Adult Mouse Bone Marrow Research Articles

A critical function for B-Raf at multiple stages of myelopoiesis

AbstractRaf kinases play an integral role in the classic mitogen-activated protein (MA3332) kinase (Raf/MEK/extracellular signal-related kinase [ERK]) intracellular signaling cascade, but their role in specific developmental processes is largely unknown. Using a genetic approach, we have identified a role for B-Raf during hematopoietic progenitor cell development and during megakaryocytopoiesis. Fetal liver and in vitro embryonic stem (ES) cell–derived myeloid progenitor development is quantitatively impaired in the absence of B-Raf. Biochemical data suggest that this phenotype is due to the loss of a normally occurring rise in B-Raf expression and associated ERK1/2 activation during hematopoietic progenitor cell formation. However, the presence of B-raf–/– ES cell–derived myeloid progenitors in the bone marrow of adult chimeric mice indicates the lack of an obligate cell-autonomous requirement for B-Raf in myeloid progenitor development. The lack of B-Raf also impairs megakaryocytopoiesis. Thrombopoietin (Tpo)–induced in vitro expansion of ES cell–derived megakaryocyte-lineage cells fails to occur in the absence of B-Raf. Moreover, this quantitative in vitro defect in megakaryocyte-lineage expansion is mirrored by chimeric mice data that show reduced B-raf–/– genotype contribution in megakaryocytes relative to its contribution in myeloid progenitors. Together, these data suggest that B-Raf plays a cell-autonomous role in megakaryocytopoiesis and a permissive role in myeloid progenitor development.

Identification and characterization of undifferentiated mast cells in mouse bone marrow

AbstractSequential immunomagnetic isolation with 2 monoclonal antibodies was used to purify and characterize an undifferentiated mast cell in adult mouse bone marrow that had not been previously recognized. This cell represents 0.02% of the cells in the bone marrow, is CD34+, CD13+, and c-kit+, and does not express FcϵRI. However, by polymerase chain reaction (PCR) the cell contains message for the α and β subunits of FcϵRI, mast cell–specific proteases, and carboxypeptidase A. Morphologically, this cell has a large nucleus, little cytoplasm, few cytoplasmic organelles, and no cytoplasmic granules. In vitro, in the presence of interleukin-3 (IL-3) and stem cell factor (SCF) these cells differentiate only into a granulated mast cell that now expresses CD13, c-kit, mast cell–specific gangliosides, FcϵRI, and binds immunoglobulin E (IgE). When injected into lethally irradiated mice, these cells are able to reconstitute the mast cell population in the spleen.

Gfi-1: another piece in the HSC puzzle

Recent work addresses the complex issue of stem cell regulation mechanisms. Both groups involved have studied the role of Gfi-1 in preserving the functional integrity of the adult hematopoietic stem cell (HSC) pool through a series of experiments, including phenotypic analysis of the primitive hematopoietic compartment, transplantation assays and cell cycle analyses. The results suggest that Gfi-1 deficiency leads to an exhaustion of HSCs in the adult mouse bone marrow, presumably through unrestricted cycling of these cells.

Tmtsp, a Novel Gene Encoding a Cell Surface Protein Highly Specific to Hematopoietic Stem and Endothelial Cells.

In the adult mouse bone marrow, hematopoietic stem cells (HSC) are highly enriched in the CD34 (−/low), c-Kit (+), Sca-1 (+), and lineage marker (−) (CD34 (−) KSL cell population. Using in silico data base search against NCBI EST database, we identified a novel cell surface molecules specific to CD34 (−) KSL HSCs. We cloned its full-length cDNAs of both mouse and human counterparts from total embryo (E15) and human spleen cDNAs, respectively. They were 2815-bp and 2953-bp long and the deduced amino acid sequences consisted of 851 and 852 amino acids, respectively. They contain a signal sequence, three Ig-like domains, a Thrombospondin type 1 (TSP1) domain, and a transmembrane region. Its domain composition shows high similarity to that of Unc5h3, a receptor for netrin, which plays a key role in neuronal axon guidance. Therefore, we designated this gene as Transmembrane Molecule with Thrombospondin Module (Tmtsp). BLAST search against the mouse genome database displayed that the mouse Tmtsp is composed of four exons and spanning over 26kbp on the chromosome 8qA2. Tmtsp was translated into a 170 kDa transmembrane protein. An alternative splicing which splices out the 3rd exon was detected in all major organs tested, leading to in-frame translation of a variant protein without TSP1 domain. RT-PCR analysis of hematopoietic cells demonstrated that Tmtsp is confined to KSL cells in expression and shows even higher expression in CD34 (−) KSL HSCs. On the other hand, Northern blot analysis detected its expression in all major organs tested and the highest expression in the lung. To specify the cells that express Tmtsp, we generated monoclonal antibodies against the extracellular domain of Tmtsp. Immunohistochemical analysis using anti-Tmtsp monoclonal antibodies revealed that its expression is tightly restricted to endothelial cells in tissues. These findings indicate that Tmtsp is a novel cell surface transmembrane molecule specific to both hematopoietic stem/progenitor cells and endothelial cells. Among a list of molecules shared by HSC and endothelial cells, Tmtsp is the first one that possesses the TSP1 domain implicated in cell adhesion. In this context, structure and expression of Tmtsp strongly suggest important roles in the development and maintenance of HSCs and endothelial cells including HSC homing and formation of vascular network.

A Novel Road Map for Blood Lineage Commitment and Development: Identification of a Lympho-Myeloid Stem/Progenitor Cell Lacking Erythro-Megakaryocytic Potential.

The recent identification of common myeloid and lymphoid progenitors (CMPs and CLPs, respectively) lends support to the classical and currently prevailing model for hematopoietic commitment and blood lineage development, suggesting that the first and decisive lineage commitment step of adult hematopoietic stem cells (HSCs) results in an immediate and complete separation of myelopoiesis and lymphopoiesis. Virtually all of multipotent (lympho-myeloid) stem and progenitor cells in adult mouse bone marrow (BM) reside in the small Lin−Sca1+kithi (LSK) compartment. We now present data demonstrating that the LSK compartment in adult BM, can be separated into three phenotypically and functionally distinct HSC subsets, based on differential expression of CD34 and flt3. Long-term HSCs (with extensive self-renewing potential) reside in the LSKCD34−flt3− fraction. The LSKCD34+ short-term HSC compartment consists of LSKCD34+flt3− cells fulfilling all criteria of short-term HSCs, being highly enriched in CFU-S activity, and capable of rapidly reconstituting myelo-erythropoiesis and thereby rescuing myeloablated mice. The short-term LSKCD34+flt3− HSCs give upon transplantation rise to LSKCD34+flt3+ cells, that although sustaining a combined lymphoid (B and T cell) and myeloid (granulocyte and monocyte) developmental potential at the single cell level, loose their ability to adapt megakaryocytic and erythroid fates in vitro as well as in vivo. These evidence for the existence of LSKCD34+flt3+ cells with granulocyte, monocyte, B and T cell, but not megakaryocyte and erythroid development potentials, are not compatible with the first lineage commitment step of HSCs resulting in strict separation of common lymphoid and common myeloid differentiation pathways. Based on the present findings we rather propose an alternative road map for blood lineage development in which LSKCD34+flt3− short-term HSCs genereate megakaryocyte-erythroid progenitors and LSKCD34+flt3+ cells upon asymmetric cell divisions.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

Isolation and clonal characterization of hematopoietic and liver stem cells.

Prospective isolation of stem cells is essential to understanding the mechanisms that control their proliferation and differentiation. Using 9 monoclonal antibodies and fluorescence-activated cell sorting (FACS), we have succeeded in prospectively identifying hematopoietic stem cells (HSCs) in adult mouse bone marrow. Mouse HSCs were exclusively enriched in CD34 negative, c-Kit Sca-1 Lineage Marker (CD34 KSL) cells representing 0.004% of bone marrow (BM) mononuclear cells. When single CD34-KSL cells were transplanted individually into a lethally irradiated mouse, 25% of the recipient mice survived and showed long-term reconstitution of the BM, providing evidence for multipotency and a self-renewal capacity of HSCs. Using a similar approach, we also prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewal capability in the c-Met CD49f c-Kit CD45 Ter119 fraction of cells isolated from day 13.5 fetal mouse liver. On cell transplantation, these cells differentiated into hepatocytes and cholangiocytes. As an alternative to the antibody based stem cell isolation, Hoechst33342 staining is useful. To understand the mechanism responsible for SP phenotype, we performed an expression cloning and identified bcrp-1/ABCG2 gene, a member of ATP binding-cassette (ABC) transporter family. Bcrp-1 is almost exclusively expressed in CD34 KSL cells among blood cells; however their expression in other tissue specific stem cells remains to be studied. With the use of FACS and monoclonal antibodies, hematopoietic and liver stem cells were prospectively isolated and characterized. HSCs could also be purified by Hoechst 33342 staining. By expression cloning, we identify bcrp-1/ABCG2 transporter as a molecule responsible for SP phenotype. Elucidation of the physiological role of bcrp-1/ABCG2 in HSCs may provide us with clues to understand the molecular mechanisms of stem cell self-renewal and differentiation.

Integrin α4β7 and its counterreceptor MAdCAM-1 contribute to hematopoietic progenitor recruitment into bone marrow following transplantation

AbstractPrevious studies have shown that α4β1 (very late activation antigen-4 [VLA-4]) and vascular cell adhesion molecule-1 (VCAM-1) play a major role in hematopoietic progenitor cell (HPC) homing to bone marrow (BM). However, the antibody used to block VLA-4 function in the mouse (hybridoma clone PS/2) is not specific to VLA-4 but inhibits both α4β1 and α4β7 integrins. Here we have evaluated the contribution of α4β7 in HPC homing to BM. LineagenegSca-1posc-kitpos cells from adult mouse BM and the factor-dependent cell progenitor (FDCP)—mix progenitor cell line express similar levels of α4β7 by flow cytometry. The α4β7 complex was functional since the chemokine CXCL12 enhanced the adhesion of FDCP-mix to immobilized mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and this was completely abrogated by anti-α4β7 (hybridoma clone DATK32) or anti-α4 integrins (PS/2). BM intravital microscopy revealed that α4β7 plays a predominant role in initial tethering and rolling but not in firm adhesion of FDCP-mix cells. Using homing assays, we demonstrate that α4β7 on HPCs contributes to about half of all α4 integrin–mediated homing activity following BM transplantation. MAdCAM-1 is likely expressed since its inhibition significantly reduced HPC homing. Although there may be other α4β7 integrin ligands involved (eg, fibronectin and VCAM-1), these data thus suggest that α4β7 and its counterreceptor MAdCAM-1 represent a novel adhesion pathway mediating HPC homing to BM.

Bone Marrow Origin of Cells with Capacity for Homing and Differentiation to Esophageal Squamous Epithelium

Our goal was to determine whether esophageal progenitor cells could be isolated from adult mouse esophagus or bone marrow and shown to home to and proliferate in the irradiated esophagus of recipient mice. Esophageal progenitor cells were isolated from adult male C3H/HeNsd or C57BL/6J green fluorescent protein (GFP(+)) mice by a serial in vitro preplate technique or the technique of side population cell sorting. When injected intravenously (i.v.), these cells homed to the 30-Gy-irradiated esophagus of GFP(-) female recipient mice and formed donor-origin esophageal foci. GFP(+) whole murine bone marrow cells injected i.v. also formed donor-origin esophageal squamous cell foci and protected recipient GFP(-) mice from upper-body irradiation in a cell dose-dependent manner. Marrow chimeric GFP(-) mice reconstituted with GFP(+) cells showed migration of GFP(+) marrow cells to the esophagus after 30 Gy irradiation. Purified esophageal progenitor cells isolated from first-generation preplate cell recipients engrafted after i.v. injection to the esophagus of second-generation-irradiated recipient mice. These data establish that esophageal progenitor cells can home to the irradiated esophagus and show limited differentiation capacity to squamous epithelium.

691. CFTR mRNA Is Expressed in Lungs of CFTR-KO Mice Following Administration of Adult Marrow Cells from Wild-Type Donor Mice

Top of pageAbstract A novel approach for treatment of cystic fibrosis and other lung diseases is suggested by recent studies demonstrating that airway and alveolar epithelium in adult mice can be repopulated with cells derived from bone marrow of adult donor mice. The mechanisms by which marrow-derived cells transform into epithelial cells are unknown. However pre-existing lung injury increases localization of bone marrow cells to lung epithelium suggesting that active repair or remodeling of lung epithelium may create a more conducive environment for bone marrow cells to lodge and transform into epithelial cells. We have previously demonstrated that donor-marrow-derived cells could localize to lungs of CFTR KO mice and that different experimentally-induced injury patterns could influence the distribution of the recruited marrow cells in the recipient lungs. We now demonstrate that wildtype CFTR mRNA is expressed in CFTR-KO lungs following administration of donor marrow cells from animals containing wtCFTR. Adult female CFTR-KO mice aged 6-12 weeks received intraperitoneal administration of naphthalene (275 mg/kg body weight of a stock solution of 10 ml/kg in sterile soybean oil) to induce airway lung epithelial injury. 3-4 days later, at the time of peak lung injury, treated and naive control CFTR KO mice received tail vein injection of 1 × 106 stromal marrow cells originally obtained from transgenic constitutive GFP-expressing donors (C57Bl/6 background) and cultured for 10 days prior to use. Mice were euthanized 1 week and 3 months after donor marrow cell administration and the lungs flash frozen in liquid N2, homogenized, and RNA extracted with Trizol. DNAse-treated purified RNA was reverse transcribed followed by PCR amplification using specific primers for murine CFTR (mCF11/mCF12). wtCFTR was observed in naphthalene-injured CFTR-KO mouse lungs collected 3 months but not 1 week after donor marrow administration. No wtCFTR expression was detected in un-injured transplanted CFTR-KO mice at either time point. Importantly, wtCFTR mRNA expression was not detected in donor marrow cells or in mature circulating leukocytes. These results suggest that donor marrow cells that traffic to lung subsequently express wtCFTR as they acquire an epithelial cell phenotype. Lung injury appears to increase this process. Importantly, these results suggest that marrow cells can directly traffic to lung and express a specific lung epithelial protein. Engraftment of the recipient marrow may not be necessary.

Regulatory elements of the vav gene drive transgene expression in hematopoietic stem cells from adult mice

Objective Previous studies have shown that the HS21/45 promoter of the vav protooncogene drives a predominant expression of exogenous transgenes in mouse hematopoietic cells, including clonogenic bone marrow (BM) progenitors. We investigated the activity of this promoter in the hematopoietic stem cell compartment of adult mice. Materials and methods Inbred Ly5.1 transgenic mice expressing a nonfunctional human CD4 marker gene (hCD4) under the control of the HS21/45 promoter were generated. BM cells from these animals were sorted based on the intensity of hCD4 expression. Fractions characterized by high, intermediate, or low/negative expression of the transgene were then assessed for their competitive repopulation ability (CRA), using unfractionated BM cells from Ly5.2 mice as a reference competitor population. Results Data showed that BM cells having a low/negative or intermediate expression of hCD4 had a very poor hematopoietic CRA. In contrast, BM cells with high hCD4 expression were characterized by a high CRA. These observations were confirmed in the short- and long-term posttransplantation of primary and secondary recipients when analyzing the lymphoid and myeloid cells of recipient mice. Conclusions Our results demonstrate for the first time that the regulatory HS21/45 sequence of the vav gene constitutes an efficient promoter for driving transgene expression in multipotent hematopoietic stem cells residing in the BM of adult mice. Thus, this promoter is proposed for the development of transgenic mice and gene therapy vectors that require restricted expression of exogenous transgenes in cells of the hematopoietic system, including primitive hematopoietic stem cells.

Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly, purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic, adipocytic, and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore, mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models.

Liver Disintegration in the Mouse Embryo Caused by Deficiency in the RNA-editing Enzyme ADAR1

ADAR1 (adenosine deaminase acting on RNA-1) is widely expressed in mammals, but its biological role is unknown. We show here by gene targeting that ADAR1 selectively edits in vivo two of five closely spaced adenosines in the serotonin 5-hydroxytryptamine subtype 2C receptor pre-mRNA of nervous tissue; and hence, site-selective adenosine-to-inosine editing is indeed a function of ADAR1. Remarkably, homozygosity for two different null alleles of ADAR1 caused a consistent embryonic phenotype appearing early at embryonic day 11 and leading to death between embryonic days 11.5 and 12.5. This phenotype manifests a rapidly disintegrating liver structure, along with severe defects in definitive hematopoiesis, encompassing both erythroid and myeloid/granuloid progenitors as well as spleen colony-forming activity from the aorta-gonad-mesonephros region and fetal liver. Probably as a consequence of these developmental impairments, ADAR1-deficient embryonic stem cells failed to contribute to liver, bone marrow, spleen, thymus, and blood in adult chimeric mice. Thus, ADAR1 subserves critical steps in developing non-nervous tissue, which are likely to include transcript editing.

Asymmetric division and lineage commitment at the level of hematopoietic stem cells: inference from differentiation in daughter cell and granddaughter cell pairs.

How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34−/low, c-Kit+, Sca-1+, and lineage marker− (CD34−KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34−KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors.

Generation of hybrid cell lines with endothelial potential from spontaneous fusion of adult bone marrow cells with embryonic fibroblast feeder.

We have previously isolated mouse embryonic cell lines with endothelial potential using a simple empirical approach. In an attempt to isolate similar cell lines from adult mouse bone marrow (BM), BM cells were cultured on mitotically inactive mouse embryonic fibroblast (MEF) feeder cells. Several cell lines with putative endothelial potential were generated. They expressed endothelial-specific genes and formed vascular-like structures when plated on matrigel. When transplanted into appropriate mouse models, they incorporated into the endothelium of the vasculature. Similar cell lines were also obtained using human or porcine BM. None of these lines induced tumor formation when transplanted into immunodeficient Rag1-/- mice. However, all the lines were aneuploid with genetic markers from BM samples and the MEF feeder, suggesting that they resulted from a non-species-specific fusion of a BM cell and mitotically inactive MEF. Together, these lines demonstrated for the first time that BM cells can also undergo fusion with commonly used mitotically inactive feeder cells. Although these fusion cell lines were culture artifacts, their derivation would be useful in understanding fusion of BM cells with other cell types, and their endothelial potential will also be useful in characterizing endothelial differentiation.

Telomerase deficiency impairs differentiation of mesenchymal stem cells

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR −/−) and wild-type (WT) mice. The MSCs isolated from mTR −/− mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR −/−MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation.

Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics

Objective The Hoechst 33342-effluxing side population (SP) of adult mouse bone marrow (BM) contains most of the hematopoietic stem cells (HSCs). Here we measured the HSC content of specific subsets of SP cells and then used a highly HSC-enriched fraction to investigate the effect of different growth factors on the initial rate of HSC proliferation in vitro and the accompanying maintenance (or loss) of HSCs in the first-division progeny. Materials and methods Staining with Rhodamine-123 (Rho) was used to subfractionate lineage marker–negative (lin −) SP cells. Cells were assayed for HSCs by examining their ability to generate sustained (>4 months) multi-lineage lympho-myeloid clones in irradiated hosts. Cultures of single lin − Rho − SP cells were used to monitor growth factor effects on HSC proliferation and function. Results More than 40% of mice injected with single lin − Rho − SP cells showed long-term lympho-myeloid reconstitution. Some clones peaked within 8 weeks but others developed more slowly apparently unrelated to the pattern of lineage representation. 3/3 clones tested repopulated secondary mice. Either Steel factor + interleukin-11 (± flt3-ligand) or Steel factor + thrombopoietin stimulated at least 75% of single lin −Rho − SP cells to divide in vitro with the same synchronous kinetics. However, in the first cocktail, the frequency of HSCs among the first-division doublets was preserved but in the latter it was greatly diminished. Conclusion Exogenous growth factors can differentially affect the ability of HSCs to execute a self-renewal division within a single cell cycle even when the kinetics of proliferation are the same.

Hematopoietic progenitors express neural genes.

Bone marrow, or cells selected from bone marrow, were reported recently to give rise to cells with a neural phenotype after in vitro treatment with neural-inducing factors or after delivery into the brain. However, we showed previously that untreated bone marrow cells express products of the neural myelin basic protein gene, and we demonstrate here that a subset of ex vivo bone marrow cells expresses the neurogenic transcription factor Pax-6 as well as neuronal genes encoding neurofilament H, NeuN (neuronal nuclear protein), HuC/HuD (Hu-antigen C/Hu-antigen D), and GAD65 (glutamic acid decarboxylase 65), as well as the oligodendroglial gene encoding CNPase (2',3' cyclic nucleotide 3'-phosphohydrolase). In contrast, astroglial glial fibrillary acidic protein (GFAP) was not detected. These cells also were CD34+, a marker of hematopoietic stem cells. Cultures of these highly proliferative CD34+ cells, derived from adult mouse bone marrow, uniformly displayed a phenotype comparable with that of hematopoietic progenitor cells (CD45+, CD34+, Sca-1+, AA4.1+, cKit+, GATA-2+, and LMO-2+). The neuronal and oligodendroglial genes expressed in ex vivo bone marrow also were expressed in all cultured CD34+ cells, and GFAP was not observed. After CD34+ cell transplantation into adult brain, neuronal or oligodendroglial markers segregated into distinct nonoverlapping cell populations, whereas astroglial GFAP appeared, in the absence of other neural markers, in a separate set of implanted cells. Thus, neuronal and oligodendroglial gene products are present in a subset of bone marrow cells, and the expression of these genes can be regulated in brain. The fact that these CD34+ cells also express transcription factors (Rex-1 and Oct-4) that are found in early development elicits the hypothesis that they may be pluripotent embryonic-like stem cells.

Participation of Adult Mouse Bone Marrow Cells in Reconstitution of Skin

Results of recent studies have indicated that bone marrow cells can differentiate into various cells of ectodermal, mesodermal, and endodermal origins when transplanted into the body. However, the problems associated with those experiments such as the long latent period, rareness of the event, and difficulty in controlling the processes have hampered detailed mechanistic studies. In the present study, we examined the potency of mouse bone marrow cells to differentiate into cells comprising skin tissues using a skin reconstitution assay. Bone marrow cells from adult green fluorescent protein (GFP)-transgenic mice were transplanted in a mixture of embryonic mouse skin cells (17.5 days post-coitus) onto skin defects made on the backs of nude mice. Within 3 weeks, fully differentiated skin with hair was reconstituted. GFP-positive cells were found in the epidermis, hair follicles, sebaceous glands, and dermis. The localization and morphology of the cells, results of immunohistochemistry, and results of specific staining confirmed that the bone marrow cells had differentiated into epidermal keratinocytes, sebaceous gland cells, follicular epithelial cells, dendritic cells, and endothelial cells under the present conditions. These results indicate that this system is suitable for molecular and cellular mechanistic studies on differentiation of stem cells to various epidermal and dermal cells.

Gated Importation of Prothymocytes by Adult Mouse Thymus Is Coordinated with Their Periodic Mobilization from Bone Marrow

The wavelike pattern of fetal T cell neogenesis is largely determined by the intermittent generation and exportation of waves of prothymocytes by the hemopoietic tissues in coordination with their gated importation by the thymus. Having previously shown that the importation of prothymocytes by the adult mouse thymus is also gated and that thymocytopoiesis proceeds in discrete (albeit overlapping) waves, we now demonstrate that prothymocytes are periodically exported in saturating numbers from the adult mouse bone marrow. Experiments in normal, radioablated, and parabiotic mice document the cyclical accumulation (3-5 wk) of prothymocytes in both the steady state and regenerating bone marrow, followed by their release into the blood approximately 1 wk before intrathymic gate opening. The results also show that circulating donor-origin thymocyte precursors can transiently ( approximately 1 wk) establish high level chimerism in the bone marrow after the mobilization of endogenous prothymocytes, presumably by occupying vacated microenvironmental niches. Hence, by analogy with the fetal state, we posit the existence of a feedback loop whereby diffusible chemokines of thymic origin regulate the production and/or release of bone marrow prothymocytes during each period of thymic receptivity. Because each resulting wave of thymocytopoiesis is accompanied by a wave of intrathymic dendritic cell formation, these coordinated events may help to optimize thymocyte selection as well as production.