Henry E. Young

Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors

This study details the profile of 13 cell surface cluster differentiation markers on human reserve stem cells derived from connective tissues. Stem cells were isolated from the connective tissues of dermis and skeletal muscle derived from fetal, mature, and geriatric humans. An insulin/dexamethasone phenotypic bioassay was used to determine the identity of the stem cells from each population. All populations contained lineage‐committed myogenic, adipogenic, chondrogenic, and osteogenic progenitor stem cells as well as lineage‐uncommitted pluripotent stem cells capable of forming muscle, adipocytes, cartilage, bone, fibroblasts, and endothelial cells. Flow cytometric analysis of adult stem cell populations revealed positive staining for CD34 and CD90 and negative staining for CD3, CD4, CD8, CD11c, CD33, CD36, CD38, CD45, CD117, Glycophorin‐A, and HLA DR‐II. Anat Rec 264:51–62, 2001.

Neuronal differentiation of stem cells isolated from adult muscle.

Lineage uncommitted pluripotent stem cells reside in the connective tissue of skeletal muscle. The present study was carried out with pluripotent stem cells (PPSCs) isolated from 6‐month old rat muscle. Before differentiation, these cells were vimentin+, CD90+, CD45−, and varied in their expression of CD34. The PPSCs were expanded as non‐adherent aggregates under similar conditions to those used to generate neurospheres from embryonic or neural stem cells. The PPSC‐derived neurospheres were positive for nestin, an early marker present in neuronal precursors, and expressed the two alternative mRNA forms of the neuroectodermal marker Pax‐6, as well as mRNA for Oct‐4, a gene related to the pluripotentiality of stem cells. To confirm their neural potential, PPSC‐derived neurospheres were plated on coated coverslips under varying conditions: Neurobasal medium with N2 or B27, and either NT3 or BDNF. After 4–6 days the cells expressed neuronal (Tuj1+, NF68), astrocytic (GFAP) and oligodendrocytic (MOSP+, MBP+) markers, both by immunocytochemistry and RT‐PCR. In addition, PPSCs were cultured as monolayers under adherent conditions, exposed to growth factors and defined differentiating conditions for 5 hr, and subsequently kept for 2 days in a maturation medium. At this point they gave rise to a mixed population of early neural progenitors (Nestin+ or NG2+), immature and mature neurons (Tuj1+ and NF145+) and myelin producing oligodendrocytes (CNPase + and MOSP+). Our study shows that PPSCs present in adult muscle can overcome germ lineage restrictions and express the molecular characteristics of brain cells. Therefore, PPSCs isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative and demyelinating diseases.

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Clonogenic analysis reveals reserve stem cells in postnatal mammals: I. Pluripotent mesenchymal stem cells

Clonal populations of lineage‐uncommitted pluripotent mesenchymal stem cells have been identified in prenatal avians and rodents. These cells reside in the connective tissue matrices of many organs and tissues. They demonstrate extended capabilities for self‐renewal and the ability to differentiate into multiple separate tissues within the mesodermal germ line. This study was designed to determine whether such cells are present in the connective tissues of postnatal mammals. This report describes a cell clone derived by isolation from postnatal rat connective tissues, cryopreservation, extended propagation, and serial dilution clonogenic analysis. In the undifferentiated state, this clone demonstrates a high nuclear‐to‐cytoplasmic ratio and extended capacity for self‐renewal. Subsequent morphological, histochemical, and immunochemical analysis after the induction of differentiation revealed phenotypic markers characteristic of multiple cell types of mesodermal origin, such as skeletal muscle, smooth muscle, fat cells, cartilage, and bone. These results indicate that this clone consists of pluripotent mesenchymal stem cells. This report demonstrates that clonal populations of reserve stem cells are present in mammals after birth. Potential roles for such cells in the maintenance, repair, and regeneration of mesodermal tissues are discussed. Anat Rec 263:350–360, 2001.

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine.

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast‐like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6‐hydroxydopamine‐lesioned Parkinsons model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult‐derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.

A population of cells resident within embryonic and newborn rat skeletal muscle is capable of differentiating into multiple mesodermal phenotypes.

We have previously shown a population of putative mesenchymal stem cells in the connective tissue surrounding embryonic avian skeletal muscle. These cells differentiate into at least five recognizable phenotypes in culture: fibroblasts, chondrocytes, myotubes, osteoblasts, and adipocytes. We have now isolated a similar population of cells from fetal and newborn rat skeletal muscle. Cells from rat leg muscle were dissected, minced, and then enzymatically digested with a collagenase‐dispase solution. The dissociated cells were plated and allowed to differentiate into two recognizable populations: myotubes and stellate mononucleated cells. The cells were then trypsinized, filtered through a 20 µm filter to remove the myotubes, frozen at −80° C, then thawed and replated. In culture the cells maintained their stellate structure. However, under treatment with dexamethasone, a nonspecific differentiating agent, seven morphologic conditions emerged: cells with refractile vesicles that stained with Sudan black B (adipocytes), multinucleated cells that spontaneously contracted in culture and stained with an antibody to myosin (myotubes), round cells whose extracellular matrix stained with Alcian blue, pH 1.0 (chondrocytes), polygonal cells whose extracellular matrix stained with Von Kossas stain (osteoblasts), cells with filaments that stained with an antibody to smooth muscle a‐actin (smooth muscle cells), cells that incorporated acetylated low density lipoprotein (endothelial cells), and spindle‐shaped cells that grew in a swirl pattern (fibroblasts). The initial population is tentatively classified as putative mesenchymal stem cells. The presence of these cells point to the existence of stem cells in the postembryonic mammal that could provide a basis for tissue regeneration as opposed to scar tissue formation during wound healing.

Isolation of embryonic chick myosatellite and pluripotent stem cells

The current study outlines the isolation and culture of two populations of cells derived from Day 11 embryonic chick leg muscle and associated connective tissues. The two populations consisted of myogenic lineage-committed stem cells (myosatellite stem cells) and lineage-uncommitted stem cells (pluripotent stem cells). After long-term culture the lineage-uncommitted stem cell population displayed differentiated phenotypes suggestive of the following adult tissues, fibroblasts, muscle, fat, cartilage, and bone.

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.

Cryopreservation of embryonic chick myogenic lineage-committed stem cells

Undifferentiated embryonic chick stem cells provide a good in vitro test system to examine the effects of recombinant growth factors on the resultant phenotypic expression of these cells. One of the major difficulties in using freshly isolated cells is variation in the proportion of stem cells recovered from different cell harvests. Variation is of particular concern when multiple cell harvests are necessary to provide cells for assaying a single growth factor. As an attempt to minimize this variation, we have examined the efficacy of cryopreserving myogenic lineage-committed stem cells derived from embryonic chick leg muscle. Percent recovery, viability, differentiation morphology, and cellular proliferation rates were compared between sister cultures of freshly isolated and cryopreserved stem cells. Embryonic chick stem cells retained their capacity to differentiate myogenically in culture when slowly frozen in and recovered from 7.5% dimethyl sulfoxide medium supplemented with horse serum and stage-specific embryo extract.

Enzyme-linked immuno-culture assay

The current study outlines a procedure, designated enzyme-linked immuno-culture assay (ELICA), that will measure phenotypic expression of cultured cells in small plate assays. Given standard curves for phenotypic expression markers and in situ DNA analysis, this procedure will quantitate (to nanogram levels) intracellular, cell surface, or extracellular phenotypic expression markers; visualize the location of the markers; and determine DNA content, all within the same well of a 24- or 96-well tissue culture plate.