Engraftment of human hematopoietic precursor cells with secondary transfer potential in SCID-hu mice

Abstract Human fetal bone fragments implanted subcutaneously in immunodeficient (SCID) mice maintain active human hematopoiesis. In this study, we show that this human hematopoietic microenvironment supports the engraftment and differentiation of HLA-mismatched, CD34+ primitive hematopoietic progenitor cells isolated from fetal and adult human bone marrow (BM). The BM CD34+ cells were depleted of CD2, CD14, CD15, CD16, glycophorin A, and CD19 lineage-committed cells (CD34+Lin-). Donor cell engraftment was manifested by the presence of B (CD19+) and myeloid (CD33+) cells of donor HLA phenotype. Successful engraftment was observed as early as 4 weeks after fetal BM donor cell injection and sustained for at least 12 weeks, with engraftment success rates of 100% (11/11 grafts) and 92% (11/12 grafts) at 8 and 12 weeks, respectively. Mixed BM chimerism of donor and endogenous cells was consistently observed in SCID-hu bones successfully engrafted with HLA-mismatched CD34+Lin- donor cells. Preconditioning of the SCID-hu bone with a single dose of sublethal (350 rad) whole body irradiation (WBI) immediately before cell injection enhanced the repopulation of the bone grafts with donor cells and, in some instances, resulted in complete repopulation. After WBI, as few as 500 fetal bone marrow CD34+Lin- cells injected in the human bone grafts resulted in donor-derived hematopoiesis. Donor progenitor cells recovered from the SCID-hu bone grafts 8 weeks postinjection had the capacity to repopulate secondary groups of HLA-disparate fetal human bones in SCID-hu mice with B and myeloid cells as well as CD34+ cells in some recipients. In addition, these cells repopulated fetal human thymus fragments in SCID mice with donor thymocytes including immature CD4+CD8+ and mature CD4+CD8- as well as CD4-CD8+ subsets. These results indicate that the fetal human bone implants of SCID-hu mice can support the maintenance of a cell population that has both multilineage potential and repopulating potential for periods of time as long as 16 weeks. The SCID-hu bone model consistently supported the engraftment of both fetal and adult CD34+Lin- cells without the administration of exogenous human cytokines to these animals. This model is currently being used to permit the isolation and characterization of candidate human hematopoietic stem cells (HSCs) and provide important information critical for human HSC therapy in humans.

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Engraftment of human hematopoietic precursor cells with secondary transfer potential in SCID-hu mice

Blood ◽

10.1182/blood.v84.8.2497.bloodjournal8482497 ◽

1994 ◽

Vol 84 (8) ◽

pp. 2497-2505 ◽

Cited By ~ 3

Author(s):

BP Chen ◽

A Galy ◽

S Kyoizumi ◽

R Namikawa ◽

J Scarborough ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Donor Cell ◽

Bone Grafts ◽

Scid Mice ◽

Human Bone ◽

Cell Injection ◽

Fetal Bone ◽

Donor Cells ◽

Cd34 Cells

Human fetal bone fragments implanted subcutaneously in immunodeficient (SCID) mice maintain active human hematopoiesis. In this study, we show that this human hematopoietic microenvironment supports the engraftment and differentiation of HLA-mismatched, CD34+ primitive hematopoietic progenitor cells isolated from fetal and adult human bone marrow (BM). The BM CD34+ cells were depleted of CD2, CD14, CD15, CD16, glycophorin A, and CD19 lineage-committed cells (CD34+Lin-). Donor cell engraftment was manifested by the presence of B (CD19+) and myeloid (CD33+) cells of donor HLA phenotype. Successful engraftment was observed as early as 4 weeks after fetal BM donor cell injection and sustained for at least 12 weeks, with engraftment success rates of 100% (11/11 grafts) and 92% (11/12 grafts) at 8 and 12 weeks, respectively. Mixed BM chimerism of donor and endogenous cells was consistently observed in SCID-hu bones successfully engrafted with HLA-mismatched CD34+Lin- donor cells. Preconditioning of the SCID-hu bone with a single dose of sublethal (350 rad) whole body irradiation (WBI) immediately before cell injection enhanced the repopulation of the bone grafts with donor cells and, in some instances, resulted in complete repopulation. After WBI, as few as 500 fetal bone marrow CD34+Lin- cells injected in the human bone grafts resulted in donor-derived hematopoiesis. Donor progenitor cells recovered from the SCID-hu bone grafts 8 weeks postinjection had the capacity to repopulate secondary groups of HLA-disparate fetal human bones in SCID-hu mice with B and myeloid cells as well as CD34+ cells in some recipients. In addition, these cells repopulated fetal human thymus fragments in SCID mice with donor thymocytes including immature CD4+CD8+ and mature CD4+CD8- as well as CD4-CD8+ subsets. These results indicate that the fetal human bone implants of SCID-hu mice can support the maintenance of a cell population that has both multilineage potential and repopulating potential for periods of time as long as 16 weeks. The SCID-hu bone model consistently supported the engraftment of both fetal and adult CD34+Lin- cells without the administration of exogenous human cytokines to these animals. This model is currently being used to permit the isolation and characterization of candidate human hematopoietic stem cells (HSCs) and provide important information critical for human HSC therapy in humans.

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Protein kinase C-α isoform is involved in erythropoietin-induced erythroid differentiation of CD34+ progenitor cells from human bone marrow

Blood ◽

10.1182/blood.v95.2.510 ◽

2000 ◽

Vol 95 (2) ◽

pp. 510-518 ◽

Cited By ~ 50

Author(s):

June Helen Myklebust ◽

Erlend B. Smeland ◽

Dag Josefsen ◽

Mouldy Sioud

Keyword(s):

Bone Marrow ◽

Protein Kinase C ◽

Protein Kinase ◽

Progenitor Cells ◽

Erythroid Differentiation ◽

Human Bone ◽

Human Bone Marrow ◽

Kinase C ◽

Pkc Isoforms ◽

Cd34 Cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases involved in many cellular responses. Although the analysis of PKC activity in many systems has provided crucial insights to its biologic function, the precise role of different isoforms on the differentiation of normal hematopoietic progenitor cells into the various lineages remains to be investigated. The authors have assessed the state of activation and protein expression of PKC isoforms after cytokine stimulation of CD34+ progenitor cells from human bone marrow. Freshly isolated CD34+ cells were found to express PKC-, PKC-β2, and PKC-ɛ, whereas PKC-δ, PKC-γ, and PKC-ζ were not detected. Treatment with erythropoietin (EPO) or with EPO and stem cell factor (SCF) induced a predominantly erythroid differentiation of CD34+ cells that was accompanied by the up-regulation of PKC- and PKC-β2 protein levels (11.8- and 2.5-fold, respectively) compared with cells cultured in medium. Stimulation with EPO also resulted in the nuclear translocation of PKC- and PKC-β2 isoforms. Notably, none of the PKC isoforms tested were detectable in CD34+ cells induced to myeloid differentiation by G-CSF and SCF stimulation. The PKC inhibitors staurosporine and calphostin C prevented EPO-induced erythroid differentiation. Down-regulation of the PKC-, PKC-β2, and PKC-ɛ expression by TPA pretreatment, or the down-regulation of PKC- with a specific ribozyme, also inhibited the EPO-induced erythroid differentiation of CD34+ cells. No effect was seen with PKC-β2–specific ribozymes. Taken together, these findings point to a novel role for the PKC- isoform in mediating EPO-induced erythroid differentiation of the CD34+ progenitor cells from human bone marrow.

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Involvement of the Retinoblastoma Protein in Monocytic and Neutrophilic Lineage Commitment of Human Bone Marrow Progenitor Cells

Blood ◽

10.1182/blood.v94.6.1971.418k34_1971_1978 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1971-1978 ◽

Cited By ~ 3

Author(s):

Gösta Bergh ◽

Mats Ehinger ◽

Inge Olsson ◽

Sten Eirik W. Jacobsen ◽

Urban Gullberg

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Differentiation ◽

Progenitor Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Lineage Commitment ◽

Flt3 Ligand ◽

Monocytic Differentiation ◽

Cd34 Cells

The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G1 arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit–granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34+ cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of -RB to liquid cultures of CD34+ cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34+ cells mediated by -RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34+ cells incubated with oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

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Involvement of the Retinoblastoma Protein in Monocytic and Neutrophilic Lineage Commitment of Human Bone Marrow Progenitor Cells

Blood ◽

10.1182/blood.v94.6.1971 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1971-1978 ◽

Cited By ~ 24

Author(s):

Gösta Bergh ◽

Mats Ehinger ◽

Inge Olsson ◽

Sten Eirik W. Jacobsen ◽

Urban Gullberg

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Differentiation ◽

Progenitor Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Lineage Commitment ◽

Flt3 Ligand ◽

Monocytic Differentiation ◽

Cd34 Cells

Abstract The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G1 arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit–granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34+ cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of -RB to liquid cultures of CD34+ cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34+ cells mediated by -RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34+ cells incubated with oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

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Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes

Blood ◽

10.1182/blood.v91.10.3693 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3693-3701 ◽

Cited By ~ 13

Author(s):

Ponnazhagan Veena ◽

Christie M. Traycoff ◽

David A. Williams ◽

Jon McMahel ◽

Susan Rice ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Progenitor Cells ◽

Cultured Cells ◽

Retroviral Vector ◽

Human Bone ◽

Human Bone Marrow ◽

Transduction Efficiency ◽

Cd34 Cells

Abstract Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

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Arsenic Primes Human Bone Marrow CD34+ Cells for Erythroid Differentiation

Bioinorganic Chemistry and Applications ◽

10.1155/2015/751013 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Yuanyuan Zhang ◽

Shasha Wang ◽

Chunyan Chen ◽

Xiao Wu ◽

Qunye Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Arsenic Trioxide ◽

Erythroid Differentiation ◽

Human Bone ◽

Human Bone Marrow ◽

Hematopoietic Progenitor Cells ◽

Therapeutic Effects ◽

Hematopoietic Progenitor ◽

Cd34 Cells

Arsenic trioxide exhibits therapeutic effects on certain blood malignancies, at least partly by modulating cell differentiation. Previousin vitrostudies in human hematopoietic progenitor cells have suggested that arsenic may inhibit erythroid differentiation. However, these effects were all observed in the presence of arsenic compounds, while the concomitant cytostatic and cytotoxic actions of arsenic might mask a prodifferentiating activity. To eliminate the potential impacts of the cytostatic and cytotoxic actions of arsenic, we adopted a novel protocol by pretreating human bone marrow CD34+ cells with a low, noncytotoxic concentration of arsenic trioxide, followed by assaying the colony forming activities in the absence of the arsenic compound. Bone marrow specimens were obtained from chronic myeloid leukemia patients who achieved complete cytogenetic remission. CD34+ cells were isolated by magnetic-activated cell sorting. We discovered that arsenic trioxide enhanced the erythroid colony forming activity, which was accompanied by a decrease in the granulomonocytic differentiation function. Moreover, in erythroleukemic K562 cells, we showed that arsenic trioxide inhibited erythrocyte maturation, suggesting that arsenic might have biphasic effects on erythropoiesis. In conclusion, our data provided the first evidence showing that arsenic trioxide could prime human hematopoietic progenitor cells for enhanced erythroid differentiation.

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Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes

Blood ◽

10.1182/blood.v91.10.3693.3693_3693_3701 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3693-3701

Author(s):

Ponnazhagan Veena ◽

Christie M. Traycoff ◽

David A. Williams ◽

Jon McMahel ◽

Susan Rice ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Progenitor Cells ◽

Cultured Cells ◽

Retroviral Vector ◽

Human Bone ◽

Human Bone Marrow ◽

Transduction Efficiency ◽

Cd34 Cells

Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

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