Comparative Blood Group Profiling of Human Erythroid Cells (EBs) Generated from Adult Blood (AB), Cord Blood (CB), Human Embryonic Stem Cells (hESC) and Induced Pluripotent Stem Cells (iPS)

Abstract Abstract 1027 Red blood cells (RBC) survive shear forces in the microvasculature because trans-membrane complexes embedded in the lipid bilayer attach their membrane to the cytoskeleton assuring its flexibility. The expression of clinically relevant red blood cell antigens present on these complexes is determined by genetic polymorphisms and their developmental regulation. Therefore, flow cytometry studies of blood group antigens may provide insights both on potential immunogenicity and on membrane structure of ex-vivo generated EBs. Blood group antigen profiles of EBs expanded ex vivo from one AB (three experiments), three CB, the H1 hESC line and one iPS line derived from mononuclear cells from a healthy donor were compared by flow cytometry using commercially available antibodies recognizing antigens present on proteins in the 4.1R [Duffy (Fya and Fy3), Kell (Kell prot, K/k, Kpa/Kpb, Jsb) and glycophorin C (GPC, Ge2)] and ankyrin R [glycophorin A (GPA, CD235a, M and EnaFS) RhAG and band 3 (Wrb)] complexes and on other important membrane proteins [glycophorin B (GPB, s and U), urea transporter (Kidd, Jk3), the complement receptor (CD35) and inhibitors of complement-mediated lysis (CD55 and CD59)]. Controls included DNA genotyping (CB, AB and H1-hESC) (HEA-Bead Chip, Immunocor, Norcross, GA) and immunophenotyping of blood red cells from the same AB and CB. Antigen expression similar to that observed on in vivo generated RBC was considered normal. EBs were generated from AB and CB at day 10 in HEMAser cultures whereas EBs from hESC and iPS were derived using previously optimized protocols. The maturation state was determined by morphological analyses and CD36/CD235a profiles. Irrespective of the stem cell source, the immunophenotype of ex-vivo expanded EBs was consistent with that predicted by genotyping. However, source specific differences in the magnitude of antigen expression and in the changes with maturation were observed (see Figure). Immature EBs from AB expressed normal levels of the antigens present on both the 4.1R (Duffy, Kell, GPC) and ankyrin R (GPA, M/N, EnaFS, RhAG and band 3) complexes. With maturation, expression of 4.1R-associated antigens remained normal while that of ankyrin R associated antigens varied (M decreased and RhAG increased). EBs from CB expressed normal levels of antigens present on the ankyrin R complex and of some of those present on the 4.1R complex (Duffy, Kell protein and GPA). However, expression of epitopes on Kell protein varied with some antigens expressed at normal levels (k and Jsb) and others (Kpa/Kpb) at levels 2x greater than normal. With maturation, CB-derived EBs maintained normal levels of ankyrin R associated antigens while those associated with complex 4.1R became barely detectable. EB from hESC expressed unbalanced levels of proteins associated with both ankyrin R (2x levels of GPA and barely detectable levels of RhAG) and 4.1R [3x levels of Duffy and 2x levels of Jsb (Kell) with normal levels of k and Kpb (Kell) antigens] complexes. The variegation in expression of different epitopes on the same protein observed with CB- and hESC-derived EBs likely reflect altered structural conformation of the complexes rather than differences in protein concentration on the membrane. EBs from iPS, as those from AB, expressed normal levels of antigens present on Ankyrin R and 4.1R complexes which increased with maturation. Irrespective of stem cell sources, EBs expressed normal levels of GPB and Kidd. EBs from AB expressed normal levels of the complement regulatory proteins tested which in the case of CD59 CD59 decreased with maturation. EBs from CB expressed normal levels of CD35 and CD59 but 2x levels of CD55 with expression of CD35 and CD55 decreasing with maturation. EBs from iPS expressed 2x levels of CD35 and CD55 and expression of these antigens was not affected by maturation. The observation that blood group antigenic profiles of ex-vivo generated EBs are consistent with those predicted by DNA-genotyping suggests that these cells are unlikely to be immunogenic for known epitopes. However, the antigen profiles of ankyrin R and 4.1R complexes were normal only for AB and iPS-derived EBs raising the possibility that antigenic deviations seen in EBs derived from CB and hESC may have immunologic or functional consequences in vivo. Disclosures: No relevant conflicts of interest to declare.

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200 TRANSPLANTATION AND IN VIVO DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN SWINE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab200 ◽

2006 ◽

Vol 18 (2) ◽

pp. 208 ◽

Cited By ~ 1

Author(s):

A. S. Lima ◽

S. A. Malusky ◽

M. R. B. Mello ◽

S. J. Lane ◽

J. R. Rivera ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Cellular Response ◽

Agricultural Research ◽

Primary Concern ◽

Adipose Derived Stem Cells ◽

Subcutaneous Adipose

A primary concern in stem cell biology is that observations made in vitro may be an artifact of the in vitro culture environment. In vitro derived stem cells can be implanted into the environment from which they are derived so that their response to physiological conditions may be observed. Several important cellular characteristics need to be examined following the cell's reintroduction to the in vivo environment, including the potential for differentiation, proliferative ability, and life span. Studying implanted stem cells will assist in determining the potential for stem cell use in clinical therapies and provide further understanding of the role adult stem cells have in the adult body. Currently, the scientific literature is lacking a detailed description of the cellular response of adipose-derived stem cells (ADSCs) reintroduced to their exact tissue of origin. Thus, the aim of this study was to evaluate porcine ADSC growth in vivo and to analyze cell differentiation in vivo following injection of undifferentiated ADSCs into subcutaneous fat. Subcutaneous adipose tissue was isolated from the back fat of male pigs (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 ADSCs were labeled with fluorescent dye (PKH26; Sigma, St. Louis, MO, USA) and sorted by flow cytometry. After sorting, positive cells were washed and re-suspended in culture medium. For transplantation, 100 �L of cell suspension in DMEM containing one of four cell concentrations (0 (control); 30 000; 300 000; and 900 000 cells) were placed in a 1-mL syringe and injected into the subcutaneous back fat of recipient pigs (n = 2). Each pig had previously been tattooed with 12 13 � 13 squares to mark injection sites. The treatments were replicated three times within each animal. Two and three weeks after transplantation, animals were euthanized, the back fat containing the transplantation site was harvested, and the cells were disaggregated as described above. The buoyant adipocytes and pelleted ADSCs cells were then analyzed by flow cytometry. The results indicated that there were dose- and time-dependent increases in labeled ADSCs and labeled adipocytes in the fat samples with increasing cell number (from 0 to 300 000 cells). There was, however, a decrease in labeled ADSCs at the 900 000-cell dose, which is likely due to excess cells being transplanted or an immune reaction. Both of these aspects are currently being evaluated. In conclusion, undifferentiated ADSCs from swine can be isolated from and returned to the subcutaneous adipose layer and differentiate into mature adipocytes. This work was supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois.

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Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning

Blood ◽

10.1182/blood.v94.7.2271.419k41_2271_2286 ◽

1999 ◽

Vol 94 (7) ◽

pp. 2271-2286 ◽

Cited By ~ 86

Author(s):

M. Rosenzweig ◽

T.J. MacVittie ◽

D. Harper ◽

D. Hempel ◽

R.L. Glickman ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Peripheral Blood ◽

Nonhuman Primates ◽

Peripheral Blood Stem Cells ◽

Hematopoietic Stem ◽

Blood Stem Cells ◽

High Level

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 μg/kg) and G-CSF (20 μg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis. CD34+ cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naı̈ve (CD45RA+and CD62L+) CD4+ and CD8+cells were the predominant phenotype of the marked CD3+ T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naı̈ve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

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Ex-Vivo Expansion of Multipotent CD133+ Stem Cells with VEGF, FLT3l and SCGF.

Blood ◽

10.1182/blood.v104.11.4147.4147 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4147-4147

Author(s):

Sonja Loges ◽

Martin Butzal ◽

Uta Fischer ◽

Ursula M. Gehling ◽

Dieter K. Hossfeld ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Stem Cell ◽

Culture System ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Facs Analysis ◽

Hematopoietic Stem ◽

Large Numbers

Abstract The rare CD133+ stem cell population contains both hematopoietic and endothelial progenitors. Successful ex-vivo expansion of this multipotent population would therefore be of great benefit in many clinical settings including stem cell transplantation and gene therapy. We developed a cell culture system containing the recombinant human cytokines vascular endothelial growth factor (VEGF), FLT3 ligand (FLT3L) and stem cell growth factor (SCGF) for ex-vivo expansion of purified human CD133+ stem cells obtained from leukapheresis products from patients pre-treated with G-CSF. FACS analysis, colony assays and NOD-SCID transplantation studies were performed to monitor stem cell and endothelial phenotype in-vitro and in-vivo. Cultivation with VEGF, FLT3L and SCGF induced a mean 2200-fold increase of total cell counts in 5 weeks. FACS analysis revealed persistence of 6–15% CD133+ stem cells indicating proliferation and survival of primitive hematopoietic stem cells. 5–6% of the proliferating cells expressed the endothelial markers CD144 (VE-Cadherin) and von-Willebrand factor (vWF). Ex-vivo expanded stem cells could be differentiated into adherent endothelial cells after withdrawal of SCGF and FLT3L allowing generation of large numbers of endothelial cells. Colony-assays showed an increase of hematopoietic and endothelial colonies after 5 weeks of ex-vivo expansion indicating simultaneous proliferation of hematopoietic and endothelial precursors under the established culture conditions (CFU-E 60-fold, CFU-GEMM 48-fold, CFU-GM 59-fold, CFU-G 99-fold, CFU-M 1356-fold and CFU-EC 1843-fold). To assess in-vivo functionality, hematopoietic stem cells expanded ex-vivo for 7, 14, 21 and 32 days were transplanted into sublethally irradiated NOD-SCID mice. For each expansion period, the mean percentage of anti-human CD45 positive bone marrow cells 3 months post-transplantation was 11, 3, 3 and 1%, respectively. Human CD45+ cells for each set of experiments contained a mean of 15, 26, 8 and 32% T-cells (CD3+), 9, 0, 7 and 21% B-cells (CD19+), 24, 2, 2 and 11% monocytes (CD14+), 21, 3, 1 and 12% granulocytes (CD33+) and 19, 37, 44 and 24% stem cells (CD34+) (d7 (n=5), d14 (n=4), d21 (n=7) and d32 (n=6) respectively). Our experiments showed multilineage engraftment of human stem cells expanded for more than 4 weeks ex-vivo. Therefore our culture system provides a tool to generate large numbers of human stem and endothelial cells for clinical purposes.

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Targeting CD96 For Antibody Based Elimination Of Leukemic Stem Cells In AML: A New Strategy In Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.3972.3972 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3972-3972 ◽

Cited By ~ 2

Author(s):

Matthias Staudinger ◽

Christian Kellner ◽

Matthias Peipp ◽

Natalie Schub ◽

Andreas Humpe ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Target Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Although the mortality of autologous stem cell transplantation in contrast to allogeneic is low, in AML patients the lack of immune surveillance as well as contamination of the transplant with residual leukemic stem cells (LSC) limits its use. Therefore, elimination of LSC by targeted therapy may represent a promising therapeutic approach. Recently, CD96 was identified as marker antigen on AML-LSC (Hosen et al., PNAS 104: 11008, 2007). Here, by addressing CD96 with magnetic cell sorting (MACS) or using antibody dependent cellular cytotoxicity (ADCC), new strategies for engineering autologous stem cell grafts or for in vivo targeting of residual AML stem cells are presented. To evaluate the efficacy of depletion of LSC by MACS technology, grafts containing hematopoietic stem cells were spiked with CD96 positive AML cells. Using biotinylated CD96 antibody TH111 raised in our laboratory in combination with anti-biotin-micro beads (Miltenyi Biotech, Bergisch Gladbach, Germany) up to a 1000-fold depletion of targeted cells was achieved. The viability, cell count and the potential of hematopoietic progenitor cells (HPC) to proliferate and differentiate were not affected by this procedure as documented by flow cytometry and colony forming assays. As residual LSC residing within the patient may also account for AML relapse after high-dose chemotherapy and subsequent SCT, eradication of AML stem cells in vivo is desirable. To target CD96+ AML-LSC by ADCC, chimeric antibodies containing wild type or affinity maturated variable regions in combination with an optimized human IgG1Fc were generated by recombinant DNA technologies. Both recombinant antibodies were expressed in Hek 293 cells enriched to homogeneity by affinity chromatography and analyzed for their functional properties. As shown by flow cytometry, the antigen binding affinity of the maturated antibody was enhanced (EC50 0.6 μg/ml vs. 2 μg/ml). Moreover, as analyzed in standard ADCC assays, NK cell mediated lytic properties against CD96-positive target cells were elevated (maximum lysis: 52%) using the affinity maturated chimeric CD96 antibody (EC50: 0.02 μg/ml vs. 0.15 μg/ml). Thus, this CD96 purging strategy avoids unwanted transplantation of AML-LSC and may help to revitalize autologous stem cell transplantation in this indication. Although, specific side effects by CD96 application will have to be considered, this may allow for an additional therapeutic avenue to eliminate in vivo residual AML-LSC in autologous as well as in allogeneic situations. Disclosures: No relevant conflicts of interest to declare.

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The OCIAD protein family: comparative developmental biology and stem cell application

The International Journal of Developmental Biology ◽

10.1387/ijdb.190038mi ◽

2020 ◽

Vol 64 (1-2-3) ◽

pp. 213-225

Author(s):

Wulligundam Praveen ◽

Saloni Sinha ◽

Rajarshi Batabyal ◽

Kajal Kamat ◽

Maneesha S. Inamdar

Keyword(s):

Stem Cells ◽

Developmental Biology ◽

Stem Cell ◽

Information Overload ◽

Ex Vivo ◽

Developmental Models ◽

Clinically Significant ◽

Insight Into ◽

Compare Analysis

Over the last two decades, an exponential growth in technologies and techniques available to biologists has provided mind-boggling quantities of data and led to information overload. Yet, answers to fundamental questions such as “how are we made?” and “what keeps us ticking?” remain incomplete. Developmental biology has provided elegant approaches to address such questions leading to enlightening insights. While several important contributions to developmental biology have come from India over the decades, this area of research remains nascent. Here, we review the journey in India, from the discovery of the ociad gene family to decoding its role in development and stem cells. We compare analysis in silico, in vivo and ex vivo, with developmental models such as Drosophila, mouse and stem cells that gave important insight into how these clinically significant genes function.

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Identification of hematopoietic stem cell subsets on the basis of their primitiveness using antibody ER-MP12

Blood ◽

10.1182/blood.v85.4.952.bloodjournal854952 ◽

1995 ◽

Vol 85 (4) ◽

pp. 952-962 ◽

Cited By ~ 37

Author(s):

JC van der Loo ◽

WA Slieker ◽

D Kieboom ◽

RE Ploemacher

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Antigen Expression ◽

Chimeric Mouse ◽

Hematopoietic Stem ◽

Colony Forming Units

Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a physical separation of primitive long-term repopulating stem cells from more mature multilineage progenitors. When used in two-color immunofluorescence with ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow (BM) cells could be identified. These subsets were isolated using magnetic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colony-forming units in culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for day-12 spleen colony-forming units (CFU-S-12), and for cells with long-term repopulating ability using a recently developed alpha-thalassemic chimeric mouse model. Cells with long-term repopulation ability (LTRA) and day-12 spleen colony-forming ability appeared to be exclusively present in the two subpopulations that expressed the ER-MP12 cell surface antigen at either an intermediate or high level, but lacked the expression of Ly- 6C. The ER-MP12med20- subpopulation (comprising 30% of the BM cells, including all lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28 CAFC (CAFC-28), 75% of the CFU-S-12, and very low numbers of CFU-C. In contrast, the ER-MP12hi20- population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primitive CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 through day 14), of which 70% were ER-MP20- and 10% to 20% ER-MP20med/hi. In addition, erythroblasts, granulocytes, lymphocytes, and monocytes could almost be fully separated on the basis of ER-MP12 and ER-MP20 antigen expression. Functionally, the presence of ER-MP12 in a long-term BM culture did not affect hematopoiesis, as was measured in the CAFC assay. Our data demonstrate that the ER-MP12 antigen is intermediately expressed on the long-term repopulating hematopoietic stem cell. Its level of expression increases on maturation towards CFU-C, to disappear from mature hematopoietic cells, except from B and T lymphocytes.

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Stem cells and lung regeneration

AJP Cell Physiology ◽

10.1152/ajpcell.00036.2020 ◽

2020 ◽

Vol 319 (4) ◽

pp. C675-C693

Author(s):

Kalpaj R. Parekh ◽

Janna Nawroth ◽

Albert Pai ◽

Shana M. Busch ◽

Christiana N. Senger ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Lung Disease ◽

Progenitor Cell ◽

Human Lung ◽

Ex Vivo ◽

Cell Types ◽

And Function ◽

Lung Regeneration

The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease.

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Human levator veli palatini muscle: A novel source of mesenchymal stem cells for use in the rehabilitation of patients with congenital craniofacial malformations

10.21203/rs.3.rs-42456/v1 ◽

2020 ◽

Author(s):

Daniela Franco Bueno ◽

Gerson Shigueru Kabayashi ◽

Carla Cristina Gomes Pinheiro ◽

Daniela Y S Tanikawa ◽

Cassio Eduardo Raposo-Amaral ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Osteogenic Potential ◽

Flow Cytometry Analysis ◽

Non Invasive ◽

Levator Veli Palatini

Abstract Background. Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stem cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent stem cells that can be isolated via non-invasive procedures. Here we analysed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during surgical rehabilitation of cleft patients during palatoplasty, represent a novel source of MSCs with osteogenic potential. Methods. We obtained levator veli palatini muscle fragments, in non-invasive procedure during surgical rehabilitation of 5 unrelated cleft palate patients (palatoplasty surgery). The levator veli palatini muscle fragments was used to obtain the mesenchymal cells using pre-plating technique in a clean rooms infrastructure and all procedures were performed at good practices of manipulation conditions. To prove that levator veli palatini muscle are mesenchymal stem cells they were induced to flow cytometry analysis and to differentiation into bone, cartilage, fat and muscle. To demonstrate the osteogenic potential of these cells in vivo a bilateral full thickness calvarial defect model was made in immunocompentent rats.Results. Flow cytometry analysis showed that the cells were positive for mesenchymal stem cell antigens (CD29, CD73, CD90), while negative for hematopoietic (CD45) or endothelial cell markers (CD31). Moreover, these cells were capable of undergoing chondrogenic, adipogenic, osteogenic and skeletal muscle cell differentiation under appropriate cell culture conditions characterizing them as mesenchymal stem cell. Defects treated with CellCeramTM scaffolds seeded with levator veli palatini muscle cells showed significantly greater bone healing compared to defects treated with acellular scaffolds. Conclusion. We have demonstrated that cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stem cells, both in vitro and in vivo. Our ﬁndings suggest that these cells may have clinical relevance in the rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

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Reducing Stem Cell Loss and Ex Vivo Differentiation during Lentivirus Gene Transfer to Murine Stem Cells - an Ultra-Short Transduction Protocol.

Blood ◽

10.1182/blood.v104.11.2113.2113 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2113-2113

Author(s):

Peter Kurre ◽

Ponni Anandakumar ◽

Vladimir A. Lesnikov ◽

Hans-Peter Kiem

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Gene Transfer ◽

Ex Vivo ◽

Lentivirus Vectors ◽

Ex Vivo Culture ◽

Marrow Cells

Abstract Most gene transfer models using Moloney murine leukemia virus (MLV) - derived vectors to target hematopoietic repopulating cells require progenitor cell enrichment and extended ex vivo culture for efficient long-term marking. Both may result in qualitative, and/or quantitative, loss of stem cells thereby limiting gene transfer rates in vivo. This can be a critical obstacle in candidate applications with exhausted autologous stem cell pools, such as Fanconi Anemia. Among the advantages of HIV-derived lentivirus vectors is their ability to transduce non dividing cells, permitting shortened ex vivo culture durations while maintaining gene transfer to long-term repopulating cells. We have previously reported long-term gene transfer rates of 12–40% after VSV-G/ lentivirus vector transduction of murine stem cells by targeting unseparated marrow cells after reduced prestimulation and a single 12 hour vector exposure (Kurre et al., Mol. Ther. 2004 Jun;9(6):914–22). We herein report studies showing maintenance of gene transfer efficiency in this model at drastically reduced ex vivo vector exposure times. In initial in vitro experiments we studied cytokine support, vector particle density, and minimum exposure duration requirements for efficient gene transfer to unseparated marrow cells. We determined that fibronectin fragment support was critical in maintaining minimum gene transfer efficiencies, even during brief 1, or 3-hour exposures. In an effort to extend these in vitro findings targeting a mixed leukocyte population and explore the feasibility in vivo, we next performed repopulation experiments in myeloablated murine recipients. Unseparated marrow cells harvested from donor animals were depleted of red blood cells, washed and immediately transduced on fibronectin fragment in the presence of murine stem cell factor. Following a 1 hour exposure to lentivector (VSV-G/RRLsin-cPPThPGK-EGFPwpre), cells were washed repeatedly, resuspended and injected into myeloablated recipients (n=10). Animals showed ready hematopoietic reconstitution and demonstrated average GFP marking of 31% (range: 17–41.2%) in peripheral blood 20 weeks after transplantation. Gene marking in secondary recipients 9 weeks after reconstitution (n=15, 3 recipient animals per donor) persisted at 29% on average (range 14.9–66%). Results also demonstrate transduction of granulocytes, B- and T-lymphocytes, as well as stable long-term GFP expression in primary and secondary animals. Copy number determination by real-time PCR in marrow cells from primary recipients shows an average of 4 proviral copies (range 2.1–8.1) per GFP-expressing cell. Our studies confirm that HIV-derived lentivirus vectors are ideally suited for the transduction of murine long-term repopulating cells. We hypothesize that ultra-short transduction actively preserves stem cell content in the inoculum. Moreover, this protocol represents an ideal platform for subsequent in vivo selection to achieve complete phenotype correction and high-level therapeutic chimerism required for some applications. We anticipate that our strategy may prove particularly useful in situations where the target stem cell quantity is greatly limited and cells are of poor ex vivo viability.

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Changes in the blood group Wright antigens are associated with a mutation at amino acid 658 in human erythrocyte band 3: a site of interaction between band 3 and glycophorin A under certain conditions [see comments]

Blood ◽

10.1182/blood.v85.2.541.bloodjournal852541 ◽

1995 ◽

Vol 85 (2) ◽

pp. 541-547 ◽

Cited By ~ 1

Author(s):

LJ Bruce ◽

SM Ring ◽

DJ Anstee ◽

ME Reid ◽

S Wilkinson ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Blood Group ◽

Antigen Expression ◽

Band 3 ◽

Glycophorin A ◽

Blood Group Antigens ◽

Cdna Sequences ◽

The Common ◽

A Site

The Wright (Wr) blood group antigens, Wra and Wrb, have been suggested to be determined by alleles of the same gene. The Wrb antigen appears to involve both red blood cell (RBC) band 3 and glycophorin A (GPA). We have examined the cDNA sequences of the band 3 and GPA of one of the two known Wr(a+b-) individuals. We show that this individual is homozygous for the mutation Glu658--<Lys in band 3, but has normal GPA. Putative heterozygotes with Wr(a+b+) RBCs have both Glu and Lys at residue 658 of band 3, whereas the common Wr(a-b+) RBC phenotype only have band 3 with Glu658. The Wra and Wrb antigens are determined by the amino acid at residue 658 of band 3 and are antithetical. Examination of the amino acid sequence and Wrb antigen expression of GPA-related hybrid glycophorins suggests that Arg61 of GPA interacts with Glu658 of band 3 to form the Wrb antigen. We suggest that the interaction is stabilized by the presence of anti-Wrb antibodies and that this site of association between GPA and band 3 may be responsible for the previously reported ability of anti-GPA antibodies to decrease the deformability of RBCs.

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