Clonal analysis of thymus-repopulating cells presents direct evidence for self-renewal division of human hematopoietic stem cells.

To elucidate the in vivo kinetics of human hematopoietic stem cells (HSCs), CD34+CD38- cells were infected with lentivirus vector and transplanted into immunodeficient mice. We analyzed the multilineage differentiation and self-renewal abilities of individual thymus-repopulating clones in primary recipients, and their descending clones in paired secondary recipients, by tracing lentivirus gene integration sites in each lymphomyeloid progeny using a linear amplification-mediated polymerase chain reaction (PCR) strategy. Our clonal analysis revealed that a single human thymus-repopulating cell had the ability to produce lymphoid and myeloid lineage cells in the primary recipient and each secondary recipient, indicating that individual human HSCs expand clonally by self-renewal division.

Direct evidence for ex vivo expansion of human hematopoietic stem cells.

To characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done.

Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment.

Hematopoiesis is maintained by specific interactions between both hematopoietic and nonhematopoietic cells. Whereas hematopoietic stem cells (HSCs) have been extensively studied both in vitro and in vivo, little is known about the in vivo characteristics of stem cells of the nonhematopoietic component, known as mesenchymal stem cells (MSCs). Here we have visualized and characterized human MSCs in vivo following intramedullary transplantation of enhanced green fluorescent protein-marked human MSCs (eGFP-MSCs) into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice.

Competitive repopulation assay of two gene-marked cord blood units in NOD/SCID/gammac(null) mice.

In multiunit cord blood transplantation, hematopoietic stem cells from each unrelated cord blood (UCB) unit competitively reconstitute the hematopoietic system in a recipient. To evaluate the fate of the progeny of each UCB unit and to determine the effects of graft-versus-graft reaction, we established a novel competitive repopulation assay using NOD/SCID/gammac(null) mice in which human T lymphocytes develop from CD34+ cells. CD34+ cells from each UCB unit were labeled with recombinant lentivirus vectors carrying genes encoding either enhanced green fluorescent protein (EGFP) or enhanced yellow fluorescent protein (EYFP).

Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction.

Bone marrow (BM) cells are reported to contribute to the process of regeneration following myocardial infarction. However, the responsible BM cells have not been fully identified. Here, we used 2 independent clonal studies to determine the origin of bone marrow (BM)-derived cardiomyocytes.

In vivo and in vitro differentiation of myocytes from human bone marrow-derived multipotent progenitor cells.

Objective: Recent studies have shown that bone marrow (BM) contains cells capable of differentiating into myocytes in vivo. However, addition of demethylation drugs has been necessary to induce myocyte differentiation from BM cells in vitro, and precise mechanisms of BM cells' conversion to myocytes and the origin of those cells have not been established. We investigated the expression of myogenic markers during differentiation and maturation of myocytes from BM-derived multipotent adult progenitor cells (MAPC) under physiological culture condition.

A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow.

To measure the ability of human hematopoietic stem cells (HSCs), the SCID-repopulating cell (SRC) assay has been widely used. Conventionally, human HSCs are transplanted into a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse via a tail vein. However, those cells must go through various obstacles until they reach the mouse marrow environment, which could explain the generally low homing efficiency in this system.

Acute Lethal Injury of Lung and Liver in Mice Transplanted with Ex Vivo-Expanded CTLs.

Clinical application of cytotoxic T lymphocytes (CTL) induced in vitro is extensively used for the treatment of viral infection and malignant diseases. We produced anti H-2d CTL in vitro from C57BL/6 (B6) splenocytes presensitized with (B6 × DBA/2) F1 (BDF1) splenocytes to establish a model system of CTL therapy. The specificity and cytotoxic activity were high enough (E/T ratio 1:1 = 38.