Hans-Jörg Bühring

A Perivascular Origin for Mesenchymal Stem Cells in Multiple Human Organs

Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-Rbeta expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.

Generation of pluripotent stem cells from adult human testis

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.

Novel Markers for the Prospective Isolation of Human MSC

Abstract:  The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)‐derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271+ population. The recognized CD271+ populations were fractionated by fluorescence‐activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU‐F. The results showed that only the CD271bright but not the CD271dim population contained CFU‐F. Two‐color flow cytometry analysis revealed that only the CD271bright population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271bright population but no other BM cells. The new MSC‐specific molecules included the platelet‐derived growth factor receptor‐β (CD140b), HER‐2/erbB2 (CD340), frizzled‐9 (CD349), the recently described W8B2 antigen, as well as cell‐surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9‐3C2F1, and HEK‐3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM‐MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.

The Basophil-Specific Ectoenzyme E-NPP3 (CD203c) as a Marker for Cell Activation and Allergy Diagnosis

Basophils are effector cells of allergic reactions. These cells produce and store a number of vasoactive and immunomodulatory mediators. During an allergic reaction, basophils can release their mediator substances into the extracellular space and thus contribute to the clinical picture and symptoms in allergy. The phenotypic hallmark of basophils is expression of high-affinity IgE receptors (FcΕRI) on their cell surface together with expression of the activation-linked molecule CD203c. This ectoenzyme is located both on the plasma membrane and in the cytoplasmic compartment of basophils. Cross-linking of the FcΕRI by an allergen or anti-IgE antibody results in a rapid upregulation of intracellular CD203c molecules to the cell surface and is accompanied by mediator release. CD203c is therefore a promising target molecule for a flow cytometry-based test to analyze sensitized individuals and patients with type I allergy. In the present article, we review the current knowledge of CD203c with special regard to its tissue distribution and regulation in basophil activation. In addition, we discuss the application of CD203c in allergy diagnosis.

Biology and Plasticity of CD133+ Hematopoietic Stem Cells

Abstract: AC133 (CD133) is a highly conserved antigen expressed on hematopoietic stem cells with unknown function. In order to further characterize CD133+ progenitor cells, we purified CD133+ stem cells using the method of magnetic activated cell sorting (MACS) from healthy adult volunteers mobilized with granulocyte colony‐stimulating growth factor (G‐CSF) to a mean purity of 94%. The purified CD133+ cells highly engrafted NOD/SCID mice. In addition, unseparated mononuclear cells or CD133+ stem cells isolated from the bone marrow of transplanted NOD/SCID mice gave rise to engraftment of secondary recipients. Upon ex vivo culture of purified CD133+ cells with FLT3/Flk2 ligand (FL) and interleukin‐6 (IL‐6), a plastic‐adherent cell population could be observed after 6 weeks in culture. These adherent cells did not express CD34 or CD133 antigens on their surface, nor did they express markers for endothelial, mesenchymal, or dendritic cells. After incubation of these adherent cells with stem cell factor (SCF), non‐adherent cells were observed which partially co‐expressed CD133, but were negative for CD34. These nonadherent CD34− cells showed a high engraftment capacity in NOD/SCID mice. From our results, we conclude that CD133 might be a marker of early progenitors with a high NOD/SCID engraftment potential. The fact that CD133+ hematopoietic progenitors can give rise to an adherent population which is CD133− and CD34− and that these cells can again give rise to a CD133+CD34− stem cell population with high NOD/SCID engraftment potential indicates the plasticity of hematopoietic precursors. CD133+ stem cells might be useful for research and for clinical application.

Chondrogenic Potential of Human Adult Mesenchymal Stem Cells Is Independent of Age or Osteoarthritis Etiology

Osteoarthritis (OA) is a multifactorial disease strongly correlated with history of joint trauma, joint dysplasia, and advanced age. Mesenchymal stem cells (MSCs) are promising cells for biological cartilage regeneration. Conflicting data have been published concerning the availability of MSCs from the iliac crest, depending on age and overall physical fitness. Here, we analyzed whether the availability and chondrogenic differentiation capacity of MSCs isolated from the femoral shaft as an alternative source is age‐ or OA etiology‐dependent. MSCs were isolated from the bone marrow (BM) of 98 patients, categorized into three OA‐etiology groups (age‐related, joint trauma, joint dysplasia) at the time of total hip replacement. All BM samples were characterized for cell yield, proliferation capacity, and phenotype. Chondrogenic differentiation was studied using micromass culture and analyzed by histology, immunohistochemistry, and quantitative reverse transcriptase‐polymerase chain reaction. Significant volumes of viable BM (up to 25 ml) could be harvested from the femoral shaft without observing donor‐site morbidity, typically containing >107 mononuclear cells per milliliter. No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA. From all tested donors, MSCs could be differentiated into the chondrogenic lineage. We conclude that, irrespective of age and OA etiology, sufficient numbers of MSCs can be isolated and that these cells possess an adequate chondrogenic differentiation potential. Therefore, a therapeutic application of MSCs for cartilage regeneration of OA lesions seems feasible.

Isolation of Highly Purified Autologous and Allogeneic Peripheral CD34+ Cells Using the CliniMACS Device

The CliniMACS CD34+ selection device was used for positive selection of apheresis products for autologous transplantation from 10 patients with malignant diseases and for allogeneic transplantation from 26 healthy donors. A total of 71 separations were performed. In 1 allogeneic donor, CD34+ progenitors were also isolated from bone marrow. Between 0.27 and 8.9 x 10(10) nucleated cells (median 4.9 x 10(10)) containing 0.09%-10.8% (median 0.67%) CD34+ progenitor cells were separated. After separation, a median number of 227 x 10(6) mononuclear cells (MNC) (51-524) were recovered, with a median viability of 99% (22%-100%) and a median purity of 97.0% (68.3%-99.7%) CD34+ cells. Depletion of T cells was extensive, with a median of 0.04% residual CD3+ cells (range <0.01%-0.92%). Residual CD19+ cells were between <0.01% and 17%, including CD34+CD19+ cells. Recovery of CD34+ cells was calculated according to the ISHAGE guidelines and ranged from 24% to 105% (median 71%). We conclude that with the CliniMACS device CD34+ cells with high purity and recovery can be isolated with concomitant effective T cell depletion in the allogeneic setting and with a high purging efficacy in the autologous setting.

Cutting Edge: Signal-Regulatory Protein β1 Is a DAP12-Associated Activating Receptor Expressed in Myeloid Cells

Signal-regulatory proteins (SIRPs) are cell-surface glycoproteins expressed on myeloid and neural cells that have been shown to recruit SH2 domain-containing protein phosphatase 1 (SHP-1) and SHP-2 and to regulate receptor tyrosine kinase-coupled signaling. One SIRP of unknown function, designated SIRPβ1, contains a short cytoplasmic domain that lacks sequence motifs capable of recruiting SHP-1 and SHP-2. Using a SIRP-specific mAb, we show that SIRPβ1 is expressed in monocytes and dendritic cells and associates with the signal transduction molecule DAP12. SIRPβ1/DAP12 complex formation was required for efficient cell-surface expression of SIRPβ1. Stimulation of this complex induced tyrosine phosphorylation, mitogen-activated protein kinase activation, and cellular activation. Thus, SIRPβ1 is a new DAP12-associated receptor involved in the activation of myeloid cells.

Expression of MUC-1 Epitopes on Normal Bone Marrow: Implications for the Detection of Micrometastatic Tumor Cells

PURPOSE The expression of the carcinoma-associated mucin MUC-1 is thought to be restricted to epithelial cells and is used for micrometastatic tumor cell detection in patients with solid tumors, including those with breast cancer. Little is known, however, about the expression of MUC-1 epitopes in normal hematopoietic cells. MATERIALS AND METHODS MUC-1 expression was analyzed by flow cytometry and immunocytology on bone marrow (BM) mononuclear cells and purified CD34+ cells from healthy volunteers, using different anti-MUC-1-specific monoclonal antibodies. In addition, Western blotting of MUC-1 proteins was performed. RESULTS Surprisingly, 2% to 10% of normal human BM mononuclear cells expressed MUC-1, as defined by the anti-MUC-1 antibodies BM-2 (2E11), BM-7, 12H12, MAM-6, and HMFG-1. In contrast, two antibodies recognizing the BM-8 and the HMFG-2 epitopes of MUC-1 were not detected. MUC-1+ cells from normal BM consisted primarily of erythroblasts and normoblasts. In agreement with this, normal CD34+ cells cultured in vitro to differentiate into the erythroid lineage showed a strong MUC-1 expression on day 7 proerythroblasts. Western blotting of these cells confirmed that the reactive species is the known high molecular weight MUC-1 protein. CONCLUSION Our data demonstrate that some MUC-1 epitopes are expressed on normal BM cells and particularly on cells of the erythroid lineage. Hence the application of anti-MUC-1 antibodies for disseminated tumor cell detection in BM or peripheral blood progenitor cells may provide false-positive results, and only carefully evaluated anti-MUC-1 antibodies (eg, HMFG-2) might be selected. Furthermore, MUC-1-targeted immunotherapy in cancer patients might be hampered by the suppression of erythropoiesis.

Identification of CD13, CD107a, and CD164 as novel basophil-activation markers and dissection of two response patterns in time kinetics of IgE-dependent upregulation.

ABSTRACTUsing two-colour flow cytometry >200 antibodies submitted to the 8th International Workshop of Human Leukocyte Differentiation Antigens (HLDA8) have been analyzed for their reactivity with resting and activated CD203c+ basophils. Four antibodies either non-reactive or weakly reactive with resting basophils exhibited an increased reactivity with basophils activated by anti-IgE-mediated cross-linking of the high affinity IgE receptor (FcεRI). These include antibodies against CD164 (WS-80160, clone N6B6 and WS-80162, clone 67D2), as well as two reagents with previously unknown specificities that were identified as CD13 (WS-80274, clone A8) and CD107a (WS-80280, clone E63-880). The activation patterns followed either the “CD203c-like” or “CD63-like” activation profile. The CD203c profile is characterized by a rapid and significant upregulation (of CD13, CD164, and CD203c), reaching maximum levels after 5-15 min of stimulation. The phosphoinositide-3-kinase (PI3K)-specific inhibitor wortmannin inhibited the upregulation of these markers whereas 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced a rapid and FcεRI-independent upregulation within 1-2 min. In the CD63 profile, maximum upregulation (of CD63 and CD107a) was detected only after 20-40 min, and upregulation by TPA reached maximum levels after 60 min. In summary, our data identify CD13, CD107a, and CD164 as novel basophil-activation antigens. Based on time kinetics of upregulation, we hypothesize that molecules of the “CD203c group” and the “CD63 group” are linked to two different mechanisms of basophil activation.