Generation of Immunodeficient Mice Bearing Human Immune Systems by the Engraftment of Hematopoietic Stem Cells v1 (protocols.io.bu9nnz5e)

Abstract Repopulation of immunodeficient mice remains the primary method to assay human hematopoietic stem cells (HSCs). Here we report that female NOD/SCID/IL-2Rgc-null mice are far superior in detecting human HSCs (Lin−CD34+CD38−CD90+CD45RA−) compared with male recipients. When multiple HSCs were transplanted, female recipients displayed a trend (1.4-fold) toward higher levels of human chimerism (female vs male: injected femur, 44.4 ± 9.3 vs 32.2 ± 6.2; n = 12 females, n = 24 males; P = .1). Strikingly, this effect was dramatically amplified at limiting cell doses where female recipients had an approximately 11-fold higher chimerism from single HSCs (female vs male: injected femur, 8.1 ± 2.7 vs 0.7 ± 0.7; n = 28 females, n = 20 males; P < .001). Secondary transplantations from primary recipients indicate that females more efficiently support the self-renewal of human HSCs. Therefore, sex-associated factors play a pivotal role in the survival, proliferation, and self-renewal of human HSCs in the xenograft model, and recipient sex must be carefully monitored in the future design of experiments requiring human HSC assays.

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Advances in Human Immune System Mouse Models for Personalized Treg-Based Immunotherapies

Frontiers in Immunology ◽

10.3389/fimmu.2021.643544 ◽

2021 ◽

Vol 12 ◽

Author(s):

Isabelle Serr ◽

Maria Kral ◽

Martin G. Scherm ◽

Carolin Daniel

Keyword(s):

Stem Cells ◽

Immune System ◽

Personalized Medicine ◽

Peripheral Blood ◽

Human Immune System ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Central Target ◽

Human Immune ◽

Cancer Immunotherapies

Immunodeficient mice engrafted with a functional human immune system [Human immune system (HIS) mice] have paved the way to major advances for personalized medicine and translation of immune-based therapies. One prerequisite for advancing personalized medicine is modeling the immune system of individuals or disease groups in a preclinical setting. HIS mice engrafted with peripheral blood mononuclear cells have provided fundamental insights in underlying mechanisms guiding immune activation vs. regulation in several diseases including cancer. However, the development of Graft-vs.-host disease restrains relevant long-term studies in HIS mice. Alternatively, engraftment with hematopoietic stem cells (HSCs) enables mimicking different disease stages, however, low frequencies of HSCs in peripheral blood of adults impede engraftment efficacy. One possibility to overcome those limitations is the use of patient-derived induced pluripotent stem cells (iPSCs) reprogrammed into HSCs, a challenging process which has recently seen major advances. Personalized HIS mice bridge research in mice and human diseases thereby facilitating the translation of immunomodulatory therapies. Regulatory T cells (Tregs) are important mediators of immune suppression and thereby contribute to tumor immune evasion, which has made them a central target for cancer immunotherapies. Importantly, studying Tregs in the human immune system in vivo in HIS mice will help to determine requirements for efficient Treg-targeting. In this review article, we discuss advances on personalized HIS models using reprogrammed iPSCs and review the use of HIS mice to study requirements for efficient targeting of human Tregs for personalized cancer immunotherapies.

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Human Hematopoietic Stem Cells Can Survive In Vitro for Several Months

Advances in Hematology ◽

10.1155/2009/936761 ◽

2009 ◽

Vol 2009 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Taro Ishigaki ◽

Kazuhiro Sudo ◽

Takashi Hiroyama ◽

Kenichi Miharada ◽

Haruhiko Ninomiya ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cultured Cells ◽

Es Cells ◽

Embryonic Stem ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Cell Sample

We previously reported that long-lasting in vitro hematopoiesis could be achieved using the cells differentiated from primate embryonic stem (ES) cells. Thus, we speculated that hematopoietic stem cells differentiated from ES cells could sustain long-lasting in vitro hematopoiesis. To test this hypothesis, we investigated whether human hematopoietic stem cells could similarly sustain long-lasting in vitro hematopoiesis in the same culture system. Although the results varied between experiments, presumably due to differences in the quality of each hematopoietic stem cell sample, long-lasting in vitro hematopoiesis was observed to last up to nine months. Furthermore, an in vivo analysis in which cultured cells were transplanted into immunodeficient mice indicated that even after several months of culture, hematopoietic stem cells were still present in the cultured cells. To the best of our knowledge, this is the first report to show that human hematopoietic stem cells can survive in vitro for several months.

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196: Ability of anti c-Kit targeting monoclonal antibody ACK-2 to target hematopoietic stem cells, and facilitate engraftment of human CD34+ engraftment and hematolymphoid development in immunodeficient mice

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2006.12.200 ◽

2007 ◽

Vol 13 (2) ◽

pp. 72

Author(s):

D.L. Kraft ◽

A. Czechowicz ◽

D. Bhattacharya ◽

I.L. Weissman

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Human Cd34

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Sustained Gene Expression in Retrovirally Transduced, Engrafting Human Hematopoietic Stem Cells and Their Lympho-Myeloid Progeny

Blood ◽

10.1182/blood.v92.1.83.413k09_83_92 ◽

1998 ◽

Vol 92 (1) ◽

pp. 83-92 ◽

Cited By ~ 62

Author(s):

Linzhao Cheng ◽

Changchun Du ◽

Catherine Lavau ◽

Shirley Chen ◽

Jie Tong ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Growth Factor Receptor ◽

Retroviral Vector ◽

Human Bone ◽

Differentiation Markers ◽

Hematopoietic Stem ◽

Immunodeficient Mice

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34+ cells lacking lineage differentiation markers (CD34+Lin−) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34+ cell population retained the Lin− phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34+Lin−NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34+Lin−NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% ± 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.

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1063. HIV Mediated Expression of Brutons Tyrosine Kinase in Hematopoietic Stem Cells Promotes B Cells Development but Not Restore Immunoglobulin Production in X-Linked Immunodeficient Mice

Molecular Therapy ◽

10.1016/s1525-0016(16)41505-4 ◽

2003 ◽

Vol 7 (5) ◽

pp. S410 ◽

Cited By ~ 1

Keyword(s):

Stem Cells ◽

B Cells ◽

Tyrosine Kinase ◽

Hematopoietic Stem Cells ◽

Immunoglobulin Production ◽

Hematopoietic Stem ◽

Immunodeficient Mice

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Beta-Globin Gene Targeting in Human Hematopoietic Stem Cells Using Cas9 Ribonucleoprotein and rAAV6

Blood ◽

10.1182/blood.v128.22.2310.2310 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2310-2310

Author(s):

Daniel Patrick Dever ◽

Matthew Porteus

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Reporter Gene ◽

Ex Vivo ◽

Globin Gene ◽

Gene Correction ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Nucleotide Mutation

Abstract The b -hemoglobinopathies including sickle cell disease (SCD) and b -thalassemia ( b -thal) affect millions of people worldwide . SCD and b -thal are caused by mutations in the b -globin gene (HBB) resulting in either abnormal sickling or severely reduced protein production, respectively. A curative strategy for the b -hemoglobinopathies would be ex vivo HBB gene correction in patient-derived hematopoietic stem and progenitor cells (HSPCs) followed by autologous hematopoietic stem cell transplantation (auto-HSCT). We report the first CRISPR/Cas9 gene-editing platform for achieving homologous recombination (HR) at the HBB gene in long-term repopulating HSCs derived from mobilized peripheral blood. We combine electroporation of Cas9 protein complexed with chemically modified sgRNAs and delivery of a HR donor by recombinant adeno-associated viral vectors, serotype 6 (rAAV6). Notably, by including a reporter gene in the HR donor, we are able to identify and purify a population of HSPCs with >90% of cells having targeted integration at the HBB gene. These cells can be identified because HR-mediated integration causes the reporter gene to be expressed at log-fold higher levels than the non-integrated reporter. When transplanted into immunodeficient mice, the purified population gives rise to engraftment of HBB-edited human cells in primary and secondary recipients, confirming the presence of long-term repopulating hematopoietic stem cells (LT-HSCs). Importantly, we show efficient correction of the SCD-causing E6V mutation in SCD patient-derived CD34+ HSPCs by either editing the nucleotide mutation or knocking in an anti-sickling b -globin cDNA. Edited SCD CD34+ cells were shown to express adult b -globin (HbA) mRNA after HSPCs were differentiated into erythrocytes in vitro, confirming intact transcriptional regulation of the edited HBB allele. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting HSCs for HR at the HBB locus to advance the development of next generation therapies for b -hemoglobinopathies. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Equity Ownership.

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