Regeneration of the germline in the annelid Capitella teleta.

The germline is essential for sexual reproduction and survival of the species. In many metazoans, the developmental potential to generate a distinct germline is segregated from somatic cell lineages early in embryogenesis, suggesting that the unique features of the germline must be established from its onset. Previous studies suggest that germ cells cannot regenerate once removed from the embryo, but few animals have been experimentally tested. We investigated the ability of the germline to regenerate in a lophotrochozoan, the segmented worm Capitella teleta, which has a stereotyped cell lineage program by deleting the germline precursor (cell 3D) in early stage embryos using an infrared laser. Larvae and juveniles resulting from germline deletions were examined for presence of multipotent progenitor cells (MPCs), stem cells that form the germ cells and somatic stem cells. In contrast to control deletions of a non-germline macromere, most larvae resulting from deletion of cell 3D lacked MPCs as assayed by expression of germline markers CapI-vasa, CapI-nanos and Ct-piwi1, but showed persistent expression of these markers in the somatic posterior growth zone. However, approximately 13% of experimental larvae had MPCs, indicative of some germline regeneration. In contrast, by two weeks post-metamorphosis, all juveniles resulting from deletion of cell 3D had MPCs, as detected by CapI-vasa expression. Furthermore, when raised to adulthood, most animals developed reproductive structures and were fertile. In another set of deletions, both the D quadrant mesodermal and germline progenitors were removed. These juveniles also regenerated MPCs. Surprisingly, this deletion caused substantial ectopic expression of CapI-vasa and CapI-nanos in other larval tissues. Our results indicate that C. teleta can regenerate the germline following removal of the germline progenitors in the early embryo. The dramatic difference in ability to regenerate the germline between the larval and adult stages suggests that there are two distinct compensation events at two phases of the life cycle: a regulative event in the early stage larva and a stem cell transition event after metamorphosis, when the animals are capable of substantial body regeneration.

Adipose-derived mesenchymal stem cells (MSCs) are a superior cell source for bone tissue engineering.

Effective bone regeneration through tissue engineering requires a combination of osteogenic progenitors, osteoinductive biofactors and biocompatible scaffold materials. Mesenchymal stem cells (MSCs) represent the most promising seed cells for bone tissue engineering. As multipotent stem cells that can self-renew and differentiate into multiple lineages including bone and fat, MSCs can be isolated from numerous tissues and exhibit varied differentiation potential. To identify an optimal progenitor cell source for bone tissue engineering, we analyzed the proliferative activity and osteogenic potential of four commonly-used mouse MSC sources, including immortalized mouse embryonic fibroblasts (iMEF), immortalized mouse bone marrow stromal stem cells (imBMSC), immortalized mouse calvarial mesenchymal progenitors (iCAL), and immortalized mouse adipose-derived mesenchymal stem cells (iMAD). We found that iMAD exhibited highest osteogenic and adipogenic capabilities upon BMP9 stimulation in vitro, whereas iMAD and iCAL exhibited highest osteogenic capability in BMP9-induced ectopic osteogenesis and critical-sized calvarial defect repair. Transcriptomic analysis revealed that, while each MSC line regulated a distinct set of target genes upon BMP9 stimulation, all MSC lines underwent osteogenic differentiation by regulating osteogenesis-related signaling including Wnt, TGF-ß, PI3K/AKT, MAPK, Hippo and JAK-STAT pathways. Collectively, our results demonstrate that adipose-derived MSCs represent optimal progenitor sources for cell-based bone tissue engineering.

Isolation of murine bone marrow hematopoietic stem and progenitor cell populations via flow cytometry.

Flow cytometry is a powerful technology that allows not only multiparameter quantitative data analysis at single cell resolution but also simultaneous cell separation of different populations of interest at high speed. It has been rapidly employed in biological research and clinical diagnostics. This technology has enabled the thorough understanding of murine hematopoiesis, especially the physiology of surface marker-defined hematopoietic stem and progenitor cell populations. The isolation of these populations has been well established over the last three decades with a large consensus among leading laboratories. Here, we describe a detailed methodology protocol of two different state-of-the-art approaches to isolate bone marrow cells and purify hematopoietic stem and progenitor cells via flow cytometry. Different gating schemes are introduced to identify well-defined populations of murine hematopoietic stem and multipotent progenitor cells.

HIV-1 Infection of Long-Lived Hematopoietic Precursors In Vitro and In Vivo.

Latent reservoirs in human-immunodeficiency-virus-1 (HIV-1)-infected individuals represent a major obstacle in finding a cure for HIV-1. Hematopoietic stem and progenitor cells (HSPCs) have been described as potential HIV-1 targets, but their roles as HIV-1 reservoirs remain controversial. Here we provide additional evidence for the susceptibility of several distinct HSPC subpopulations to HIV-1 infection in vitro and in vivo. In vitro infection experiments of HSPCs were performed with different HIV-1 Env-pseudotyped lentiviral particles and with replication-competent HIV-1. Low-level infection/transduction of HSPCs, including hematopoietic stem cells (HSCs) and multipotent progenitors (MPP), was observed, preferentially via CXCR4, but also via CCR5-mediated entry. Multi-lineage colony formation in methylcellulose assays and repetitive replating of transduced cells provided functional proof of susceptibility of primitive HSPCs to HIV-1 infection. Further, the access to bone marrow samples from HIV-positive individuals facilitated the detection of HIV-1 gag cDNA copies in CD34+ cells from eight (out of eleven) individuals, with at least six of them infected with CCR5-tropic HIV-1 strains. In summary, our data confirm that primitive HSPC subpopulations are susceptible to CXCR4- and CCR5-mediated HIV-1 infection in vitro and in vivo, which qualifies these cells to contribute to the HIV-1 reservoir in patients.

Characterization of traumatized muscle-derived multipotent progenitor cells from low-energy trauma.

BACKGROUND: Multipotent progenitor cells have been harvested from different human tissues, including the bone marrow, adipose tissue, and umbilical cord blood. Previously, we identified a population of mesenchymal progenitor cells (MPCs) isolated from the traumatized muscle of patients undergoing reconstructive surgery following a war-related blast injury. These cells demonstrated the ability to differentiate into multiple mesenchymal lineages. While distal radius fractures from a civilian setting have a much lower injury mechanism (low-energy trauma), we hypothesized that debrided traumatized muscle near the fracture site would contain multipotent progenitor cells with the ability to differentiate and regenerate the injured tissue. METHODS: The traumatized muscle was debrided from the pronator quadratus in patients undergoing open reduction and internal fixation for a distal radius fracture at the Walter Reed National Military Medical Center. Using a previously described protocol for the isolation of MPCs from war-related extremity injuries, cells were harvested from the low-energy traumatized muscle samples and expanded in culture. Isolated cells were characterized by flow cytometry and q-RT-PCRs and induced to adipogenic, osteogenic, and chondrogenic differentiation. Downstream analyses consisted of lineage-specific staining and q-RT-PCR. RESULTS: Cells isolated from low-energy traumatized muscle samples were CD73+, CD90+, and CD105+ that are the characteristic of adult human mesenchymal stem cells. These cells expressed high levels of the stem cell markers OCT4 and NANOG 1-day after isolation, which was dramatically reduced over-time in monolayer culture. Following induction, lineage-specific markers were demonstrated by each specific staining and confirmed by gene expression analysis, demonstrating the ability of these cells to differentiate into adipogenic, osteogenic, and chondrogenic lineages. CONCLUSIONS: Adult multipotent progenitor cells are an essential component for the success of regenerative medicine efforts. While MPCs have been isolated and characterized from severely traumatized muscle from high-energy injuries, here, we report that cells with similar characteristics and multipotential capacity have been isolated from the tissue that was exposed to low-energy, community trauma.

Regeneration in the Segmented Annelid Capitella teleta.

The segmented worms, or annelids, are a clade within the Lophotrochozoa, one of the three bilaterian superclades. Annelids have long been models for regeneration studies due to their impressive regenerative abilities. Furthermore, the group exhibits variation in adult regeneration abilities with some species able to replace anterior segments, posterior segments, both or neither. Successful regeneration includes regrowth of complex organ systems, including the centralized nervous system, gut, musculature, nephridia and gonads. Here, regenerative capabilities of the annelid Capitella teleta are reviewed. C. teleta exhibits robust posterior regeneration and benefits from having an available sequenced genome and functional genomic tools available to study the molecular and cellular control of the regeneration response. The highly stereotypic developmental program of C. teleta provides opportunities to study adult regeneration and generate robust comparisons between development and regeneration.

Lineage Tracing and Cell Potential of Postnatal Single Progenitor Cells In Vivo.

Understanding the contribution of adult neural progenitor cells (NPCs) and their lineage potential is a great challenge in neuroscience. To reveal progenitor diversity and cell-lineage relationships of postnatal NPCs in the subventricular zone (SVZ), we performed in vivo lineage-tracing genetic analysis using the UbC-StarTrack. We determined the progeny of single SVZ-NPCs, the number of cells per clone, the dispersion of sibling cells, and the cell types within clones. Long-term analysis revealed that both the cell-dispersion pattern and number of cells comprising clones varied depending on the glial/neuronal nature of sibling cells. Sibling-olfactory interneurons were primarily located within the same layer, while sibling-glial cells populated SVZ-adjacent areas. Sibling astrocytes and interneurons did not form big clones, whereas oligodendroglial-lineage clones comprised the largest clones originated in adult brains. These results demonstrate the existence of SVZ postnatal bipotential progenitors that give rise to clones widely dispersed across the olfactory bulb and SVZ-adjacent areas.

Extracellular matrix-regulated neural differentiation of human multipotent marrow progenitor cells enhances functional recovery after spinal cord injury.

BACKGROUND CONTEXT: Recent advanced studies have demonstrated that cytokines and extracellular matrix (ECM) could trigger various types of neural differentiation. However, the efficacy of differentiation and in vivo transplantation has not yet thoroughly been investigated. PURPOSE: To highlight the current understanding of the effects of ECM on neural differentiation of human bone marrow-derived multipotent progenitor cells (MPCs), regarding state-of-art cure for the animal with acute spinal cord injury (SCI), and explore future treatments aimed at neural repair. STUDY DESIGN: A selective overview of the literature pertaining to the neural differentiation of the MSCs and experimental animals aimed at improved repair of SCI. METHODS: Extracellular matrix proteins, tenascin-cytotactin (TN-C), tenascin-restrictin (TN-R), and chondroitin sulfate (CS), with the cytokines, nerve growth factor (NGF)/brain-derived neurotrophic factor (BDNF)/retinoic acid (RA) (NBR), were incorporated to induce transdifferentiation of human MPCs. Cells were treated with NBR for 7 days, and then TN-C, TN-R, or CS was added for 2 days. The medium was changed every 2 days. Twenty-four animals were randomly assigned to four groups with six animals in each group: one experimental and three controls. Animals received two (bilateral) injections of vehicle, MPCs, NBR-induced MPCs, or NBR/TN-C-induced MPCs into the lesion sites after SCI. Functional assessment was measured using the Basso, Beattie, and Bresnahan locomotor rating score. Data were analyzed using analysis of variance followed by Student-Newman-Keuls (SNK) post hoc tests. RESULTS: Results showed that MPCs with the transdifferentiation of human MPCs to neurons were associated with increased messenger-RNA (mRNA) expression of neuronal markers including nestin, microtubule-associated protein (MAP) 2, glial fibrillary acidic protein, ßIII tubulin, and NGF. Greater amounts of neuronal morphology appeared in cultures incorporated with TN-C and TN-R than those with CS. The addition of TN-C enhanced mRNA expressions of MAP2, ßIII tubulin, and NGF, whereas TN-R did not significantly change. Conversely, CS exposure decreased MAP2, ßIII tubulin, and NGF expressions. The TN-C-treated MSCs significantly and functionally repaired SCI-induced rats at Day 42. Present results indicate that ECM components, such as tenascins and CS in addition to cytokines, may play functional roles in regulating neurogenesis by human MPCs. CONCLUSIONS: These findings suggest that the combined use of TN-C, NBR, and human MPCs offers a new feasible method for nerve repair.

Adult mesenchymal stem cells and cell surface characterization - a systematic review of the literature.

Human adult mesenchymal stem cells (MSCs) were first identified by Friedenstein et al. when observing a group of cells that developed into fibroblastic colony forming cells (CFU-F). Ever since, the therapeutic uses and clinical applications of these cells have increased research and interest in this field. MSCs have the potential to be used in tissue engineering, gene therapy, transplants and tissue injuries. However, identifying these cells can be a challenge. Moreover, there are no articles bringing together and summarizing the cell surface markers of MSCs in adults. The purpose of this study is to summarize all the available information about the cell surface characterization of adult human MSCs by identifying and evaluating all the published literature in this field. We have found that the most commonly reported positive markers are CD105, CD90, CD44, CD73, CD29, CD13, CD34, CD146, CD106, CD54 and CD166. The most frequently reported negative markers are CD34, CD14, CD45, CD11b, CD49d, CD106, CD10 and CD31. A number of other cell surface markers including STRO-1, SH2, SH3, SH4, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-I, DP, EMA, DQ (MHC Class II), CDIO5, Oct 4, Oct 4A, Nanog, Sox-2, TERT, Stat-3, fibroblast surface antigen, smooth muscle alpha-actin, vimentin, integrin subunits alpha4, alpha5, beta1, integrins alphavbeta3 and alphavbeta5 and ICAM-1 have also been reported. Nevertheless, there is great discrepancy and inconsistency concerning the information available on the cell surface profile of adult MSCs and we suggest that further research is needed in this field to overcome the problem.