PubMed Health⌕ Search

Biomedical subjects

M C Yoder

Publications and source records attributed to M C Yoder.

At least 19 recordsLinked to original sources
PubMed1998

Identification of human and mouse hematopoietic stem cell populations expressing high levels of mRNA encoding retrovirus receptors.

One obstacle to retrovirus-mediated gene therapy for human hematopoietic disorders is the low efficiency of gene transfer into pluripotent hematopoietic stem cells (HSC). We have previously shown a direct correlation between retrovirus receptor mRNA levels in mouse HSC and the efficiency with which they are transduced. In the present study, we assayed retrovirus receptor mRNA levels in a variety of mouse and human HSC populations to identify HSC which may be more competent for retrovirus transduction. The highest levels of amphotropic retrovirus receptor (amphoR) mRNA were found in cryopreserved human cord blood HSC. The level of amphoR mRNA in Lin- CD34(+) CD38(-) cells isolated from frozen cord blood was 12-fold higher than the level in fresh cord blood Lin- CD34(+) CD38(-) cells. In mice, the level of amphoR mRNA in HSC from the bone marrow (BM) of mice treated with stem cell factor and granulocyte-colony stimulating factor was 2.8- to 7.8-fold higher than in HSC from the BM of untreated mice. These findings suggest that HSC from frozen cord blood and cytokine-mobilized BM may be superior targets for amphotropic retrovirus transduction compared with HSC from untreated adult BM.

Animals↗
PubMed1998

c-Kit and CD38 are expressed by long-term reconstituting hematopoietic cells present in the murine yolk sac.

Murine fetal liver (FL) and adult bone marrow (BM) hematopoietic stem cells (HSCs) are characterized by cell surface expression of CD38 and c-kit. Because murine yolk sac (YS) HSC activity precedes the initiation of FL hematopoiesis, we investigated whether YS-derived HSCs also expressed c-kit and CD38. c-Kit+ CD38+ lineage- cells derived from day 9 YS as well as adult BM were found to be enriched in high proliferative potential colony-forming cells. c-Kit+ CD38+ lineage- YS or adult BM cells were capable of long-term reconstitution (>6 months) of busulfan-conditioned newborn or lethally irradiated adult mice, respectively. In contrast, c-kit+ CD38- lineage- populations from both tissues were enriched in lineage-committed progenitors and had no long-term HSC activity. We concluded that c-kit and CD38 are cell surface markers of HSCs expressed throughout murine ontogeny.

ADP-ribosyl Cyclase↗
PubMed1998

Adeno-associated virus type 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression.

Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593-1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.

Cell Line↗
PubMed1998

Adeno-associated virus type 2-mediated gene transfer: correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo.

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3' end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the beta-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34+ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1+ lin- primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.

Animals↗
PubMed1998

Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and acquired human diseases affecting cells of erythroid lineage.

Bone Marrow Cells↗
PubMed1997

In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.

The murine yolk sac, being the first site of embryonic blood cell production, has long been theorized to contain the migrating hematopoietic stem cells (HSC) that seed the liver and initiate hematopoiesis on day 10.0 postcoitus (pc). However, it remains controversial whether yolk sac cells isolated before day 11.0 pc possess any long-term repopulating HSC activity upon transplantation into adult recipient mice. We hypothesized that failure to demonstrate engraftment of day <11.0 yolk sac cells in adult hosts may result from an inability of yolk sac cells to home to the active adult hematopoietic sites (spleen and bone marrow). In the present studies, we transplanted yolk sac cells into conditioned newborn mice in whom the liver, as well as the spleen and bone marrow, concomitantly function as a site of blood cell formation. We report that yolk sac cells isolated from day 9.0 pc embryos provide long-term multilineage reconstitution for at least 11 months in primary conditioned newborn mice and for at least 6 months in secondary recipients. Donor yolk sac HSC progeny repopulated mature peripheral blood, thymus, spleen, and bone marrow lymphoid, myeloid, and erythroid compartments. Thus, day 9.0 pc yolk sac HSC can contribute to definitive multilineage hematopoiesis in transplanted recipients. Determination of HSC activity in the day 9.0 pc murine yolk sac suggests that yolk sac HSC are available to seed the liver on day 10.0 pc when definitive hematopoiesis is initiated.

Animals↗
PubMed1997

Adeno-associated virus 2-mediated gene transfer in vivo: organ-tropism and expression of transduced sequences in mice.

Adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus, is gaining attention as a vector for its potential use in human gene therapy. However, few studies have examined the safety and the efficacy of this vector system in vivo. We report here that recombinant AAV vectors, when directly injected intravenously in mice, accumulated predominantly in liver cells, suggesting that AAV may possess in vivo organ-tropism for liver. The transduced lacZ reporter gene was expressed in hepatocytes in the liver and, at the level examined, did not appear to induce any detectable cytotoxic T lymphocyte response against beta Gal. AAV-mediated transduction of murine hematopoietic progenitor cells ex vivo followed by transplantation into lethally irradiated syngeneic mice also revealed high-efficiency gene transfer into progeny cells without any observable cytotoxicity or deleterious effect. The transduced reporter gene sequences were also expressed in mice in vivo. The AAV-based vectors may thus prove useful as a potentially safe alternative to the more commonly used retrovirus- and adenovirus-based vector systems.

Animals↗
PubMed1997

Engraftment of embryonic hematopoietic cells in conditioned newborn recipients.

Yolk sac hematopoiesis is characterized by restricted hematopoietic cell differentiation. Although multipotent hematopoietic progenitor cells have been identified in the early yolk sac, long-term multilineage repopulating (LTMR) hematopoietic stem cell (HSC) activity has not been demonstrable before day 11 postcoitus (PC) using standard transplantation assays. In the present study, day-10 PC yolk sac hematopoietic cells were infused into myeloablated congenic newborn pups and donor cell engraftment and multilineage reconstitution of peripheral blood cells for at least 11 months in primary recipients was observed. In contrast, transplantation of day-10 PC yolk sac cells into congenic adult recipients did not result in engraftment despite pretransplant conditioning of the recipients or use of recipients that were genetically deficient in stem cells. Although fresh yolk sac cells were incapable of reconstitution when injected into adult recipient mice, yolk sac donor-derived cells residing in the bone marrow of primary newborn transplant recipients were capable of efficient reconstitution of conditioned secondary recipient adult mice. Primary newborn and secondary adult recipient animals engrafted with yolk sac cells were observed to have normal peripheral blood white blood cell counts. Lymphocyte subsets in peripheral blood, thymus, and spleen were also similar to control animals. The distribution and frequency of lineage-restricted progenitors derived from bone marrow of secondary transplant recipients were normal. These results indicate that day-10 PC yolk sac HSCs are capable of engrafting and reconstituting the hematopoietic system of conditioned newborn but not adult recipient animals. Furthermore, the ability of the yolk sac HSCs to differentiate into all hematopoietic lineages in these recipients strongly suggests that the local cellular microenvironment plays a prominent role in regulating yolk sac HSC differentiation.

Animals↗
PubMed1997

Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac.

The site of origin of lymphohematopoietic stem cells (HSC) that initiate definitive blood cell production in the murine fetal liver is controversial. Contrary to reports that the preliver yolk sac does not contain definitive HSC, we observed that CD34+ day 9 yolk sac cells repopulated multiple blood cell lineages in newborn hosts for at least 1 year. Furthermore, 100 CD34+c-Kit+ day 9 yolk sac or para-aortic splanchnopleura (P-Sp) cells, known to give rise to embryonic HSC, similarly repopulated hematopoiesis in recipient hosts. Surprisingly, 37-fold more CD34+c-Kit+ cells reside in the day 9 yolk sac than in the P-Sp. In sum, definitive HSC are coexistent, but not equal in number, in the murine yolk sac and P-Sp prior to fetal liver colonization.

Animals↗
PubMed1997

A recombinant human fibronectin fragment facilitates retroviral mediated gene transfer into human hematopoietic progenitor cells.

Gene transfer into hematopoietic progenitor cells is increased when these cells are adherent to the carboxy-terminal chymotryptic fragment of human plasma fibronectin (FN30/35) containing the heparin binding domain (HBD) and the alternatively spliced type three connecting segment (IIICS) region. We report herein the production of a recombinant human fibronectin fragment comprised of the HBD and IIICS regions expressed by Sf9 insect cells following recombinant baculovirus infection. The resulting isolated peptide, HuBacFN, facilitated gene transfer into human hematopoietic cell lines and primary human hematopoietic progenitors to a level achieved with the purified human plasma FN30/35 peptide.

Base Sequence↗
PubMed1997

Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation.

Although the adeno-associated virus type 2 (AAV) is known to possess a broad host range that transcends the species barrier, we suggested in an earlier study that AAV infection of human cells is receptor mediated (S. Ponnazhagan et al., J. Gen. Virol. 77:1111-1122, 1996). In the present studies, we investigated the ability of AAV to infect primary human hematopoietic progenitor cells capable of multilineage differentiation. Bone marrow-derived CD34+ cells from 12 hematologically normal volunteer donors were infected with a recombinant AAV containing the beta-galactosidase gene under the control of the cytomegalovirus immediate-early promoter (vCMVp-lacZ). Whereas 15 to 80% of the cells from approximately 50% of the donors showed various levels of lacZ gene expression, the expression was undetectable in cells from the remaining donors. However, if cells from both sets of donors were stimulated with various combinations of cytokines to induce differentiation into myeloid and lymphoid lineages following AAV infection, then the level of expression of the transduced gene increased up to 20-fold over a period of 14 days. The results of virus-binding assays suggested that the observed difference between the two groups was due to the differential susceptibility of CD34+ cells to AAV infection rather than to differences in transcription and translation of the transduced gene. To corroborate these results, CD34+ cells from the two donor groups, KB (human nasopharyngeal carcinoma) cells, and M07e (human megakaryocytic leukemia) cells were infected with vCMVp-lacZ. KB cells served as a positive control for AAV infection, and M07e cells served as a negative control. Whereas abundant hybridization to the single-stranded viral DNA on Southern blots was detected in KB and CD34+ cells that were positive for lacZ gene expression, little activity was detected in M07e and CD34+ cells that did not show expression of the lacZ gene. These results suggest that the levels of expression of the putative cellular receptor for AAV vary widely in CD34+ cells from different donors. These studies have implications for the potential use of AAV vectors in human gene therapy involving primary human primitive hematopoietic stem and progenitor cells.

Antigens, CD34↗
PubMed1997

Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo.

Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, is gaining attention as a vector for potential use in human gene therapy. We and others have described AAV-mediated beta-globin gene transfer and expression in established human and murine erythroleukemia cell lines in vitro. However, successful AAV-mediated globin gene transduction of hematopoietic stem cells and long-term expression in vivo in progeny cells have not been documented. We report here that infection of murine hematopoietic bone marrow cells ex vivo with a recombinant AAV vector containing the genomic copy of a normal human globin gene followed by transplantation of these cells into lethally irradiated congenic mice resulted in efficient gene transfer into hematopoietic cells with long-term repopulating ability as detected by the presence of the human globin gene sequences in bone marrow and spleen in primary recipient mice for at least 6 months. Long-term expression of the human globin gene was also detected in bone marrow, but not in spleen, in primary recipient mice. Furthermore, in secondary-transplant experiments, we were also able to document the presence as well as expression of the transduced human globin gene in mouse bone marrow for up to 3 months. These results provide further support for potential use of the AAV-based vector system in gene therapy of human hemoglobinopathies in general and sickle-cell anemia and beta-thalassemia in particular.

Animals↗
PubMed1997

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.

Animals↗
PubMed1997

A1 adenosine receptors potently regulate heart rate in mammalian embryos.

A1 adenosine receptors (A1ARs) have been recently shown to be expressed in rodent embryonic hearts at very early stages of development. To determine the functional significance of fetal cardiac A1AR expression during embryogenesis, murine fetal heart preparations were studied between postconceptual days 9 and 12. Dose-response curves generated using a variety of adenosine agonists revealed that A1AR activation potently regulated fetal heart rates. The A1AR agonist, N6-cyclopentyladenosine, inhibited heart rates in a dose-dependent manner (half-maximal effective concentration = 3.6 x 10(-8) M) and stopped fetal cardiac contractions in 63% of preparations. In contrast, A2a and A2b receptor activation did not alter heart rates, and activation of A3 receptors produced modest declines in heart rates. Endogenous adenosine also acted tonically to suppress fetal heart rates, as demonstrated by the A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine, increasing heart rates, whereas the adenosine reuptake blocker dipyridamole lowered fetal heart rates. Pertussis toxin treatment blocked A1AR action, showing that A1AR action was G protein mediated. Using drugs that alter cAMP levels and ion channel action, we were able to show that A1AR action involves events mediated by cAMP, ATP-dependent K, L-type calcium, sodium, and chloride channels, and the pacemaker current. These data show that adenosine and A1ARs potently regulate mammalian heart rates via multiple effector systems at very early stages of prenatal development.

Adenosine↗
PubMed1996

Differential expression of insulin-like growth factor binding proteins in murine hematopoietic stromal cell lines.

The expression of the insulin-like growth factor (IGF) system in the hematopoietic microenvironment during ontogeny was studied utilizing immortalized murine cell lines established from several sites of hematopoiesis. Conditioned media was obtained from tissue cultures of murine yolk sac endoderm and mesoderm, fetal liver and adult bone marrow stromal cell lines. IGF-I and -II were quantified by radioimmunoassay and RT-PCR. The presence of insulin-like growth factor binding proteins (IGFBPs) in conditioned media was determined by Western ligand blot and Western immunoblot. IGF-I and IGF-II were present in conditioned media from every stromal cell line and differential expression of the IGFBPs was found among the hematopoietic sites. Stimulation of the cell lines with interleukin-1 alpha altered the IGFBPs in yolk sac endoderm and bone marrow conditioned media. We report that IGF-I and -II are expressed in stromal cell lines obtained from different ontogenic sites of hematopoiesis and IGFBPs are differentially expressed by these sites. The expression of IGFBPs, but not IGFs, is in part regulated by interleukin-1 alpha.

Animals↗
PubMed1996

Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential.

The objective of ex vivo expansion of primitive hematopoietic progenitor cells (HPC) is to increase the number of progeny cells possessing hematopoietic potential similar to the original HPC. In the context of bone marrow (BM) transplantation in mice, this implies that expanding a number of HPC sufficient for long-term rescue of one lethally irradiated animal should generate enough cells to rescue more than one lethally irradiated recipient. In the present study, Sca-1+Lin- cells from male C57Bl/6 mice were expanded in vitro with stem cell factor (SCF), interleukin-1alpha (IL-1alpha), IL-3, and IL-6 and used to transplant lethally irradiated syngeneic female recipients. Expanded cells were tracked in vitro with the fluorescent membrane dye PKH2, which becomes evenly distributed among dividing daughter cells, and fractionated on day 7 into Sca-1+ cells which did not divide (Sca-1+PKH2bright), those which had divided 1 to 2 times (Sca-1+PKH2moderate), or those which had divided four or more times (Sca-1+PKH2dim). Grafts of expanded cells consisted of either the same number of fresh cells proven to rescue lethally irradiated animals [3X10(3) cells; referred to as one repopulating dose (1 RD)] or the expansion equivalent (EE) of these cells. One EE of cells represented 3X10(3) multiplied by the fold increase in the number of cultured cells on day 7. All animals transplanted with 3X10(3) freshly isolated Sca-1+Lin- cells survived long-term. Only 53% of animals receiving 1 EE of all cultured day-7 cells survived. One RD from all three PKH2 fractions (bright, moderate, and dim) of day-7 cultured Sca-1+ cells failed to rescue more than 30% of lethally irradiated recipients. Comparable survival rates were obtained when 1 EE of Sca-1+PKH2dim or only 4 RD of Sca-1+PKH2bright cells were used as grafts, suggesting that a larger frequency of long-term repopulating cells may have been retained within the fraction of Sca-1+ cells undergoing minimal or no proliferation in culture. Engraftment of male ex vivo expanded cells in recipients was confirmed by polymerase chain reaction (PCR) analysis with Y chromosome-specific primers. When analyzed for their cell cycle status, Sca-1+PKH2bright cells were mostly quiescent, whereas a higher percentage of Sca-1+PKH2dim cells were in active phases of cell cycle. These data suggest that ex vivo expansion does not augment the number of BM repopulating HPC and that ex vivo expansion generates classes of progenitor cells with different BM repopulating potentials depending on their proliferative history. These studies also suggest that the cell cycle status of graft cells may affect the ability of these cells to engraft in myeloablated hosts.

Animals↗
PubMed1996

Isolation of primitive human bone marrow hematopoietic progenitor cells using Hoechst 33342 and Rhodamine 123.

In addition to possessing multilineage differentiation and self-renewal capabilities, pluripotent hematopoietic stem cells are believed to be mitotically quiescent and metabolically inactive. Fractions of human bone marrow (BM) CD34+ cells can be further enriched for primitive hematopoietic progenitor cells (HPC) by using a number of cell-surface markers. All of these fractions, however, contain cells that are still heterogeneous as far as their metabolic and mitotic activities are concerned. We therefore used Hoechst 33342 (Hst) to identify quiescent cells and Rhodamine 123 (Rh123) to identify metabolically inactive cells. CD34+HstdimRh123dim (CD34+d/d) and CD34+HstbrightRh123bright (CD34+b/b) cells were isolated by flow cytometry to examine the hematopoietic functions of mitotically and metabolically homogeneous progenitors. Cell-cycle status, progenitor cell content, maintenance of in vitro hematopoiesis, and long-term hematopoietic culture-initiating cell (LTHC-IC) content of CD34+d/d and CD34+b/b cells were compared with CD34+HLA-DR- cells, a well-defined phenotype of primitive HPC. Whereas 99.2 +/- 0.5% of freshly isolated CD34+d/d cells were in G0/G1 phase of the cell cycle, only 74.4 +/- 11.5% of CD34+b/b and 75.6 +/- 1.1% of CD34+HLA-DR- cells were in G0/G1. The number of multipotential progenitors (colony-forming units-granulocyte/erythroid/ macrophage/megakaryocyte [CFU-GEMM]) detected in CD34+d/d cells was twice that observed in CD34+HLA-DR- cells and eight times that in CD34+b/b cells. In stromal cell-free long-term cultures maintained for 10 weeks, production of assayable progenitors in cultures initiated with CD34+d/d cells exceeded that detected in CD34+HLA-DR- cultures by more than three-fold. Only in CD34+d/d cultures were high proliferative potential colony-forming cell (HPP-CFC)-derived colonies detected over a period of 6 weeks. Limiting dilution analysis revealed that the frequency of LTHC-IC was highest among CD34+d/d cells (7.2 +/- 3.3%), followed by a frequency of 4.5 +/- 4.8% for CD34+HLA-DR- cells and 2.2 +/- 3.5% for CD34+b/b cells. The primitive nature of HPC identified by CD34, Hst, and Rh123 was confirmed by the ability of as few as 200 murine marrow cells isolated by this technique to radioprotect and fully reconstitute lethally irradiated recipients. These results indicate that Hst and Rh123 staining can be used in combination with CD34 immunofluorescence to isolate a quiescent subpopulation of human primitive hematopoietic progenitor cells. Cells isolated by this technique appear to have functional properties associated with stem cells, suggesting that they may be ideal candidates for studies requiring primitive HPC, such as ex vivo expansion and somatic gene therapy.

Adult↗