Cindy L. Miller

The marrow homing efficiency of murine hematopoietic stem cells remains constant during ontogeny

OBJECTIVEnSeveral recent studies have established the potential clinical utility of hematopoietic stem cells (HSCs) not only for marrow rescue but also for regenerating diseased or damaged nonhematopoietic tissues. These findings have focused renewed interest in understanding the in vivo trafficking patterns of HSCs from different sources. Previous experiments have suggested that the half-life of HSCs in the circulation is short, although the actual proportion that return to the bone marrow (BM) following transplantation has not been previously quantitated. The present study was undertaken to measure this fraction and compare the values obtained for functionally defined HSCs from adult murine BM and day-14 fetal liver (FL).nnnMETHODSnThe number of HSCs that could be recovered from the BM of lethally irradiated mice 24 hours after intravenous injection of Ly-5 congenic BM or FL cells was determined by limiting-dilution competitive repopulating unit (CRU) assays in secondary mice.nnnRESULTSnThe marrow seeding efficiency of both adult BM- and FL-CRU able to produce lymphoid and myeloid progeny for 5-26 weeks posttransplant was approximately 10%. FL-CRU generated clones that were approximately threefold larger than those produced by BM-CRU. Interestingly, clones produced by homed HSCs were approximately twofold smaller than those produced by freshly isolated HSCs. Differences were also seen in the proportions of lymphoid vs myeloid progeny generated by fresh and homed HSCs.nnnCONCLUSIONSnThese data suggest common mechanisms regulating the BM homing of long-term repopulating HSCs throughout ontogeny despite subtle differences in the size and composition of the clones they generate.

Human and Mouse Hematopoietic Colony-Forming Cell Assays

Hematopoietic stem cells present in small numbers in certain fetal organs during development and in adult bone marrow produce a heterogeneous pool of progenitors that can be detected in vitro using colony-forming cell (CFC) assays. Hematopoietic progenitor cells, when cultured in a semisolid methylcellulose-based medium that is supplemented with suitable growth factors, proliferate and differentiate to produce clonal clusters (colonies) of maturing cells. The CFCs are then classified and enumerated in situ by light microscopy. Protocols for the detection and enumeration of myeloid multipotential progenitors and committed progenitors of the erythroid, monocyte, and granulocyte lineages in samples from human peripheral blood, bone marrow, and cord blood as well as mouse fetal liver and bone marrow are described.

Characterization of Mouse Hematopoietic Stem and Progenitor Cells

The unit describes functional assays for the quantification of mouse hematopoietic stem cells and progenitor cells. The competitive repopulating unit (CRU) assay detects transplantable mouse hematopoietic stem cells with the capacity to regenerate all of the blood cell lineages for extended time periods in vivo. The long‐term culture‐initiating cell (LTC‐IC) assay, founded on the bone marrow long‐term culture system, measures primitive hematopoietic progenitors based on their capacity to produce myeloid progeny for at least four weeks. Colony‐forming cell (CFC) assays, performed in semisolid medium cultures to assess mouse pre‐B, megakaryocyte, erythroid, granulocyte‐monocyte, and multipotential hematopoietic progenitors are also described. These assays are powerful tools for evaluating human stem cell (HSC) and progenitor content in various hematopoietic tissues, during development as well as in the adult animal, and in cell populations manipulated ex vivo. Curr. Protoc. Immunol. 80:22B.2.1‐22B.2.31.

Quantitation of Murine and Human Hematopoietic Stem Cells by Limiting-Dilution Analysis in Competitively Repopulated Hosts

In designing functional assays for the various classes of hematopoietic cells described in this book, one needs to consider the properties of the cell to be measured which must be incorporated into the assay design, and the end points to allow its specific detection. The most primitive hematopoietic stem cells (HSC) in mouse and man are characterized by two cardinal properties that distinguish them from more mature clonogenic cells and their terminally differentiated progeny. Firstly, HSCs are pluripotent: they are characterized by the potential to differentiate into all of the eight major lineages of lymphoid, myeloid, and erythroid cells in vivo (1-3). Secondly, HSCs are able to self-renew, or generate daughter stem cells in vivo and in vitro that are functionally identical to the stem cell that gave rise to them (3-5). These hallmark properties of HSCs are measured empirically by their potential to regenerate and maintain lymphocytes, granulocytes, and erythrocytes upon transplantation into lethally irradiated or immunocompromised primary and secondary hosts. However, functional assays for primitive HSCs must also consider the fact that differentiated cells present in the hematopoietic organs at different times after bone marrow transplantation are derived from different types of precursors (6), and particularly at later times, cannot be assumed to be of donor origin (7). Support for this concept derives from the relatively recent demonstration in mice that most, if not all, spleen colonies detectable ≈ 2 wk after transplantation originate.

Long-Term Culture-Initiating Cell Assays for Human and Murine Cells

In normal adults, the majority of primitive hematopoietic cells are concentrated in the bone marrow, where they are in contact with a variety of molecules that influence their cell-cycle status, viability, motility, and differentiation. These include components of the extracellular matrix, soluble and bound growth-promoting factors and inhibitors, and adhesion molecules that mediate direct interactions between cells. The long-term culture (LTC) system initially developed to support the continued production of myeloid cells, (1-3) and subsequently for the production of lymphoid cells (4-7) has provided a unique approach for the investigation of the regulation and maintenance of early hematopoietic progenitors under conditions that reproduce many aspects of the marrow microenvironment. The LTC system has also provided a basis for the development of powerful assay procedures for quantitating and distinguishing cells at discrete stages of early hematopoietic cell differentiation.

Human Long-Term Culture Initiating Cell Assay

The long-term culture initiating cell (LTC-IC) assay, founded on the bone marrow long-term culture (LTC) system, measures primitive hematopoietic stem cells (termed LTC-IC) based on their capacity to produce myeloid progeny for at least 5 weeks. Adaptations of the LTC system including the use of stromal cell lines, application of limiting dilution analysis, and estimation of average hematopoietic progenitor output per LTC-IC under defined conditions have made it possible to accurately determine LTC-IC content in minimally separated and highly purified cell populations from human hematopoietic tissue sources such as bone marrow, peripheral blood, cord blood, fetal liver as well as cord blood and mobilized peripheral blood. Methodologies for measuring human LTC-IC using bulk cultures, limiting dilution analysis, and single cell cultures are described.

Ex vivo expansion of human and murine hematopoietic stem cells.

The last decade has seen major advances in our knowledge of the molecular control of hematopoiesis, widespread access to cytokines, and the development of practical assays for quantitating highly primitive hematopoietic cells. This progress has now made feasible the predictable manipulation of hematopoietic stem cells (HSC) and progenitors for a variety of experimental and clinical applications. Nevertheless, our understanding of events that induce and/ or block the differentiation of primitive hematopoietic cells is still very limited. Therefore, it is not surprising that procedures for expanding HSC populations ex vivo are based largely on a small set of empirical observations. The incentive to improve this situation is provided by many clinical situations in which the number of stem cells available for particular types of transplants is inadequate, or where HSC amplification may be useful as part of a purging strategy to reduce the potential burden of malignant cells in an autograft. Cell division with retention of stem cell integrity could also facilitate the generation in vitro of many specific types of differentiated cells (e.g., dendritic cells) and is a requirement for retroviral-mediated gene therapy. Progress in each of these rapidly evolving areas has recently been reviewed in greater depth elsewhere (1-5).

Colony Forming Cell Assays for Human Hematopoietic Progenitor Cells

Hematopoietic stem cells (HSCs) present in small numbers in adult bone marrow (BM), peripheral blood (PB) and umbilical cord blood (CB) produce a heterogeneous pool of progenitors that can be detected in vitro using colony forming cell (CFC) assays. Hematopoietic progenitor cells proliferate and differentiate to produce colonies of maturing cells when cultured in a semisolid methylcellulose-based medium that is supplemented with suitable growth factors and other supplements. The colonies are then classified and enumerated in situ by light microscopy or an automated imaging instrument. CFC assays are important tools in basic hematology research but are also used by clinical cell processing laboratories to measure the progenitor cell content of BM, CB and mobilized PB (MPB) preparations used for cell transplantation. Standard CFC assays for human progenitor cells require a culture period of at least 14 days to enable optimal outgrowth and differentiation of the maximum number of CFCs in a cell preparation. In this chapter protocols are described for the detection and enumeration of myeloid multipotential progenitors and committed progenitors of the erythroid, monocyte, and granulocyte lineages in samples from human PB, MPB, BM, and CB. In addition protocols are described for a modified version of the CFC-assay that allows accurate enumeration of total CFC numbers in CB or MPB after a culture period of only 7 days, but without distinction of colony types.

Long-Term Culture-Initiating Cell Assay for Mouse Cells

The long-term culture-initiating cell (LTC-IC) assay is a well-established in vitro assay used to enumerate primitive mouse hematopoietic stem cells (HSCs) and relies on the two cardinal functions of HSCs: ability to self-renew and differentiation capacity. LTC-ICs present in minimally processed and purified cell suspensions and cocultured on a supportive feeder layer are detected by their sustained ability to produce hematopoietic progenitors (colony forming cells) after ≥ 4 weeks in culture. Refinements including the use of a defined stromal cell line, and extending the in vitro culture to 6 weeks allow detection of LTC-IC at similar frequencies to transplantable HSCs quantified using in vivo assays.

A defined, feeder-independent medium for human embryonic stem cell culture

Human embryonic stem cells (hESC) are derived from the epiblast tissue of the inner cell mass and are capable of giving rise to cells from all three germ layers - ectoderm, mesoderm, and endoderm. Although the basic techniques to create and culture hESC are well established, limitations remain in many of the procedures used for their culture. In particular, most existing hESC lines have been cultured with components derived from animal serum and using mouse embryonic fibroblast feeder layers. However, the use of serum or feeder layers induces variability in these culture systems, and limits their application in the clinical setting. Accordingly, there is considerable interest in the development of serum-free and feeder-independent hESC culture systems. mTeSR™1 is a fully-defined, serum-free medium for the derivation and maintenance of hESCs cultured on BD Matrigel™ hESC-qualified Matrix(Ludwig et al. Nat Methods 3(8):637-46). mTeSR™1 has been demonstrated to support the undifferentiated culture of multiple hESC lines derived by independent sources, with examples including H1, H9 CA1, and SA121. Cells grown for multiple passages in mTeSR™1 maintain characteristic undifferentiated colony morphology, and FACS analysis confirms high expression of pluripotency-associated markers (Oct4, SSEA-3, SSEA4, TRA-1-60, and TRA-1-81), and the absence of expression of the differentiation marker SSEA-1. RT-PCR analysis of cells cultured in mTeSR™1 also validate the continued expression of Oct4 and Nanog and the lack of expression of multiple differentiation markers. Furthermore, hESC cultures maintained in mTeSR™1 retain a high proliferative capacity and normal karyotype after extended culture. Finally, the teratoma formation assay verified the preserved potential of these cells to subsequently differentiate into tissues derived from all three germ layers. Together these results establish mTeSR™1 as an optimal, standardized medium for researchers culturing hESC.