Obtain human cord blood, bone marrow or mobilized peripheral blood cells after informed consent in conformity with. Hematopoietic stem cell protocols hematopoietic stem cell protocols theilgaard. The third main topic is the manipulation of stem cells by in vitro culture and genetic modification using. Bcca protocol summary for myeloablative conditioning. Hematopoietic stem cell protocols methods in molecular. Hematopoietic stem cell transplant hsct the term bmt has been change to hematopoietic stem cell transplant hsct since the introduction of peripheral blood and umbilical cord stem cells hscts become an established standard treatment for malignant and nonmalignant blood disorders.
We collect hematopoietic progenitor stem cells in a minimally manipulated fashion as defined by the foundation for the accreditation of hematopoietic cell therapy fahct with a minimal cell dose of at least 2. Hematopoietic stem cells residing within mouse bonemarrow were first discovered by till and mcculloch in the 1960s and indicated that 1 hematopoiesis could be studied as a quantitative science, 2 clonal hematopoietic cells in the marrow existed that could give rise to mixed myeloid progeny. Use fbs directly without the heat inactivation step for op9 culture, hematopoietic differentiation, expansion of myeloid progenitors, and the generation of mature myelomonocytic cells from human pluripotent stem cells. Lysosomal cross correction by hematopoietic stem cell. Hscs divide to form more hsc selfgeneration or to form cells committed either to lymphocyte formation common lymphoid progenitors, clps or to the formation of myeloid cells common myeloid progenitors, cmps. Hematopoietic stem cells hscs can be found in bone marrow, peripheral blood, and umbilical cord blood, and are important in studies of blood disorders and bloodrelated malignancies. Bunting isolation of quiescent murine hematopoietic stem cells by homing properties tarja a. Updating and building upon previous editions, hematopoietic stem cell protocols, third edition provides uptodate protocols from leading stem cell. Traditional and advanced cell cultures in hematopoietic stem cell. Since hematopoietic stem cells are an extremely rare population in the marrow approximately 110 000 bone marrow cells, the identification of hematopoietic stem cells in the marrow is still challenging.
Although hematopoietic stem cell hsc therapy for hematological diseases can lead to a good outcome from the clinical point of view, the limited number of ideal donors, the comorbidity of patients and the increasing number of elderly patients may limit the application of this therapy. Hematopoietic stem cell transplantation hsct can be used to treat malignant and nonmalignant diseases. Here, we present the most complete analysis of the transcriptional program of definitive hsc ontogeny to date, gleaned from rigorously characterized hematopoietic stem and progenitor cell hspc populations isolated from over 2,500 murine embryos and adult mice. Allogeneic hematopoietic stem cell transplantation. Conditioning therapy with intravenous busulfan and cyclophosphamide iv bucy2 for hematologic malignancies prior to allogeneic stem cell transplantation. Hscs give rise to all mature blood and immune cells. Abstract the unit describes functional assays for the quantification of mouse hematopoietic stem cells and progenitor cells. Hematopoietic stem cell transplants, including peripheral blood, bone marrow, and cord blood transplants are used most often to treat cancers affecting the blood or immune system. The younger the stem cell source, the better this is for recipients, especially in cases in which children require a stem cell transplant 42,63. Hematopoietic stem cell protocols, european journal of. Hematopoietic stem cell expansion medium sigmaaldrich. Protocols for hematopoietic stem cell expansion from. Hematopoietic stem cell characterization and isolation ncbi. Hematopoietic stem cell culture protocols sigmaaldrich.
Hematopoietic stem cell transplant introduction hematopoietic stem cell transplants, including peripheral blood, bone marrow, and cord blood transplants are used most often to treat cancers affecting the blood or immune system. Functional mouse hematopoietic stem cells hscs are expanded 236 to 899fold ex vivo using a fully defined albuminfree culture system. A team of leading researchers supply this volume with indepth, readily reproducible methods for effective characterization of hsc and their developmental potential. A second edition might be anticipated to include chapters on gene microarray. A novel target to optimize hematopoietic stem cell transplantation protocols in hematological malignancies and rare genetic disorders stefania crippa 1, ludovica santi 1, roberto bosotti 1, giulia porro 1 and maria ester bernardo 1,2, 1 san ra aeletelethon institute for gene therapy srtiget, san ra aele scienti. Hematopoietic stem cells hscs thermo fisher scientific.
Current protocols in stem cell biology wiley online library. Very low risk of transmitting clinically important infections through latent viruses, such as the cytomegalovirus cmv and the epstein barr virus 61,62. These techniques for studying a variety of stem cell topics, including blood formation, bloodbased disease, and blood. Analysis and isolation of stem cells using flow cytometry. Hematopoietic stem cells hscs are tissuespecific stem cells that exhibit remarkable selfrenewal capacity and are responsible for the lifelong mainte nance of. Hematopoietic stem cell an overview sciencedirect topics. Hematopoietic stem cell hsc research took hold in the 1950s with the demonstration that intravenously injected bone marrow cells can rescue irradiated mice from lethality by reestablishing blood cell production. Hematopoietic stem cell protocols hematopoietic stem cell protocols. The approaches employ molecular and cell biology techniques, along with animal model systems, and include hematopoietic stem cell expansion protocols and methods for the purification and genetic modification of stem cells. Enrichment of hematopoietic stem cells with slam and lsk markers for the detection of hematopoietic stem cell function in normal and trp53 null mice. Hematopoietic stem cells have an enormous clinical importance for bone marrow transplantation, hematopoietic gene therapy, solid organ transplantation, and somatic tissue regeneration. Hematopoietic stem cell transplantation for nonmalignant. Mm9620 to alert providers that cms has expanded national coverage of allogeneic hsct in an.
Hematopoietic stem cell phenotyping cells of the hematopoietic system are well characterized with respect to surface marker expression, which is often used to isolate and characterize subsets of cells during hematopoiesis. Allogeneic t cells that express an anticd19 chimeric antigen receptor induce remissions of b cell malignancies that progress after allogeneic hematopoietic stem cell transplantation without causing graftversus host disease clinical trial of allogeneic t cells genetically engineered to express a chimeric antigen receptor car targeting. Analysis of hematopoietic stem progenitor cell metabolism. This therapeutic modality is based on using immunoablation followed by reinfusion of hematopoietic progenitor cells to regenerate naive tlymphocytes. The hematopoietic stem cell hsc field has rapidly grown in the past several years as new technologies have been developed and the older tried and true methods have been used in new ways. Hscs can be generated from induced pluripotent stem cells ipscs, which requires the understanding of the. Updating and building upon previous editions, hematopoietic stem cell protocols, third edition provides uptodate protocols from leading stem cell researchers. It may be autologous the patients own stem cells are used, allogeneic the stem cells come from a donor or syngeneic from an identical twin. Hematopoietic stem cells hscs are rare 1 per 10 5 bonemarrow cells, small mononuclear cells that tend to be noncycling or to have long cell cycles. Since 2015, our institution has utilized a secondary chamber mononuclear cell mnc protocol on the spectra optia apheresis system. Pdf protocols for hematopoietic stem cell expansion from. Mesenchymal stromal cells mscs are crucial elements in the bone marrow bm niche where they provide physical support and secrete soluble factors to control and maintain hematopoietic stem progenitor cells hspcs. Hematopoietic stem cells hscs are defined by the capabilities of.
Hematopoietic stem cells from induced pluripotent stem. Knockout of ptpn21 results in decreased cellular stiffness, enhanced deformability and motility, stem and progenitor cell egress, and defective hematopoietic reconstitution capabilities. Major advances in the isolation of hsc and progenitor subsets have been sparked by the gene expression array. Sharkis mobilization of hematopoietic stem and progenitor cells in mice simon n. Longterm ex vivo expansion of mouse hematopoietic stem. Ptpn21 controls hematopoietic stem cell homeostasis and. Hematopoietic stem cells macrophages tunneling nanotubes lysosomal cross. Characterization of mouse hematopoietic stem and progenitor cells. The transcriptional landscape of hematopoietic stem cell. Hematopoietic stem cells hscs surface during embryogenesis leading to the genesis of the hematopoietic system, which is vital for immune function, homeostasis balance, and inflammatory responses in the human body. Hematopoietic stem cells an overview sciencedirect topics. Hematopoietic stem cell protocols bone marrow transplantation. The purification strategies described above recognize a rare subset of cells. Video articles in jove about hematopoietic stem cells include directed differentiation of primitive and definitive hematopoietic progenitors from human pluripotent stem cells, clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche coculture, isolation of murine embryonic hemogenic endothelial cells.
The longterm goal of my research is to identify novel strategies to ameliorate aginginduced decline in hematopoietic stem cell hsc function. In hematopoietic stem cell protocols, a group of leading stemcell investigators describe in detail the key laboratory methods for investigating this critically important cell type. Hscs can be found in adult bone marrow, peripheral blood, and umbilical cord blood. Hematopoietic stem cells hscs are multipotent, selfrenewing progenitor cells that develop from mesodermal hemangioblast cells. Ping zhou, sarah hohm, ben capoccia, louisa wirthlin, david hess, dan link et al.
Exact numbers depend on the assay used as well as on the genetic background studied. This indepth volume presents a clear view of the landscape of assays available to the stem cell researcher working in the growing hematopoietic stem cell hsc field. Hematopoietic stem cells hscs, the source of these hematopoietic cells, are currently a focus area in. Hematopoiesis is the process of blood cell formation, which initiates from hematopoietic stemprogenitor cells hspcs and is responsible for the generation of all adult blood. Ptpn21, a protein tyrosine phosphatase highly expressed in hscs, plays an important role in maintaining cellular mechanics and hsc retention in the niche. This indepth volume presents a clear view of the landscape of assays available to the stem cell researcher. Introduction hematopoietic stem cells hscs are primitive cells capable of both self renewal and producing progenitor cells that can differentiate into all the cells of the hematopoietic system figure 1.
Updating and building upon the thorough first editions foundation, hematopoietic stem cell protocols, second edition provides uptodate protocols developed in the hsc field. In this respect, hematopoietic stem cells differ from other types of stem cells, such as embryonic stem cells, which can undergo multiple cell divisions in culture while still retaining their stem cell properties. Hematopoietic stem and progenitor cells can be cultured under defined conditions designed either to promote selfrenewal and increase the number of primitive cells, or to promote lineage specific expansion and differentiation into mature blood cell types. Peripheral hematopoietic stem cell hsc collections are needed for autologous hematopoietic stem cell transplantation hsct. All differentiated blood cells from the lymphoid and myeloid lineages arise from hscs. Autologous stem cells come from the person who will be. Understanding the journey of human hematopoietic stem cell. Hematopoietic stem cells, niches and differentiati. Both types of cell products are being explored for their utility as cell therapeutics and. Given their role in the bm niche and hspc support, mscs have been employed in the clinical setting to expand exvivo hspcs, as well as to facilitate hspc engraftment in vivo. Hematopoietic stem cell transplantation hematopoietic stem cell a progenitor cell in the bone marrow that can replicate itself as well as produce precursor cells of the various blood cell lineages.