Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2685-2685
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Adel Ersek ◽  
James A. Thomson ◽  
Rachel L. Lewis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Human Dna ◽  
Cd34 Cells ◽  
Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Purified T Depleted Peripheral Blood and Bone Marrow Cd34 Transplantation from Haploidentical Mother to Child with Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3106-3106
Author(s):  
Pietro Sodani ◽  
Buket Erer ◽  
Javid Gaziev ◽  
Paola Polchi ◽  
Andrea Roveda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
Therapeutic Option ◽  
B Cell Lymphoma ◽  
Marrow Transplant ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant. We have employed a new preparative regimen for the transplant in fourteen thalassemic children aged 3 to 12 years (median age 5 years) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day -59 until day-11, fludarabine (FLU) 30 mg/m 2 from day -17 to day -11, busulphan (BU) 14 mg/kg starting on day -10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs system), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease(GVHD) prophylaxis during the first two months after the bone marrow transplantation. Results. Thirteen patients are alive. Four patients rejected the transplant and are alive with thalassemia One patients died six months after bone marrow transplant for central nervous system diffuse large B cell lymphoma EBV related. Nine patients are alive disease free with a median follow up of 30 months (range12–47). None of the seven patients showed AGVHD and CGVHD. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients without genotipically or phenotipically HLA identical donor.

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

Induced Hematopoietic Differentiation Of Common Marmoset Embryonic Stem Cells By LYL1 Can Give Rise To Hematopoietic Stem Cells Having The Enhanced Bone Marrow Reconstitution Capability

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1192-1192
Author(s):  
Hirotaka Kawano ◽  
Tomotoshi Marumoto ◽  
Takafumi Hiramoto ◽  
Michiyo Okada ◽  
Tomoko Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host’s hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro. The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point. Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures: No relevant conflicts of interest to declare.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

Different kinases acting on stathmin (oncoprotein, Op18) in human healthy and malignant hematopoietic stem cells

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 17527-17527
Author(s):  
H. Lannert ◽  
T. Able ◽  
S. Leicht ◽  
R. Saffrich ◽  
V. Eckstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Proteome Analysis ◽  
Becton Dickinson ◽  
Hematopoietic Stem ◽  
Cd34 Cells

17527 Background: Stathmin/Op18 is a cytosolic phosphoprotein which regulates the dynamics of microtubules. This regulation is important in mitosis during cell division and in the migration of cells in modification of the cytoskeleton. The process of tumor proliferation and metastasis is characterized by high rates of mitosis and migration into distant tissues. Stathmin itself is regulated by kinases through phosphorylation of mainly 4 different serin sides. In this study, we investigated stathmin- and its kinases expression in native hematopoietic CD34+ stem cells (HSCs) from bone marrow (BM) in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated donors and leukemic CD34+ cells from patients with AML. Methods: Mononuclear cells were isolated by a standard Ficoll-Hypaque gradient separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. In comparative proteome analysis, we detected the protein expression of stathmin in mPBSCs, AML CD34+ cells, and in native HSCs from BM. We performed microarray-based gene expression profiles of these cells and focused on kinases regulating stathmin’s activity. Furthermore, we monitored stathmin and its relevant kinases by FACS analyses of the enriched cell fractions and by fluorescence microscopy of bone marrow smears and cytospins. Results: In this study, we have shown in comparative proteome analysis (Q-TOF-MS/MS) that stathmin is expressed in G-CSF mobilized hematopoietic stem cells for the first time and in AML cells. In microarray analysis we indentified up- and down-regulated kinases: MAPK, PAK1, PKC beta/zeta, MEKK3 and CDKs. Accordingly, we demonstrated in FACS analyses and in immunofluorescence microscopy the high intracellular expression of PKCzeta in AML cells and MEKK3 as well PAK1 in mPBSCs. Conclusions: Our findings show that G-CSF stimulates Stathmin expression in mPBSCs and plays a key role in migration into peripheral blood. Furthermore, we show the different expression of kinases acting on stathmin in mPBSCs and AML cells. Consequently, stathmin and its relevant kinases promise to become a future target in therapies of malignant processes. No significant financial relationships to disclose.

scholarly journals In vivo administration of stem cell factor to mice increases the absolute number of pluripotent hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 445-455 ◽  
Author(s):  
DM Bodine ◽  
NE Seidel ◽  
KM Zsebo ◽  
D Orlic
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Stem Cell Factor ◽  
Absolute Number ◽  
Hematopoietic Stem ◽  
The Absolute ◽  
In Vivo Administration

We have examined the effects of administration of stem cell-factor (SCF) on the number and distribution of pluripotent hematopoietic stem cells (PHSC) in normal mice. Using the competitive repopulation assay we found that in vivo administration of SCF increases the absolute number of PHSC per mouse threefold. The increased numbers of PHSC are found in the peripheral blood and spleen of the SCF-treated animals. The spleen and peripheral blood stem cells completely repopulated the erythroid, myeloid, and lymphoid lineages of irradiated or W/Wv hosts, similar to bone marrow PHSC. PHSC from the peripheral blood of SCF- treated mice have a lineage marker-negative, c-kit-positive phenotype that is indistinguishable from that of bone marrow PHSC. The increase in the absolute number of spleen PHSC is associated with efficient gene transfer to these cells without prior treatment with 5-fluorouracil. This is a US government work. There are no restrictions on its use.

In Vivo Selection of Wild-Type Hematopoietic Stem Cells in a Murine Model of Fanconi Anemia

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 2151-2158 ◽  
Author(s):  
Kevin P. Battaile ◽  
Raynard L. Bateman ◽  
Derik Mortimer ◽  
Jean Mulcahy ◽  
R. Keaney Rathbun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Serial Transplantation

Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.

scholarly journals Human embryonic stem cell–derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 2180-2183 ◽  
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Rachel L. Lewis ◽  
Xinghui Tian ◽  
Dan S. Kaufman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Xenograft Model ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Hematopoietic Activity

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.

Retroviral-Mediated Gene Transfer of CD18 into Hematopoietic Stem Cells in Dogs with Canine Leukocyte Adhesion Deficiency Reverses the Severe Deficiency Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3169-3169
Author(s):  
Mehreen Hai ◽  
Thomas R. Bauer ◽  
Yu-chen Gu ◽  
Laura M. Tuschong ◽  
Robert A. Sokolic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Leukocyte Adhesion ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Post Infusion ◽  
Low Levels

Abstract Background: Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific, large-animal model for the human disease leukocyte adhesion deficiency (LAD). Puppies with CLAD, like children with LAD, experience recurrent life-threatening bacterial infections due to the inability of their leukocytes to adhere and migrate to sites of infection. Mutations in the gene encoding the leukocyte integrin CD18 are responsible for both CLAD and LAD. Allogeneic bone marrow or hematopoietic stem cell transplantation is currently the only curative therapy for LAD. We recently reported the results of non-myeloablative allogeneic transplants in CLAD dogs and showed that very low levels of CD18+ donor-derived neutrophils (less than 300/microliter) were sufficient to reverse the CLAD disease phenotype. These results indicated that CLAD dogs may be amenable to treatment using gene therapy, where there are frequently low numbers of transduced cells. We report the results of retroviral- mediated transduction in autologous hematopoietic stem cells with the canine CD18 gene. Method: Bone marrow was harvested and CD34+ selected from four dogs with CLAD at approximately 3–4 months of age. The purified CD34+ cells were either used immediately or were frozen and subsequently thawed. Cells were pre-stimulated with cSCF, hFlt3-L, hTPO and cIL-6 for approximately 24 hours, then exposed to two rounds of supernatant from the retroviral vector PG13/MSCV-cCD18 for 24 hours each on recombinant fibronectin. At the end of the transduction, the cells were infused back into the animal that had been conditioned with 200 cGy total body irradiation. Post-transplant immunosuppression consisted of cyclosporine given at a dose of 30 mg/kg from day -1 to day 35, then 15 mg/kg from day 36 to day 60, and mycophenolate mofetil at a dose of 20 mg/kg from day 0 to day 28. Peripheral blood samples, as well as pus samples from one animal, were analyzed by flow cytometry at designated time points post-transplant. Results: The four dogs who received autologous, gene-corrected cells have been followed for 7–12 weeks post-infusion. The number of CD18+ CD34+ cells infused per dog ranged from 0.2 to 0.55 x 106 cells/kg. The post-infusion percentage of CD18+ neutrophils in each dog was 0.09%, 0.13%, 0.62% and 0.02% at 12, 10, 8 and 6 weeks respectively. Clinically all four treated CLAD dogs are alive with marked improvement of their CLAD disease. These dogs are now 6–7 months of age. These results contrast with those seen in untreated CLAD dogs who uniformly die or are euthanized within the first few months of life. The reversal of the severe CLAD phenotype despite the very low levels of CD18+ neutrophils in the peripheral blood is likely due to the selective egress of CD18+ neutrophils into the tissue since one treated CLAD dog who had less than 1% CD18+ neutrophils in the blood had nearly 10% CD18+ neutrophils in pus collected from an inflammatory dental lesion. Conclusion: These data suggest that a non-myeloablative conditioning regimen coupled with a minimal immunosuppressive regimen may enable sufficient CD18+ autologous gene-corrected cells to engraft and result in reversal of the severe CLAD phenotype.

Expression of a HoxC4 Retroviral Vector Mediates Expansion of Murine Hematopoietic Stem Cells with Normal Hematopoiesis in Transplanted Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2788-2788
Author(s):  
Lilia Stepanova ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

Abstract Homeobox (Hox) transcription factors are important regulators of hematopoietic cell proliferation and differentiation. Of them, HoxB4 is of particular interest because overexpression promotes rapid expansion of mouse hematopoietic stem cells (HSCs) without causing neoplastic transformation. Despite the effects of HoxB4 overexpression on HSCs, mice that are homozygous for HoxB4 gene deletion have only subtle defects in HSCs and progenitor cells. We hypothesized that other paralogs of HoxB4 may also be capable of inducing HSC expansion could thereby compensate for loss of HoxB4 function. To test this hypothesis, we have studied the effects of retroviral overexpression of a HoxC4 gene in murine progenitors and HSCs. The murine HoxC4 cDNA was cloned and inserted into an MSCV vector that co-expresses an IRES-YFP reporter gene. We transduced murine bone marrow cells with a MSCV-HoxC4-YFP vector and compared the secondary replating efficiency of myeloid colonies (CFU-Cs) to that seen using either a MSCV-HoxB4-GFP or an MSCV-GFP vector. This assay tests for progenitor cell self-renewal which is increased using HoxB4-expressing vectors. Cells transduced with the MSCV-HoxC4-YFP vector formed 20–40 times more secondary CFU-Cs than with cells transduced with the MSCV-GFP control vector. This increase in CFU-C replating efficiency was equivalent to that seen with the MSCV-HoxB4-IRES-GFP vector. To test the in vivo effects of the MSCV-HoxC4-YFP vector on self-renewal of HSCs, we transplanted lethally irradiated mice with a mixture of cells; 20% transduced with the MSCV-HoxC4-YFP vector and 80 % mock-transduced. Peripheral blood analysis of the transplanted recipients up to 28 weeks post-transplantation showed that the percentage of cells transduced with the MSCV-HoxC4-YFP vector was 70–85% in both lymphoid and myeloid cells in the peripheral blood. A similar degree of chimerism was noted in concurrent controls using the MSCV-HoxB4-GFP vector. In contrast, the percentages of peripheral blood cells transduced with the MSCV-GFP vector was only 15–25%, paralleling the input ratios of transplanted cells. Secondary transplantation experiments showed stable levels of chimerism in both HoxC4 and HoxB4 groups, indicating that the expansion seen with the MSCV-HoxC4-YFP vector occurred at the HSC level. These results indicate that retroviral-mediated expression of HoxC4, like HoxB4, can cause significant expansion of HSCs in vivo. Because several other Hox genes can cause hematopoietic abnormalities and leukemia when expressed from a retroviral vector, we transplanted lethally irradiated mice with 4x106 cells that were transduced with the MSCV-HoxC4-YFP vector and monitored the animals for survival and complete blood counts. Now, at 33 weeks post transplantation, no tumor formation was observed in mice expressing either the HoxB4 or the HoxC4 vector, and peripheral blood counts have remained normal. Our results show that retroviral overexpression of HoxC4 can induce a significant expansion of the HSCs in vivo, and suggest that expression of HoxC4 may compensate for the loss of HoxB4 in knockout mice. We are currently analyzing the effects of HoxA4 and HoxD4 to determine if they share the same functional characteristics, and are also determining whether HoxB4 and HoxC4 are modulating the same downstream genes using microarray analysis of transduced murine bone marrow cells.

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