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Isolation of a stem cell for neurons and glia from the mammalian neural crest.

We have isolated mammalian neural crest cells using a monoclonal antibody to the low affinity NGF receptor, and established conditions for the serial propagation of these cells in clonal culture to assess their developmental potential. This analysis indicates that, first, single mammalian neural crest cells are multipotent, able to generate at least neurons and Schwann cells like their avian counterparts. Second, multipotent neural crest cells generate multipotent progeny, indicating that they are capable of self-renewal and therefore are stem cells. Third, multipotent neural crest cells also generate some clonal progeny that form only neurons or glia, suggesting the production of committed neuroblasts and glioblasts. Manipulation of the substrate alters the fate of the multipotent cells. These findings have implications for models of neural crest development in vivo, and establish a system for studying the generation of cellular diversity by a multipotent stem cell in vitro.

Animals↗

Isolation of neural crest-derived stem cells from rat embryonic mandibular processes.

BACKGROUND INFORMATION: Substantial evidence indicates the existence of NCSCs (neural crest-derived stem cells) in embryonic mandibular processes; however, they have not been fully investigated or isolated. The aim of the present study was to isolate stem cells from mandibular process during embryonic development by MACS (magnetic-activated cell sorting). The findings show that the cells are multipotent and self-renewing. RESULTS: LNGFR (low-affinity nerve-growth-factor receptor)+ cells were isolated from rat embryonic mandibular processes by MACS. The cells were grown in clonal culture by limiting dilution to assess their developmental potential. Clone analysis indicated that, first, LNGFR+ cells are multipotent, being able to generate at least neurons and Schwann cells, similar to peripheral neural crest stem cells. Secondly, multipotent LNGFR+ cells generate multipotent progenies, indicating that they are capable of self-renewal and therefore are stem cells. Thirdly, manipulation of the medium supplementation alters the fate of the isolated LNGFR+ cells. CONCLUSIONS: These results indicate that LNGFR antibodies label NCSCs with high specificity and purity, and suggest that positive selection using these antibodies may become the method of choice for obtaining multipotent cells from rat embryonic mandibular processes for tissue engineering or regenerative therapeutic use.

Animals↗

[Adult stem cells and possible mechanisms of its differentiation--editorial].

Adult stem cells are the multi-potential cells, which exist in fetal and adult tissues. It can reproduce itself (undergo self-renewal) or give rise to more specialized (differentiated) cells. Under certain inducing conditions, adult stem cells can acquire the ability to differentiate into different tissue cells. Multipotent adult progenitor cells (MAPC), an alternative name of adult stem cell given by Catherine Verfaillie, existing in bone marrow, can differentiate into cells with characteristics of mesodermal, neuroectodermal, and endodermal lineages in vitro at the single-cell level. MAPC can also contribute to most cell types when injected into the blastocyst. Adult stem cell differentiation implies that different cell lineages are derived from a single initial cell; all differentiated cell types are functional in vitro and in vivo; and engraftment is robust and persistent in the physiological and pathological situations. The possible mechanisms may underlie the differentiation: various tissue-specific stem cells are present in different organs; adult stem cells would be reprogrammed when removed from their usual microenvironment and introduced into a different niche that imparts signals to activate a novel genetic program needed for the new cell fate. And true multi-potential stem cells persist in postnatal life. In the future, multi-potent adult stem cells might then be used for therapies of degenerative or genetic disorders of multiple different organs.

Adult Stem Cells↗

Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population.

In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. A central question is whether the generation of progenitor cells during limb regeneration and mammalian tissue repair occur via separate or overlapping mechanisms. Limb regeneration depends on the formation of a blastema, from which the new appendage develops. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. We describe a multipotent Pax7+ satellite cell population located within the skeletal muscle of the salamander limb. We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. Activation of salamander satellite cells occurs in an analogous manner to how the mammalian myofiber mobilizes stem cells during skeletal muscle tissue repair. Thus, limb regeneration and mammalian tissue repair share common cellular and molecular programs. Our findings also identify satellite cells as potential targets in promoting mammalian blastema formation.

Adipocytes↗

Induction of globin mRNA expression by interleukin-3 in a stem cell factor-dependent SV-40 T-antigen-immortalized multipotent hematopoietic cell line.

Erythropoiesis requires the stepwise action on immature progenitors of several growth factors, including stem cell factor (SCF), interleukin 3 (IL-3), and erythropoietin (Epo). Epo is required to sustain proliferation and survival of committed progenitors and might further modulate the level of expression of several erythroid genes, including globin genes. Here we report a new SCF-dependent immortalized mouse progenitor cell line (GATA-1 ts SCF) that can also grow in either Epo or IL-3 as the sole growth factor. When grown in SCF, these cells show an "open" chromatin structure of the beta-globin LCR, but do not significantly express globin. However, Epo or IL-3 induce globin expression and are required for its maintainance. This effect of IL-3 is unexpected as IL-3 was previously reported either to be unable to induce hemoglobinization, or even to antagonize it. This suggests that GATA-1 ts SCF cells may have progressed to a stage in which globin genes are already poised for expression and only require signal(s) that can be elicited by either Epo or IL-3. Through the use of inhibitors, we suggest that p38 may be one of the molecules modulating induction and maintenance of globin expression.

Animals↗

Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells.

OBJECTIVE: To compare the hematopoietic support provided by telomerized human mesenchymal stem cells (MSCs) and telomerized MSC-derived stromal cells. METHODS: We transfected the human telomerase catalytic subunit (hTERT) gene into primary MSCs to establish hTERT-transduced MSCs (hTERT-MSCs). Stromal induction of hTERT-MSCs was performed by replacing the culture medium with Dexter-type culture medium. Hematopoietic support was examined by coculture with cord blood CD34(+) cells. RESULTS: The hTERT-MSCs were morphologically identical with the primary MSCs and expressed surface antigens including CD105, CD73, and CD166. hTERT-MSCs showed a similar doubling time as primary MSCs and continued to proliferate to over 80 population doublings (PD), although the primary MSCs underwent crisis in vitro at 16 PD. The osteogenic, chondrogenic, adipogenic, neurogenic, and stromal differentiation potential of hTERT-MSCs were maintained up to at least 40 PD. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-C) upon 12-day coculture with the hTERT-MSC-derived stromal cells were nearly the same as those upon 12-day coculture with hTERT-MSCs (CD34, 33.0-fold+/-2.8-fold vs 36.1-fold+/-1.7-fold of the initial cell number; CFUs, 344.4-fold+/-62.5-fold vs 239.3-fold+/-87.0-fold; CFU-mix, 368.4-fold+/-113.7-fold vs 341.3-fold+/-234.3-fold). However, on day 18 of coculture, the number of cobblestone areas (CA) observed beneath the stromal cells was 15 times higher than that beneath hTERT-MSCs (CA, 146.9+/-54.6 vs 9.4+/-8.1, p<0.01). CONCLUSION: Stromal induction of hTERT-MSCs exclusively enhanced the support of CA formation provided by hTERT-MSCs. Our human hTERT-MSCs will be useful for elucidating the mechanism of the formation of CAs.

Adult↗

In vitro analysis of multipotent mesenchymal stromal cells as potential cellular therapeutics in neurometabolic diseases in pediatric patients.

Multipotent mesenchymal stromal cells (MSCs) play an important role in stromal support for hematopoietic stem cells, immune modulation, and tissue regeneration. We investigated their potential as cellular therapeutic tools in neurometabolic diseases as a growing number of affected children undergo to bone marrow transplantation. MSCs were isolated from bone marrow aspirates and expanded ex vivo under various culture conditions. MSCs under optimal good medical practice (GMP)-conform culture conditions showed the typical morphology, immunophenotype, and plasticity. Biochemically, the activities of beta-hexosaminidase A, total beta-hexosaminidase, arylsulfatase A (ASA), and beta-galactosidase measured in MSCs were comparable to those in fibroblasts of healthy donors. These four enzymes were interesting for their expression in MSCs, as each of them is defective, respectively, in well-known neurometabolic diseases. We found that MSCs released significant amounts of ASA into the media. In coculture experiments, fibroblasts from patients with metachromatic leukodystrophy, who are deficient for ASA, took up a substantial amount of ASA that was released into the media from MSCs. Mannose-6-phosphate (M6P) inhibited this uptake, which was in accordance with the M6P receptor-mediated uptake of lysosomal enzymes. Taken together, we show that MSCs produce appreciable amounts of lysosomal enzyme activities, making these cells first-choice candidates for providing metabolic correction when given to enzyme-deficient patients. With the example of ASA, it was also shown that an enzyme secreted from MSCs is taken up by enzyme-deficient patient fibroblasts. Given the plasticity of MSCs, these cells represent an interesting add-on option for cellular therapy in children undergoing bone marrow transplantation for lysosomal storage diseases and other neurometabolic diseases.

Bone Marrow Transplantation↗

Human mesenchymal stem cells modulate allogeneic immune cell responses.

Mesenchymal stem cells (MSCs) are multipotent cells found in several adult tissues. Transplanted allogeneic MSCs can be detected in recipients at extended time points, indicating a lack of immune recognition and clearance. As well, a role for bone marrow-derived MSCs in reducing the incidence and severity of graft-versus-host disease (GVHD) during allogeneic transplantation has recently been reported; however, the mechanisms remain to be investigated. We examined the immunomodulatory functions of human MSCs (hMSCs) by coculturing them with purified subpopulations of immune cells and report here that hMSCs altered the cytokine secretion profile of dendritic cells (DCs), naive and effector T cells (T helper 1 [T(H)1] and T(H)2), and natural killer (NK) cells to induce a more anti-inflammatory or tolerant phenotype. Specifically, the hMSCs caused mature DCs type 1 (DC1) to decrease tumor necrosis factor alpha (TNF-alpha) secretion and mature DC2 to increase interleukin-10 (IL-10) secretion; hMSCs caused T(H)1 cells to decrease interferon gamma (IFN-gamma) and caused the T(H)2 cells to increase secretion of IL-4; hMSCs caused an increase in the proportion of regulatory T cells (T(Regs)) present; and hMSCs decreased secretion of IFN-gamma from the NK cells. Mechanistically, the hMSCs produced elevated prostaglandin E2 (PGE(2)) in co-cultures, and inhibitors of PGE(2) production mitigated hMSC-mediated immune modulation. These data offer insight into the interactions between allogeneic MSCs and immune cells and provide mechanisms likely involved with the in vivo MSC-mediated induction of tolerance that could be therapeutic for reduction of GVHD, rejection, and modulation of inflammation.

Cell Communication↗

Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells.

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Animals↗

Lymphoid-restricted development from multipotent candidate murine stem cells: distinct and complimentary functions of the c-kit and flt3-ligands.

The two tyrosine kinase receptors, c-kit and flt3, and their respective ligands KL and FL, have been demonstrated to play key and nonredundant roles in regulating the earliest events in hematopoiesis. However, their precise roles and potential interactions in promoting early lymphoid commitment and development remain unclear. Here we show that most if not all murine Lin(-/lo)Sca1(+)c-kit(+) bone marrow (BM) cells generating B220(+)CD19(+) proB-cells in response to FL and interleukin-7 (IL-7) also have a myeloid potential. In contrast to FL + IL-7, KL + IL-7 could not promote proB-cell formation from Lin(-/lo)Sca1(+)c-kit(+) cells. However, KL potently enhanced FL + IL-7-stimulated proB-cell formation, in part through enhanced recruitment of FL + IL-7-unresponsive Lin(-/lo)Sca1(+)c-kit(+) progenitors, and in part by enhancing the growth of proB-cells. The enhanced recruitment (4-fold) in response to KL occurred exclusively from the Lin(-/lo)Sca1(+)c-kit(+)flt3(-) long-term repopulating stem cell population, whereas KL had no effect on FL + IL-7-stimulated recruitment of Lin(-/lo)Sca1(+)c-kit(+)flt3(+) short-term repopulating cells. The progeny of FL + IL-7-stimulated Lin(-/lo)Sca1(+)c-kit(+) cells lacked in vitro and in vivo myeloid potential, but efficiently reconstituted both B and T lymphopoiesis. In agreement with this FL, but not KL, efficiently induced expression of B220 and IL-7 receptor-alpha on Lin(-/lo)Sca1(+)c-kit(+)flt3(+) cells. Thus, whereas KL appears crucial for recruitment of FL + IL-7-unresponsive candidate (c-kit(+)flt3(-)) murine stem cells, FL is essential and sufficient for development toward lymphoid restricted progenitors from a population of (c-kit(+)flt3(+)) multipotent short-term reconstituting progenitors.

Animals↗

Adipose-derived adult stem cells for cartilage tissue engineering.

Tissue engineering is a promising therapeutic approach that uses combinations of implanted cells, biomaterial scaffolds, and biologically active molecules to repair or regenerate damaged or diseased tissues. Many diverse and increasingly complex approaches are being developed to repair articular cartilage, with the underlying premise that cells introduced exogenously play a necessary role in the success of engineered tissue replacements. A major consideration that remains in this field is the identification and characterization of appropriate sources of cells for tissue-engineered repair of cartilage. In particular, there has been significant emphasis on the use of undifferentiated progenitor cells, or "stem" cells that can be expanded in culture and differentiated into a variety of different cell types. Recent studies have identified the presence of an abundant source of stem cells in subcutaneous adipose tissue. These cells, termed adipose-derived adult stem (ADAS) cells, show characteristics of multipotent adult stem cells, similar to those of bone marrow derived mesenchymal stem cells (MSCs), and under appropriate culture conditions, synthesize cartilage-specific matrix proteins that are assembled in a cartilaginous extracellular matrix. The growth and chondrogenic differentiation of ADAS cells is strongly influenced by factors in the biochemical as well as biophysical environment of the cells. Furthermore, there is strong evidence that the interaction between the cells, the extracellular biomaterial substrate, and growth factors regulate ADAS cell differentiation and tissue growth. Overall, ADAS cells show significant promise for the development of functional tissue replacements for various tissues of the musculoskeletal system.

Adipocytes↗

Yield of human adipose-derived adult stem cells from liposuction aspirates.

BACKGROUND: Primary cultures of isolated human adipose-derived adult stem (ADAS) cells are multipotent and differentiate in vitro along the adipocyte, chondrocyte, neuronal, osteoblast, and skeletal muscle pathways. METHODS: We examined the ADAS cell yield per unit volume of liposuction tissue, and their surface protein phenotype by flow cytometry. Adipogenesis was assessed by Oil Red O staining and ELISA analysis of leptin secretion. RESULTS: The donor population was 87.5% female (n=18) with a mean age (+/-SD) of 44+/-10 years and body mass index (BMI) of 24.9+/-2.7. The mean cell yield was 404 000+/-206 000 cells per milliliter of lipoaspirate (n=18). Linear regression analysis of the cells derived from the female donors demonstrated a significant negative correlation between the number of cells obtained per milliliter of lipoaspirate with the BMI but not the age of the donor. The undifferentiated ADAS cells were homogeneously positive for the cell-surface markers CD10, CD13, CD29, CD44, CD49e, CD59, CD90, and HLA-ABC, and homogeneously negative for the cell surface markers CD11b, CD45, and HLA-DR. The absence of the panhematopoietic marker, CD45, indicates that the ADAS cells do not derive from circulating BM hematopoietic stem cells. Adipocyte differentiation led to a 5.1-fold increase in Oil Red O staining, and a 196-fold increase in leptin secretion levels. Culture of the cells in the presence of antibiotic and fungizone did not alter the undifferentiated ADAS cell immunophenotype based on flow cytometry, or their adipocyte differentiation based on leptin secretion. DISCUSSION: The ability to isolate a consistently homogeneous population of undifferentiated adult stem cells from adipose tissue of multiple donors supports their potential utility in future tissue-engineering applications.

Adipose Tissue↗

Adipose-derived adult stem cells: isolation, characterization, and differentiation potential.

Adipose tissue is an abundant, accessible, and replenishable source of adult stem cells that can be isolated from liposuction waste tissue by collagenase digestion and differential centrifugation. These adipose-derived adult stem (ADAS) cells are multipotent, differentiating along the adipocyte, chondrocyte, myocyte, neuronal, and osteoblast lineages, and can serve in other capacities, such as providing hematopoietic support and gene transfer. ADAS cells have potential applications for the repair and regeneration of acute and chronically damaged tissues. Additional pre-clinical safety and efficacy studies will be needed before the promise of these cells can be fully realized.

Adipose Tissue↗

Human mesenchymal stem cells: insights from a surrogate in vivo assay system.

Mesenchymal stem cells (MSC) are multipotent cells that have been isolated from the bone marrow of multiple species that can be induced to differentiate into at least bone, cartilage, and adipose tissue in vitro. Using a model of in utero cellular transplantation in which human MSC are transplanted into fetal lambs, we have shown that MSC can engraft in multiple tissues and persist for over one year. Furthermore, these cells can differentiate into cardiac and skeletal myocytes, bone marrow stromal cells, adipocytes, thymic epithelial cells, and chondrocytes. These observations lend support to the potential utility of MSC for cellular and/or gene therapy in the treatment of a variety of congenital or acquired diseases such as osteogenesis imperfecta, muscular dystrophy, lysosomal storage diseases, and for enhancement of bone marrow transplantation.

Animals↗

Astrocytes as stem cells: nomenclature, phenotype, and translation.

Recently discovered multipotent astrocytic stem cells are discussed in light of current nomenclature for glial precursor and lineage-associated cells in the developing, postnatal, and adult mammalian brain. Defining the phenotype of any immature cell in the nervous system is a challenge, and a position is stated that includes the need for categorizing cells within a continuum of differentiation potential. The possibility for dedifferentiating glial cells into clonogenic stem-like cells offers numerous possibilities for translating knowledge and technology from this subfield of stem cell biology to regenerative medicine. Along with the need for developing a new lexicon for defining the cellular players that contribute to the generation of glia and neurons in the developing and mature central nervous system, the relationships also need to be established among potency, repopulation attempts, and tumorigenesis of cells meeting the criteria of glial stem cells. Finally, it is possible that understanding the normal differentiation, de- and transdifferentiation potential of glial stem-like cells in the mature central nervous system will provide insights into the possible use of these cells, or biogenic factors associated with their growth and differentiation, in therapeutic approaches for a variety of neurological disorders.

Animals↗

BCR-ABL alters the proliferation and differentiation response of multipotent hematopoietic cells to stem cell factor.

Chronic myeloid leukaemia (CML), a hematopoietic stem cell disorder is characterized by the expression of BCR-ABL. To investigate the effects of BCR-ABL on multipotent hematopoietic cells, a temperature sensitive BCR-ABL tyrosine kinase was expressed in the cell line, FDCP-Mix. BCR-ABL mediated an increase in c-kit expression that correlated with an enhanced mitogenic response to SCF. This was not observed in the absence of Bcr-Abl kinase activity or presence of the BCR-ABL inhibitor STI571, which also inhibits c-kit. When cultured in a combination of SCF plus G-CSF the FDCP-Mix cells undergo neutrophilic differentiation over a 7-10 day period. When BCR-ABL was active there was a marked inhibition of cell maturation compared to control cells in which BCR-ABL was either inactive or not present. However, BCR-ABL did not block differentiation as the cells eventually undergo terminal maturation. These data argue that BCR-ABL is directly responsible for the enhanced response to SCF reported in CML progenitor cells. Furthermore, although the primary effect of STI571 is via direct inhibition of BCR-ABL, STI571 additionally reduces the enhanced response to SCF. Thus there are two sites of STI571 action of potential importance in Bcr-Abl expressing cells.

Antineoplastic Agents↗

Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation.

Multipotent murine stem cell lines (FDC-Pmix) depend on IL-3 for self-renewal and proliferation and can be induced to differentiate into multiple hematopoietic lineages. Single FDC-Pmix cells infected with retroviral vectors expressing GM-CSF are induced to differentiate into granulocytes and macrophages. This results in a complete loss of clonogenic cells if IL-3 is not exogenously supplied; however, multipotent variants can be selected that do not terminally differentiate if cells are kept in the presence of IL-3. Unidirectional and synchronous granulocyte and macrophage differentiation accompanied with loss of self-renewal capacity is induced when IL-3 is removed. Our data indicate that activation of the GM-CSF receptor induces differentiation of stem cells by an instructive mechanism that can be blocked by the activated IL-3 receptor. A model of how receptors can induce proliferation and cell-specific differentiation by two separate pathways is discussed.

Animals↗