In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 466-466
Author(s):  
Jennifer L Gori ◽  
Jason M Butler ◽  
Devikha Chandrasekaran ◽  
Brian C Beard ◽  
Daniel J Nolan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Nonhuman Primate ◽  
Fold Increase ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model. Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.

scholarly journals Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1497-1504 ◽  
Author(s):  
VF Quesniaux ◽  
GJ Graham ◽  
I Pragnell ◽  
D Donaldson ◽  
SD Wolpe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Hematopoietic Progenitors ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

Exhaustion of Donor Hematopoietic Stem Cells in Lethally-Irradiated Hosts Can Be Sbstantially Mitigated by Targeting p18INK4C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1200-1200
Author(s):  
Hui Yu ◽  
Youzhong Yuan ◽  
Xianmin Song ◽  
Feng Xu ◽  
Hongmei Shen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Total Period ◽  
Over Expression

Abstract Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

Long-Term In Vivo Expression from a Self-Inactivating Lentiviral Vector Flanked by a Chromatin Insulator Element.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1289-1289
Author(s):  
Ping Xia ◽  
Richard Emmanuel ◽  
Kuo Isabel ◽  
Malik Punam
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vector ◽  
Gfp Expression ◽  
Hematopoietic Stem ◽  
Insulator Element ◽  
Gfp Fluorescence

Abstract We have previously shown that self-inactivating lentiviral vectors infect quiescent hematopoietic stem cells (HSC), express long-term, resist proviral silencing in HSC and express in a lineage specific manner. However, their random integration into the host chromosome results in variable expression, dependent upon the flanking host chromatin (Mohamedali et al, Mol. Therapy 2004). Moreover, the recent occurrence of leukemogenesis from activation of a cellular oncogene by the viral enhancer elements calls for safer vector designs, with expression cassettes that can be ‘insulated’ from flanking cellular genes. We analyzed the role of the chicken β-globin locus hypersensitive site 4 insulator element (cHS4) in a self-inactivating (SIN) lentiviral vector in the RBC progeny of hematopoietic stem cells (HSC) in long term in vivo. We designed an erythroid-specific SIN-lentiviral vector I8HKGW, expressing GFP driven by the human ankyrin gene promoter and containing two erythroid-specific enhancer elements and compared it to an analogous vector I8HKGW-I, where the cHS4 insulator was inserted in the SIN deletion to flank the I8HKGW expression cassette at both ends upon integration. First, murine erythroleukemia (MEL) cells were transduced at &lt;5% transduction efficiency and GFP+ cells were sorted to generate clones. Single copy MEL clones showed no difference in the mean GFP fluorescence intensity (MFI) between the I8HKGW+ and the I8HKGW-I+ MEL clones. However, there was a reduction in the chromatin position effect variegation (PEV), reflected by reduced coefficient of variation of GFP expression (CV) in I8HKGW-I clones (n=115; P&lt;0.01), similar to in vitro results reported by Ramezani et al (Blood 2003). Next, we examined for expression and PEV in the RBC progeny of HSC, using the secondary murine bone marrow transplant model. Lethally irradiated C57Bl6 (CD45.2) mice were transplanted with I8HKGW and I8HKGW-I transduced B6SJL (CD45.1) Sca+Lin- HSC and 4–6 months later, secondary transplants were performed. Mice were analyzed 3–4 months following secondary transplants (n=43). While expression from both I8HKGW and I8HKGW-I vectors appeared similar in secondary mice (46±6.0% vs. 48±3.6% GFP+ RBC; MFI 31±2.6 vs. 29±1.4), there were 0.37 vs. 0.22 copies/cell in I8HKGW and I8HKGW-I secondary recipients, respectively (n=43), suggesting that the probability of GFP expression from I8HKGW-I vectors was superior when equalized for vector copy. The CV of GFP fluorescence in RBC was remarkably reduced to 55±1.7 in I8HKGW-I vs. 196±32 in I8HKGW RBC (P&lt;0.001). We therefore, analyzed these data at a clonal level in secondary CFU-S and tertiary CFU-S. The I8HKGW-I secondary CFU-S had more GFP+ cells (32.4±4.4%) vs. I8HKGW CFU-S (8.1±1.2%, n=143, P&lt;0.1x10E-11). Similarly, I8HKGW-I tertiary CFU-S also had more GFP+ cells (25±1.8%) vs. I8HKGW CFU-S (6.3±0.8%, n=166, P&lt;0.3x10E-10). We also plated bone marrow from secondary mice in methylcellulose and analyzed GFP expression in individual BFU-E. The I8HKGW-I tertiary BFU-E had more GFP+ cells (28±3.9%) vs. I8HKGW BFU-E (11±5%, n=50, P&lt;0.03) with significantly reduced CV (67 vs 125, n=50, P&lt;6.6X10E-7). Taken together, the ‘insulated’ erythroid-specific SIN-lentiviral vector increased the probability of expression of proviral integrants and reduced PEV in vivo, resulting in higher, consistent transgene expression in the erythroid cell progeny of HSC. In addition, the enhancer blocking effect of the cHS4, although not tested here, would further improve bio-safety of these vectors for gene therapy for RBC disorders.

Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 796-803 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Aylin Bonifacino ◽  
Taihe Lu ◽  
Brian Agricola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Drug Treatment ◽  
Blood Cells ◽  
Rhesus Macaques ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

Abstract One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

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