Existence of reserve quiescent stem cells in adults, from amphibians to humans.

Several theories have been proposed to explain the phenomenon of tissue restoration in amphibians and higher order animals. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed stem cells, and activation of quiescent stem cells. Young and colleagues demonstrated that connective tissues throughout the body contain multiple populations of quiescent lineage-committed progenitor stem cells and lineage-uncommitted pluripotent stem cells. Subsequent cloning and cell sorting studies identified quiescent lineage-uncommitted pluripotent mesenchymal stem cells, capable of forming any mesodermal cell type, and pluripotent epiblastic-like stem cells, capable of forming any somatic cell type. Based on their studies, they propose at least 11 categories of quiescent reserve stem cells resident within postnatal animals, including humans. These categories are pluripotent epiblastic-like stem cells, pluripotent ectodermal stem cells, pluripotent epidermal stem cells, pluripotent neuronal stem cells, pluripotent neural crest stem cells, pluripotent mesenchymal (mesodermal) stem cells, pluripotent endodermal stem cells, multipotent progenitor stem cells, tripotent progenitor stem cells, bipotent progenitor stem cells, and unipotent progenitor stem cells. Thus, activation of quiescent reserve stem cells, i.e., lineage-committed progenitor stem cells and lineage-uncommitted pluripotent stem cells, resident within the connective tissues could provide for the continual maintenance and repair of the postnatal organism after birth.

Karyotypic analysis of adult pluripotent stem cells.

Three categories of precursor cells have been identified in postnatal mammals: tissue-committed progenitor cells, germ layer lineage-committed stem cells and lineage-uncommitted pluripotent stem cells. Progenitor cells are the immediate precursors of differentiated tissues. Germ layer lineage stem cells can be induced to form multiple cell types belonging to their respective ectodermal, mesodermal, and endodermal embryological lineages. Pluripotent stem cells will form somatic cell types from all three primary germ layer lineages. Progenitor cells demonstrate a finite life span before replicative senescence and cell death occur. Both germ layer lineage stem cells and pluripotent stem cells are telomerase positive and display extensive capabilities for self-renewal. Stem cells which undergo such extensive replication have the potential for undergoing mutations that may subsequently alter cellular functions. Gross mutations in the genome may be visualized as chromosomal aneuploidy and/or chromosomes that appear aberrant. This study was designed to determine whether any gross genomic mutations occurred within the adult pluripotent stem cells. Karyotypic analysis was performed using pluripotent stem cells purified from adult male rats using established procedures. Giemsa Banding was used in conjunction with light microscopy to visualize metaphase chromosome spreads. To date over 800 metaphase spreads have been analyzed. We found that the metaphase spreads averaged 42 chromosomes and concluded that these pluripotent stem cells isolated from adult rats have a normal karyotype.

Change in synthesis of sulfated glycoconjugates during muscle development, maturation and aging in embryonic to senescent CBF-1 mouse.

Previous biochemical and morphological studies have demonstrated a change in the synthetic pattern of sulfated proteoglycans during skeletal musculogenesis in the embryonic chick. These studies revealed that a transition occurs in both composition and deposition of sulfated glycoconjugates that parallels the developmental state of the tissue. The current study was undertaken to ascertain whether this transition in the embryonic chick is a conserved developmental process during musculogenesis in the mouse. Leg musculature from embryonic, newborn, juvenile, adolescent, young adult, mature adult and senescent mice, radiolabeled in vivo with [35S]sulfate, was analyzed for relative size and composition of newly synthesized sulfated macromolecules. The data reveal a transition in the synthesis of sulfated proteoglycans and glycoproteins that parallels the myogenic differentiative state of the mouse leg muscle. Embryonic mouse leg musculature synthesizes relatively large proteoglycans consisting of large chondroitin sulfate glycosaminoglycan chains. Subsequently, these major newly synthesized proteoglycans are replaced synthetically by smaller molecules composed of mixtures of dermatan sulfate, chondroitin sulfate and heparan sulfate glycosaminoglycans (newborn through 2 weeks); dermatan sulfate, heparan sulfate and chondroitin sulfate glycosaminoglycans (13 months) and heparan sulfate and dermatan sulfate glycosaminoglycans (25-26 months). The sulfated glycoproteins demonstrate a reciprocal synthetic pattern. Early in development sulfated glycoproteins form a small proportion of the newly synthesized sulfated material. With increasing developmental and maturational age, the proportion of sulfated glycoproteins increases. This continues until they become the predominant sulfated moieties synthesized by senescent mouse muscle. The results from this study thus extend observations initially made in chick to muscle development in the mouse and, therefore, suggest that the transition in synthesis of sulfated glycoconjugates is a conserved developmental process during musculogenesis.

Comparison of fixatives for maximal retention of radiolabeled glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated [35S]-sulfate.

Previous studies have used [35S]-sulfate as a specific marker to autoradiographically localize sulfated glycosaminoglycans, proteoglycans, and glycoproteins. Embryonic chicks were labeled with [35S]-sulfate, followed by previously reported routine fixation and processing techniques. Subsequent processing revealed loss of radiolabeled macromolecules and retention of unincorporated label in the tissue, using these procedures. Biochemical analysis after various fixation and processing procedures demonstrated that an additional agent, such as cetylpyridinium chloride, was necessary in the fixative to retain the highly aqueous soluble sulfated macromolecular components. Molecular sieve chromatography was used to monitor digestate solutions for the identity of glycosaminoglycans and proteoglycans as indicated by selective enzymatic removal. Retained unincorporated [35S]-sulfate could be completely removed by rinsing the tissue in dehydration solutions containing exogenous sodium sulfate. This new procedure ensures the quantitative retention of sulfate labeled macromolecules in fixed tissue with the complete removal of unincorporated radiotracer, both of which are necessary for meaningful autoradiography.

Experience with fully 3D PET and implications for future high-resolution 3D tomographs.

The aim of this paper is to report on experience with 3D positron emission tomography (PET) in our institute where we have three 3D scanners, of which two operate exclusively in 3D mode (ECAT ART, EXACT 3D). Fully 3D PET requires attention to a number of factors which are not as problematic in 2D PET. Firstly, 3D tomographs designed for whole-body acquisition suffer from a large single-photon field of view, extending well beyond the coincidence field of view. Single photons from outside the coincidence field of view increase the dead time and random coincidence rates, and contribute scattered events. For brain studies, we have extended the lead side shielding at the ends of the tomograph to mitigate against these effects, and this has dramatically improved the count rate performance. This approach is not as effective for whole-body scanning. In addition, operating in 3D without septa necessitates new approaches to transmission scanning, as measurements using positron emitters such as 68Ge/68Ga have the unfavourable characteristics of high dead time and high scatter. Both of our fully 3D scanners use 137Cs for single-photon transmission measurements, although the data are treated differently. On the ECAT ART, a combination of physical and electronic collimation effectively reduces transmission scatter to acceptable levels. On the EXACT 3D physical collimation is not as readily implemented and therefore segmentation and reassignment of the histogrammed attenuation (mu) values is employed to produce unbiased attenuation correction factors in 3D. Many of the lessons learnt with these BGO (bismuth germanate) based tomographs will be applicable to the next generation of systems using faster detectors such as lutetium oxyorthosilicate (LSO).

Histochemical analysis of newly synthesized and accumulated sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg.

The leg musculature from 11, 14, and 17 day chick embryos was analyzed histochemically to investigate the temporal and spatial distribution of various types of sulfated glycosaminoglycans present during skeletal muscle development. Types of glycans were identified by selective degradation with specific glycosidases and nitrous acid coupled with Alcian blue staining procedures for sulfated polyanions and with [35S]sulfate autoradiography. On day 11, radiolabeled chondroitin sulfate glycosaminoglycans are localized extracellularly in both the myogenic and connective tissue cell populations. By day 17, incorporation of [35S]sulfate into chondroitin sulfate is substantially reduced, although Alcian blue-stained chondroitin sulfate molecules are still detectable. With increasing age and developmental state of the tissues, radiolabeled and stained dermatan sulfate and heparan sulfate progressively increase in relative quantity compared to chondroitin sulfate both in muscle and in associated connective tissue elements. These changes in glycosaminoglycans correlate well with similar changes previously determined biochemically and further document the alterations in extracellular matrix components during embryonic skeletal myogenesis.

Pluripotent mesenchymal stem cells reside within avian connective tissue matrices.

Recent studies have noted the presence of putative stem cells derived from the connective tissues associated with skeletal muscle, heart, and dermis. Long-term continuous cultures of these cells from each tissue demonstrated five distinct phenotypes of mesodermal origin, i.e. muscle, fat, cartilage, bone, and connective tissue. Clonal analysis was performed to determine whether these morphologies were the result of a mixed population of lineage-committed stem cells or the differentiation of pluripotent stem cells or both. Putative stem cells from four tissues (skeletal muscle, dermis, atria, and ventricle) were isolated and cloned. Combined, 1158 clones were generated from the initial cloning and two subsequent subclonings. Plating efficiency approximated 5.8%. Approximately 70% of the 1158 clones displayed a pure stellate morphology, while the remaining clones contained a mixture of stellate, chondrogenic- or osteogenic-like morphologies or both. When cultured in the presence of dexamethasone, cells from all clones differentiated in a time- and concentration-dependent manner into muscle, fat, cartilage, and bone. These results suggest that pluripotent mesenchymal stem cells are present within the connective tissues of skeletal muscle, dermis, and heart and may prove useful for studies concerning the regulation of stem cell differentiation, wound healing, and tissue restoration, replacement and repair.

Differentiation factors induce expression of muscle, fat, cartilage, and bone in a clone of mouse pluripotent mesenchymal stem cells.

Growth factors have been used experimentally to accelerate wound healing by increasing scar tissue formation at a wound site. These studies suggest that stimulation of fibroblastic differentiation and proliferation are essential components of adult tissue repair. Recent studies report the presence of mesenchymal stem cells within granulation tissue and as connective tissue-resident stem cells. This suggests that mesenchymal stem cells as well as fibroblasts may contribute to wound healing and repair. To determine the potential for mesenchymal stem cells to contribute to nonfibrogenic tissue repair, a clonal population of murine mesenchymal stem cells was examined with dexamethasone, a general differentiation agent, and muscle morphogenetic protein, a specific differentiation-inducing agent. Dexamethasone induced the expression of phenotypic markers for fat, cartilage, and bone in the stem cells. Muscle morphogenetic protein induced the expression of mRNAs for the muscle specific regulatory genes MyoD1 and myogenin in these cells. These results suggest that pluripotent mesenchymal stem cells within connective tissue compartments and granulation tissue have the potential to contribute to functional tissue restoration, rather than contributing solely to fibrogenic scar tissue formation during tissue repair.

Initial characterization of small proteoglycans synthesized by embryonic chick leg muscle-associated connective tissues.

Muscle development in the embryonic chick involves the interfacing of two different cell types: myogenic cells forming the contractile network and connective tissue cells which wrap this network into discrete morphologies. Previous reports from this laboratory detailed the characterization of large extracellular matrix proteoglycans synthesized by skeletal muscle. This report describes the initial characterization of small muscle-associated connective tissue proteoglycans synthesized in embryonic chick leg in ovo and by embryonic leg muscle-associated secondary fibroblasts in vitro by CsCl equilibrium density gradient centrifugation. Analysis of the D1 gradient fractions from the in ovo and in vitro material revealed that the proteoglycans were of relatively small size. These molecules eluted from Sepharose CL-2B through a Kav range of 0.6-0.8. Analysis of alkaline borohydride-released glycosaminoglycan chains by Sepharose CL-6B, Sephadex G-25, and thin layer chromatography demonstrated a mixture of three proteoglycan families composed of chondroitin sulfate, dermatan sulfate, and heparan sulfate glycosaminoglycans.

Effect of selected denervations on glycoconjugate composition and tissue morphology during the initiation phase of limb regeneration in adult Ambystoma.

This study was undertaken to assess the effects of various quantities of neural tissue on the temporal relationship of matrix glycoconjugates to the regeneration morphology. 1) Denervation before amputation revealed that a threshold level of nervous tissue was necessary to activate a regeneration response from the tissue, i.e., appearance of regeneration-specific morphologies and glycoconjugates. 2) Denervation after amputation demonstrated that the level of neural tissue necessary to maintain these responses was below the level necessary to activate the regeneration response. If neural tissue was completely removed there was a concomitant loss of regenerate morphologies and glycoconjugates. 3) Bilateral amputation of a neurogenically intact limb and its completely denervated contralateral limb revealed that the regeneration response was a localized phenomenon during the first 30 days after amputation. After 30 days the regeneration response appeared within the previously degenerated denervating limb. The results suggest that the factors controlling the regenerative response in adult Ambystoma are large diffusible substances that can be transported by the circulation and can affect the regenerative response in remote, previously activated, tissues.

Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma.

The present study identifies, localizes, and reports the relative composition of specific glycosaminoglycans within tissue matrices during the initiation phase of limb regeneration. The regenerate tissues were harvested and assayed morphologically, histochemically, and chemically. We observed 1) a population of cells interspersed among the cells of the dermis, epimysium, perimysium, perichondrium, and periosteum. 2) This population was distinguishable by a unique pattern of glycoconjugate staining, i.e., intracellular and pericellular heparan sulfate and glycoproteins and extracellularly associated hyaluronate and glycoproteins. 3) Cells with these staining characteristics aggregated to a position directly beneath the apical epidermal cap. 4) Extracellular hyaluronate and glycoproteins colocalized with undifferentiated tissues. And 5) extracellular chondroitin sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans colocalized with differentiated tissues. The correlations of distinct glycoconjugate compositions with specific regeneration morphologies suggest the possibility that these components may be related to the phenotypic expression of tissues during regeneration.

Environmental conditions prerequisite for complete limb regeneration in the postmetamorphic adult land-phase salamander, Ambystoma.

Historically, postmetamorphic adult land-phase salamanders have been shown to exhibit minimal to nonexistent limb regeneration. Hence, it has been generally accepted that these forms have lost the intrinsic capacity to regenerate a limb. Due to the experimental protocols used, an alternate explanation is also possible: that this intrinsic capacity cannot be expressed when the salamanders are maintained under adverse laboratory environmental conditions. Therefore, this study addresses two questions: 1) What are the optimal environmental conditions for long-term survival of adult land-phase salamanders; and 2) will complete limb regeneration occur in these salamanders if they are maintained under survival conditions. A mixed population of adult Ambystoma were tested under varying conditions of habitat, temperature, humidity, photoperiod, and food source. Complete limb regeneration was possible in 100% of four species of adult postmetamorphic land-phase Ambystoma salamanders given the proper environmental laboratory conditions of a peat moss and potting soil habitat with a controlled temperature of 25 degrees C +/- 5 degrees C, 70% or greater humidity, a 12/12 light/dark photoperiod, a diet including nightcrawlers released into their respective terraria, and an extended observation time of up to 370 days postamputation (dpa). Regeneration was completed during the following range periods for the adult salamanders: A. annulatum, 324 to 370 dpa; A. maculatum, 255 to 300 dpa; A. texanum, 215 to 250 dpa; and A. tigranum, 155 to 180 dpa.

Gross morphological analysis of limb regeneration in postmetamorphic adult Ambystoma.

Due to the great disparity between regeneration times for the larval salamander (40 days), axolotl (30+ days), newt (44 days), and adult salamander (155 to 370 days), a staging system was devised so correlative comparisons could be made between regenerative model systems. The sequence was based on two criteria: 1) the stages should be similar to previously reported sequences for the newt, axolotl, and larval salamander, and 2) the stages must be readily recognizable by examination of the external morphology in the living state. Postmetamorphic adult land-phase Ambystoma were amputated through the forearm, placed within survival conditions, and observed until regeneration was completed. Of the initial 160 salamanders, 157 (98%) progressed through 11 clearly defined stages of regeneration. A side-by-side comparison of the staging sequence for land and aquatic phase urodeles is given along with a summary of key external morphological characteristics for the adult salamander. It was noted that as the length of time for regeneration decreased, the relative ratio of the nerves innervating the limb (spinal nerves 4, 5, and 6) increased for the four species of Ambystoma examined: A. annulatum, 324 to 370 days postamputation (dpa) with a 1:1:1 neural tissue ratio; A. maculatum, 255 to 300 dpa with a 2:2:1 ratio; A. texanum, 215 to 250 dpa with a 2:2:2 ratio; and A. tigranum, 155 to 180 dpa with a 2:3:3 ratio.

Effects of prenatal ethanol exposure on the hippocampal neurochemistry of albino rats at 90 days of postnatal age.

OBJECTIVE: Our purpose was to test the hypothesis that prenatal ethanol exposure alters the hippocampal muscarinic cholinergic neurochemistry of albino rats. STUDY DESIGN: Ethanol was administered in a liquid diet to pregnant albino Sprague-Dawley rats. Liquid diet control animals received the same diet in which ethanol was replaced by an isocaloric amount of maltose-dextrin. Chow-fed control animals were fed laboratory chow as desired. Progeny were killed at 90 days of age, and their hippocampi were analyzed for muscarinic cholinergic receptors by use of tritiated quinuclidinyl benzilate. RESULTS: Prenatal ethanol exposure produced a statistically significant decrease in the number of muscarinic receptors in males. Similar trends were noted in females, but the results were not statistically significant. CONCLUSION: Prenatal ethanol treatment caused long-lasting alterations in the muscarinic cholinergic receptors of the hippocampus in male rats.

Histological analysis of limb regeneration in postmetamorphic adult Ambystoma.

Previous investigation into the regenerative ability of postmetamorphic adult land phase Ambystoma has revealed that these species have the capacity to completely regenerate a limb, given optimal environmental conditions, and the gross morphological characteristics of limb regeneration in these species compared favorably with the external regeneration morphology of aquatic phase forms. The present study concerns a histological and histochemical examination of the regenerating limb tissues and their respective extracellular and intracellular tissue matrices. Postmetamorphic adult Ambystoma were amputated through the forearm, placed within optimal environmental conditions, and allowed to regenerate. The tissues were harvested at designated intervals after amputation and prepared for light microscopic examination. The limb tissues were assayed histologically for similarities to and differences from previously established regeneration morphologies. It was noted that specific correlations (i.e., apical epidermal cap formation, but outgrowth and elongation, palette formation, and digit formation) existed between regeneration histologies in these species and those previously reported for the aquatic urodeles, newt, axolotl, and larval salamander. By utilizing the histological and histochemical characteristics of the tissue, the regenerate limb was divided into five tissue units: epidermal, blastemal, soft, hard, and neuro/vascular. Based on the unique morphology of their extracellular matrices and respective histochemical staining patterns, four distinct blastemal regions were delineated within the blastemal units: subregenerate epidermal blastema, soft-tissue blastema, hard-tissue blastema, and core blastema. Histochemically, changing patterns of highly sulfated, weakly sulfated, and carboxylated polysaccharides and glycosylated compounds were located within both the extra- and intracellular stump and regenerate tissue matrices during regeneration. In addition, these patterns of intra- and extracellular macromolecular material correlated to previous reports of similar-type compounds assayed during regeneration in aquatic urodeles. With this in mind, the adult land phase Ambystoma can be considered an appropriate model system for studies concerning normal limb regeneration.

Formation of abdominal adhesions is inhibited by antibodies to transforming growth factor-beta1.

Transforming growth factor-beta (TGF-beta) is an important factor in regulating the inflammatory response and the production of extracellular matrix by fibroblasts. These two processes are linked in the formation of fibrous adhesions after abdominal surgery. When the mesothelium is injured a fibrin strand is produced which is populated first by inflammatory cells then by fibroblasts which secrete extracellular matrix forming a permanent adhesion. TGF-beta promotes both chemotaxis of monocytes and the production of extracellular matrix by fibroblasts. We have used a model of abdominal adhesions in rats in which a circle of peritoneum is dissected and then sutured into place again. After 2 weeks the rats are euthanized and the adhesions are scored. Six groups of 10 rats each underwent this surgery. Group I served as the operative control. Group II was treated with saline which was injected immediately after surgery and on Days 1 and 2 after surgery (vehicle control). Using the same protocol with saline as vehicle, the other four groups of rats were treated with nonspecific IgG (150 microgram per day), anti-TGF-beta (panspecific, 167 microgram per day), anti-TGF-beta1 (67 microgram per day), or anti-TGF-beta2 (50 microgram per day). The rats injected with anti-TGF-beta1 had significantly lower adhesion scores (P < 0.05) than the controls. Rats injected with anti-TGF-beta2 or anti-TGF-beta (panspecific) did not differ significantly from the control saline-injected rats. The results indicate that specifically reducing levels of TGF-beta1 alone can be effective in preventing abdominal adhesions.

A population of cells isolated from rat heart capable of differentiating into several mesodermal phenotypes.

A population of stem cells has been isolated from embryonic avian and neonatal rat skeletal muscle. These cells differentiate into several mesodermal phenotypes in culture upon treatment with dexamethasone. This study reports the isolation of a similar population of stem cells from another mesodermal tissue, the heart. Hearts were excised from 3- to 5-day- old rats, minced, and treated with a collagenase-dispase solution. Single cells were collected by centrifugation, washed, and plated in dishes. The cells were grown to confluence, trypsinized, and frozen at -80 degrees C in 7.5% dimethylsulfoxide. After at least 24 hr, the cells were thawed and plated in 24-well plates and treated with media containing dexamethasone at concentrations of 10(-6)-10(-10) M for 4 weeks. Control cultures contained mononucleated cells with a stellate morphology. Treatment with dexamethasone resulted in the appearance of several mesodermal phenotypes. Bone and cartilage nodules were identified with von Kossa and Alcian blue staining respectively. Adipocytes were identified using Sudan black B stain. Smooth muscle cells were identified by an anti-smooth muscle alpha-actin antibody, and skeletal myotubes were stained with anti-myosin antibody. Large binuclear cells with obvious fibers were noted and stained with anti-desmin. These binuclear cells appeared in both the control and the dexamethasone-treated cultures and were tentatively identified as cardiomyocytes. These data strongly suggest the existence of a population of mesenchymal stem cells in neonatal rat heart.

Recombinant human bone morphogenetic proteins-2 and -4 induce several mesenchymal phenotypes in culture.

Bone morphogenetic protein has previously been shown to induce the formation of cartilage and bone in vivo. We have isolated a population of mesenchymal stem cells from rat skeletal muscle capable of forming multiple mesodermal morphologies in vitro. These cells were treated with recombinant human bone morphogenetic proteins-2 and -4 to determine the differentiation-inducing activities of bone morphogenetic protein on these cells. The mesenchymal stem cells were cultured in medium with 10% preselected horse serum containing 0 to 100 ng/ml recombinant human bone morphogenetic proteins-2 or -4 for a maximum of 4 weeks. Control cultures maintained the stellate morphology of mesenchymal stem cells. Cultures treated with recombinant human bone morphogenetic protein-2 exhibited discrete cartilage nodules and mineralized bone nodules. The first increase in chondrogenesis was seen at 0.5 ng/ml. Cultures treated with recombinant human bone morphogenetic protein-4 also exhibited an increase in chondrogenesis at the higher concentration of 2 ng/ml. Skeletal myotubes and adipocytes also appeared in cultures treated with either bone morphogenetic protein. Mesenchymal stem cells do respond to inductive factors, but bone morphogenetic proteins-2 and -4 were not specific for the induction of cartilage and bone.

Muscle morphogenetic protein induces myogenic gene expression in Swiss-3T3 cells.

Myogenesis is thought to be regulated by the MyoD family of regulatory genes, which includes MyoD, myogenin, MRF- 4/myf-6, and myf-5. In situ hybridization studies of vertebrate skeletal muscle development have shown the colocalization of the MyoD family of regulatory genes to specific stages of muscle development. Although many studies have analyzed the regulatory role of these genes during myogenesis, there have been few reports dealing with the activation of these myogenic regulatory genes by exogenous agents. We have previously shown that muscle morphogenetic protein induces myogenesis in clonal populations of avian pluripotent stem cells. The current study was designed to examine the ability of muscle morphogenetic protein to induce myogenesis in a clonal population derived from the established fibroblastic Swiss-3T3 cell line. Swiss-3T3 cells were cloned to generate separate cell populations, tested for pluripotency, propagated through 690 cell doublings, retested for pluripotency, treated with muscle morphogenetic protein, and examined for the induction of gene expression using probes for the transcription products of MyoD and myogenin. Muscle morphogenetic protein induced the expression of mRNAs for MyoD and myogenin, suggesting a role for this compound as an exogenous activator of myogenesis.

Bioactive factors affect proliferation and phenotypic expression in progenitor and pluripotent stem cells.

Progenitor and pluripotent stem cells reside within connective tissue compartments. They are also present in granulation tissue. This study examined the effects of treating these two cell populations with eight bioactive factors. Cells were assayed for DNA content as a measure of proliferation and for tissue-specific phenotypic markers as measures of lineage progression and lineage commitment. Platelet-derived endothelial growth factor and insulin-like growth factor-II did not induce proliferation in either population. However, dexamethasone, insulin, insulin-like growth factor-I, muscle morphogenetic protein, platelet-derived growth factor-AA, and platelet-derived growth factor-BB stimulated proliferation in one or both cell populations. Platelet-derived growth factor-BB was the most potent stimulator of proliferation in either population. Phenotypic expression markers were induced in the progenitor cells by insulin, insulin-like growth factor-I, insulin-like growth factor-II, dexamethasone, and muscle morphogenetic protein. However, only dexamethasone and muscle morphogenetic protein induced phenotypic expression markers in the pluripotent cells. Platelet-derived endothelial cell growth factor, platelet-derived growth factor-AA, and platelet-derived growth factor-BB did not induce phenotypic expression markers in progenitor or pluripotent cells. This study suggests the potential for using progenitor and pluripotent cells as an in vitro model to ascertain the effects of various bioactive factors on stem cells potentially involved in tissue maintenance and repair.