreased dramatically. Thus, Dorsky et al. provided key evidence that Wnt signaling plays an essential early role in pigment cell formation.

MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

Early events in kidney organogenesis involve reciprocal interactions between the ureteric bud and the metanephric mesenchyme, which lead to remodeling of the extracellular matrix. This remodeling involves matrix metalloproteases (MMPs), but the specific roles of individual MMPs in kidney development are not completely understood. Here, we analyzed MMP9-deficient mice at the first step of kidney development and found that MMP9 deficiency delayed embryonic kidney maturation and increased apoptosis ex vivo by 2.5-fold. These early defects resulted in a 30% decrease in nephron number, a 20% decrease in adult kidney weight, and altered kidney function and morphology at 12 mo. The membrane form of stem cell factor (SCF) increased, whereas the activated form of the SCF receptor, c-kit, decreased in MMP9-deficient embryonic kidneys. In organotypic culture, MMP9-deficient kidneys failed to secrete SCF, and addition of recombinant SCF partially rescued both apoptosis and the branching defect. In conclusion, these data show that MMP9 protects mesenchymal cells from apoptosis during kidney development and stimulates ureteric bud branching morphogenesis, most likely by releasing the soluble form of SCF, suggesting that normal renal development requires MMP9.

Mechanism of Action of l-CDB-4022, a Potential Nonhormonal Male Contraceptive, in the Seminiferous Epithelium of the Rat Testis

The present study was conducted to elucidate the possible molecular mechanisms involved in the antispermatogenic activity of l-CDB-4022, an indenopyridine. In this study 45-d-old male Sprague-Dawley rats were treated with a single oral dose of l-CDB-4022 (2.5 mg/kg) or vehicle, and blood and testes were collected at various time points. The rate of body weight gain was not affected, but a significant loss of testes weight was induced by l-CDB-4022. Serum hormones were assayed using specific RIAs or ELISAs, and testicular protein and RNA were analyzed by Western blotting and RT-PCR, respectively. There was a significant decrease in inhibin B and concomitant increase in FSH in serum from l-CDB-4022-treated rats, but serum levels of activin A, testosterone, and LH were unchanged. Western analysis of testicular lysates from l-CDB-4022-treated rats exhibited phosphorylation of ERK1/2 at 4 h and later time points. Loss of nectin/afadin complex occurred at 48 h, but there was an increase in levels of integrin-beta1, N-cadherin, alpha-catenin, and beta-catenin protein at 24 h and later time points. Increase in expression of Fas ligand and Fas receptor was detected 8 and 24 h after l-CDB-4022 treatment. The ratio of the membrane to soluble form of stem cell factor mRNA was decreased. Immunohistochemical analysis of testicular sections indicated a dramatic disruption of the Sertoli cell microtubule network in l-CDB-4022-treated rats. Collectively, these results suggest that l-CDB-4022 activates the MAPK pathway, reduces expression of prosurvival factors such as the membrane form of stem cell factor, alters expression of Sertoli-germ cell adherens junction proteins, disrupts Sertoli cell microtubule structure, and induces the proapoptotic factor, Fas, culminating in germ cell loss from the seminiferous epithelium.

Targeted retroviral transduction of c-kit+ hematopoietic cells using novel ligand display technology

AbstractGene therapy for a wide variety of disorders would be greatly enhanced by the development of vectors that could be targeted for gene delivery to specific populations of cells. We describe here high-efficiency targeted transduction based on a novel targeting strategy that exploits the ability of retroviruses to incorporate host cell proteins into the surface of the viral particle as they bud through the plasma membrane. Ecotropic retroviral particles produced in cells engineered to express the membrane-bound form of stem cell factor (mbSCF) transduce both human cell lines and primary cells with high efficiency in a strictly c-kit (SCF receptor)-dependent fashion. The availability of efficient targeted vectors provides a platform for the development of a new generation of therapies using in vivo gene delivery. (Blood. 2004;104: 2697-2703)

Primordial germ cells: what does it take to be alive?

Specification of primordial germ cells (PGCs) in the proximal epiblast enables about 45 founder PGCs clustered at the base of the allantoic bud to enter the embryo by active cell movement. Specification of the PGC lineage depends on paracrine signals derived from the somatic cell neighbors in the extraembryonic ectoderm. Secretory bone morphogenetic proteins (BMP) 4, BMP8b, and BMP2 and components of the Smad signaling pathway participate in the specification of PGCs. Cells in the extraembryonic ectoderm induce expression of the gene fragilis in the epiblast in the presence of BMP4, targeting competence of PGCs. The fragilis gene encodes a family of transmembrane proteins presumably involved in homotypic cell adhesion. As PGCs migrate throughout the hindgut, they express nanos3 protein. In the absence of nanos3 gene expression, no germ cells are detected in ovary and testis. During migration and upon arrival at the genital ridges, the population of PGCs is regulated by a balanced proliferation/programmed cell death or apoptosis. Paracrine and autocrine mechanisms, involving transforming growth factor-beta1 and fibroblast growth factors exert stimulatory or inhibitory effects on PGCs proliferation, modulated in part by the membrane-bound form of stem cell factor. Apoptosis requires the participation of the pro-apoptotic family member Bax, whose activity is balanced by the anti-apoptotic family member Bcl21/Bcl-x. In addition, a loss of cell-cell contacts in vitro results in the apoptotic elimination of PGCs. It needs to be determined whether apoptosis is triggered by a failure of PGC to establish and maintain appropriate cell-cell contacts with somatic cells or whether undefined survival factors released by adjacent somatic cells cannot reach physiological levels to satisfy needs of the expanding population of PGCs.

Fibrosis in ocular allergic inflammation: recent concepts in the pathogenesis of ocular allergy.

Mast cells and eosinophils are the main effector cells in allergic inflammation, but there is now compelling evidence that fibroblasts are also important players in the inflammatory response. In fact, they respond to different stimuli and release several mediators that modulate mast-cell and eosinophil functionality. In several allergic conditions such as vernal keratoconjunctivitis, asthma and atopic dermatitis the chronic presence of the inflammatory process has been associated with fibrosis and tissue remodeling, which in turn could cause irreversible alterations in the organ anatomy and functions. This review will discuss current advances in mast cell, eosinophil and fibroblast interactions in terms of their importance in the perpetuation of allergic inflammation and in contributing to the fibrosis and/or remodeling process in ocular allergy. As a main example of allergic ocular diseases associated with fibrosis, vernal keratoconjunctivitis is discussed in the light of recent findings. Several studies have recently shown that fibroblasts can modulate the functions of mast cells and eosinophils through the membrane form of stem cell factor and granulocyte-macrophage colony-stimulating factor, respectively. On the other hand, fibroblasts can be affected by inflammatory mediators derived from mast cells and eosinophils, such as transforming growth factor beta and nerve growth factor and by the T helper type 2 cytokines, IL-4 and IL-13, and vernal keratoconjunctivitis-derived fibroblasts display altered functions. Considerable useful information has been gained about the role of mast cells, eosinophils and fibroblasts in the perpetuation of allergic inflammation and tissue fibrosis and/or remodeling in general, and specifically in ocular allergy.

Non-IgE–dependent activation of human lung– and cord blood–derived mast cells is induced by eosinophil major basic protein and modulated by the membrane form of stem cell factor

The allergic reaction begins with the antigen-induced aggregation of occupied high-affinity IgE receptors expressed on mast cell surface, their activation, and the release of proinflammatory mediators that cause the "early phase" of this process. In addition, mast cell activation induces the onset of a "late phase" reaction characterized by the tissue infiltration of inflammatory cells, mainly eosinophils. We have hypothesized that during the late phase mast cells interact with and are activated by eosinophils. Here we report that highly purified human lung mast cells became responsive to eosinophil major basic protein (MBP) when in coculture with human lung fibroblasts. In addition, cord blood-derived mast cells maintained in coculture with 3T3 fibroblasts released more histamine and prostaglandin D(2) (PGD(2)) compared with cells maintained in suspension. The fibroblast-derived membrane form of stem cell factor (SCF) was found to be involved in the mast cell increased responsiveness to MBP. In fact, cord blood-derived mast cells cocultured with 3T3 in the presence of antisense for SCF or cocultured with fibroblasts that do not express the membrane form of SCF were inhibited in their histamine-releasing activity toward MBP. In addition, this form of SCF induced the expression of a pertussis toxin-sensitive G(i) protein, G(i3) that interacts with MBP to trigger mast cell non-IgE-dependent activation in a manner similar to other cationic compounds such as compound 48/80. Mast cell responsiveness to eosinophil mediators is a potentially novel evidence for an alternative pathway of allergen-independent activation able to contribute to the perpetuation of allergy.

Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal.

Located within the tunica muscularis of the gastrointestinal (GI) tract are networks of cells known as interstitial cells of Cajal (ICC). ICC are critical for important basic functions of GI motility such as generation and propagation of slow-wave pacemaker activity and reception of regulatory inputs from the enteric nervous system. We have developed a novel procedure to identify and isolate individual ICC from freshly dispersed cell preparations of the murine small intestine and gastric fundus and to determine differential transcriptional expression We have compared the expression profiles of pacemaker ICC isolated from the murine small intestine (IC-MY) and ICC involved in neurotransmission from the gastric fundus (IC-IM). We have also compared expression profiles between ICC and smooth muscle cells (SMC) and between freshly isolated ICC and cultured ICC. Cultured ICC express smooth muscle myosin, whereas freshly dispersed ICC do not. All cell types express muscarinic receptor types M(2) and M(3), neurokinin receptors NK(1) and NK(3), and inhibitory receptor VIP-1, whereas only cultured ICC and SMC express VIP-2. Both cultured and freshly dispersed IC-IM and IC-MY express the soluble form of stem cell factor, whereas SMC from the gastric fundus express only the membrane-bound form.

Requirement of endogenous stem cell factor and granulocyte-colony-stimulating factor for IL-17-mediated granulopoiesis.

IL-17 is a novel, CD4+ T cell-restricted cytokine. In vivo, it stimulates hematopoiesis and causes neutrophilia consisting of mature granulocytes. In this study, we show that IL-17-mediated granulopoiesis requires G-CSF release and the presence or induction of the transmembrane form of stem cell factor (SCF) for optimal granulopoiesis. However, IL-17 also protects mice from G-CSF neutralization-induced neutropenia. G-CSF neutralization completely reversed IL-17-induced BM progenitor expansion, whereas splenic CFU-GM/CFU-granulocyte-erythrocyte-megakaryocyte-monocyte was only reduced by 50% in both Sl/Sld and littermate control mice. Thus, there remained a significant SCF/G-CSF-independent effect of IL-17 on splenic granulopoiesis, resulting in a preservation of mature circulating granulocytes. IL-17 is a cytokine that potentially interconnects lymphocytic and myeloid host defense and may have potential for therapeutic development.

Effects of feeder cells and growth factors on the proliferation of mouse primordial germ cells

There are various kinds of stem cell which have the ability to self-renew under appropriate microenvironments and growth factors. In vitro culture of stem cells may provide a tool for elucidating the mechanism of self-renewal of these cells. Hematopoietic B cell progenitor cell proliferation is supported on the born marrow-derived cell line, ST2. Embryonic stem (ES) cells derived from mouse blastocysts acquire their ability for self-renewal and pluripotency on mouse embryonic fibroblast cells (MEF). In the present study, we examined effects of these 2 different kinds of feeder cells, in the presence of growth factors, on the ability of mouse primordial germ cells (PGCs) collected at 8.5 d post coitum (dpc) to proliferate and acquire of pluripotency. The addition of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF), ST2, but not MEF, supported the proliferation of the PGCs for at least 5 d in culture. The presence of either LIF or bFGF renders a lower ability to support the proliferation of PGCs. Reverse transcription-PCR analysis shows that both feeder cells transcribed the soluble form of stem cell factor (SCF) but not membrane-bound SCF, which is an established factor related to the proliferation and pluripotency of PGCs. These results suggest that both hematopoietic stem cells and PGCs make use of the common growth factors for their growth in culture. In addition, the inability of MEF to affect proliferation of PGCs may suggest that the initial culture conditions for the proliferation could be different between ES cells and PGCs in the mouse.

Long-Term Culture of Chronic Myelogenous Leukemia Marrow Cells on Stem Cell Factor-Deficient Stroma Favors Benign Progenitors

Long-term culture of marrow from patients with chronic myelogenous leukemia (CML) has been reported to favor the outgrowth of bcr/abl- progenitor cells in some patients. We examined the effect of the presence of soluble or transmembrane forms of stem cell factor (SCF) in long-term cultures of CML marrow. CD34-enriched cells from CML patients in advanced chronic phase or accelerated phase were plated on immortalized fetal liver stromal cells from homozygous SCF-deficient SI/SI mice (SI/SI4) with or without the addition of soluble human SCF, SI/SI4 cells expressing high levels of the transmembrane form of human SCF (SI/SIh220), or primary human allogeneic stroma. Cells were removed from cultures and plated weekly in colony assays. The clonagenic cell output from cultures completely lacking SCF was lower over the first 2 to 3 weeks, but by 5 weeks was similar to the clonagenic cell output from the other culture conditions. Analysis of bcr/abl transcripts from individual colonies showed a lower percentage of malignant progenitors present in long-term cultures completely deficient in SCF than under the other culture conditions, particularly compared with primary human stroma-containing long-term cultures. SCF may specifically favor malignant versus benign progenitor cells present in the marrow of CML patients, and an abnormal proliferative response to SCF in very primitive cells may be an underlying defect in the pathophysiology of this disease.

Long-Term In Vivo Expression of a Murine Adenosine Deaminase Gene in Rhesus Monkey Hematopoietic Cells of Multiple Lineages After Retroviral Mediated Gene Transfer Into CD34+ Bone Marrow Cells

Abstract Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.

Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.