C. Donaldson

Adult bone marrow is a rich source of human mesenchymal ‘stem’ cells but umbilical cord and mobilized adult blood are not

Summary. In postnatal life, mesenchymal stem cells (MSC) self‐replicate, proliferate and differentiate into mesenchymal tissues, including bone, fat, tendon, muscle and bone marrow (BM) stroma. Possible clinical applications for MSC in stem cell transplantation have been proposed. We have evaluated the frequency, phenotype and differentiation potential of MSC in adult BM, cord blood (CB) and peripheral blood stem cell collections (PBSC). During culture, BM MSC proliferated to confluence in 10–14 d, maintaining a stable non‐haemopoietic phenotype, HLA class‐1+, CD29+, CD44+, CD90+, CD45–, CD34– and CD14 through subsequent passages. Using the colony forming unit fibroblasts assay, the estimated frequency of MSC in the BM nucleated cell population was 1 in 3·4 × 104 cells. Both adipogenic and osteogenic differentiation of BM MSC was demonstrated. In contrast, CB and PBSC mononuclear cells cultured in MSC conditions for two passages produced a population of adherent, non‐confluent fibroblast‐like cells with a haemopoietic phenotype, CD45+, CD14+, CD34–, CD44–, CD90– and CD29–. In paired experiments, cultured BM MSC and mature BM stroma were seeded with CB cells enriched for CD34+. Similar numbers of colony‐forming units of granulocytes–macrophages were produced by MSC‐based and standard stroma cultures over 10 weeks. We conclude that adult BM is a reliable source of functional cultured MSC, but CB and PBSC are not.

Chromosome translocations and covert leukemic clones are generated during normal fetal development

Studies on monozygotic twins with concordant leukemia and retrospective scrutiny of neonatal blood spots of patients with leukemia indicate that chromosomal translocations characteristic of pediatric leukemia often arise prenatally, probably as initiating events. The modest concordance rate for leukemia in identical twins (≈5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and/or genetic events are required for clinically overt leukemia development. This notion leads to the prediction that chromosome translocations, functional fusion genes, and preleukemic clones should be present in the blood of healthy newborns at a rate that is significantly greater than the cumulative risk of the corresponding leukemia. Using parallel reverse transcriptase–PCR and real-time PCR (Taqman) screening, we find that the common leukemia fusion genes, TEL-AML1 or AML1-ETO, are present in cord bloods at a frequency that is 100-fold greater than the risk of the corresponding leukemia. Single-cell analysis by cell enrichment and immunophenotype/fluorescence in situ hybridization multicolor staining confirmed the presence of translocations in restricted cell types corresponding to the B lymphoid or myeloid lineage of the leukemias that normally harbor these fusion genes. The frequency of positive cells (10−4 to 10−3) indicates substantial clonal expansion of a progenitor population. These data have significant implications for the pathogenesis, natural history, and etiology of childhood leukemia.

Bone marrow-derived mesenchymal stem cells

Human mesenchymal stem cells (MSCs) contribute to the regeneration of mesenchymal tissues, and are essential in providing support for the growth and differentiation of primitive hemopoietic cells within the bone marrow microenvironment. Techniques are now available to isolate human MSCs and manipulate their expansion in vitro under defined culture conditions without change of phenotype or loss of function. Mesenchymal stem cells have generated a great deal of interest in many clinical settings, including that of regenerative medicine, immune modulation and tissue engineering. Studies have already demonstrated the feasibility of transplanted MSCs providing crucial new cellular therapy. In this review, many aspects of the MSC will be discussed, with the main focus being on clinical studies that describe the potential of MSCs to treat patients with hematological malignancies who are undergoing chemotherapy and/or radiotherapy.

Impact of obstetric factors on cord blood donation for transplantation

Recent reports have shown that low nucleated cell dose significantly decreases survival after cord blood transplantation. Prior to starting clinical cord blood banking we investigated the impact of obstetric factors on cell dose and volume of cord blood donations. Cord blood was obtained from 114 normal full‐term deliveries. Mean volume collected was 93.5 ml, mean total nucleated cell count (TNC) was 13.1 × 108. Statistical analysis was by backwards stepwise regression. Significant factors affecting nucleated cell yield were volume of blood collected (P < 0.001), length of gestation (P < 0.0001), time from delivery of the infant to cord clamping (P = 0.018) and total length of labour (P = 0.002). In clinical cord blood banking we have successfully used these findings for pre‐collection assessment of placentae. Out of 476 cord blood donations subsequently collected for banking, only 29 (6.1%) have been discarded due to low volume. The mean TNC of the 409 banked units following volume reduction was 10.1 × 108. Despite careful optimization of collection, processing and storage techniques, cell dose still limits cord blood transplantation to smaller recipients.

Chemotherapy-induced mesenchymal stem cell damage in patients with hematological malignancy

Hematopoietic recovery after high-dose chemotherapy (HDC) in the treatment of hematological diseases may be slow and/or incomplete. This is generally attributed to progressive hematopoietic stem cell failure, although defective hematopoiesis may be in part due to poor stromal function. Chemotherapy is known to damage mature bone marrow stromal cells in vitro, but the extent to which marrow mesenchymal stem cells (MSCs) are damaged by HDC in vivo is largely unknown. To address this question, the phenotype and functional properties of marrow MSCs derived from untreated and chemotherapeutically treated patients with hematological malignancy were compared. This study demonstrates a significant reduction in MSC expansion and MSC CD44 expression by MSCs derived from patients receiving HDC regimens, thus implicating potential disadvantages in the use of autologous MSCs in chemotherapeutically pretreated patients for future therapeutic strategies. The clinical importance of these HDC-induced defects we have observed could be determined through prospective randomized trials of the effects of MSC cotransplantation on hematopoietic recovery in the setting of HDC with and without hematopoietic stem cell rescue.

Is in vitro expansion of human cord blood cells clinically relevant

Peripheral blood recovery after cord blood (CB) transplantation is delayed compared with marrow. Expansion of CB haemopoietic cells has been investigated with the aim of reducing cytopenia following transplantation. Mature cells, post-progenitors, progenitors and long-term culture-initiating cells (LTC-IC) from expansion products may contribute to peripheral blood recovery. We investigated the increase in total nucleated cells, colony-forming cells (CFC), CD34+ cells and LTC-IC by limiting dilution after a 14 day culture of CB CD34+ cells (5 × 103/ml) with SCF, IL-3, IL-6, GM-CSF and G-CSF all at 10 ng/ml. On average, nucleated cells increased 2500-fold, CD34+ cells 39-fold and CFU-GM 49-fold with maintenance of BFU-E. The more primitive LTC-IC expanded on average 2.5-fold but effects on long-term marrow-repopulating cells (LTRC) during culture are unknown. A practical application of in vitro expansion of CB might be to expand a 20% aliquot of a CB donation and infuse the remainder unmanipulated. This could provide a 5- to 7-fold increase in progenitor cells, an estimated 1570-fold increase in post-progenitor cells and maintenance of LTC-IC compared to an untreated donation. Combined with in vivo post-transplant growth factor therapy this could prompt early peripheral blood recovery after CB transplantation, without significant loss of LTC-IC or donor lymphocytes.

The CD34+CD38neg population is significantly increased in haemopoietic cell expansion cultures in serum-free compared to serum-replete conditions: Dissociation of phenotype and function

Expansion of haemopoietic stem cells is proposed to combat graft failure in adult recipients following cord blood (CB) transplantation. Cultures are traditionally performed in medium containing FCS, but to transfer expansion to the clinic, ‘good manufacturing practice’ (GMP) standards are required. This study evaluated expansion cultures in culture bags and serum-free (SF) conditions, to comply with GMP, by analysing sub-populations of CD34+ cells, colony-forming cells (CFC) and long-term culture initiating cells (LTC-IC). CD34+cell analysis has previously been used to measure clonogenic capacity and the CD34+CD38neg surface phenotype to measure primitive cell numbers. In this study, comparison of expansion in serum-replete medium with that in SF conditions demonstrated a lack of expression of CD38 on CD34+ cells in the absence of serum. These findings must be considered in clinical studies using in vitro expansion in SF conditions, and the CD34+CD38neg phenotype should not be used to confirm maintenance, or expansion, of primitive progenitor cells. Bone Marrow Transplantation (2001) 27, 365–371.

Multiple myeloma: the bone marrow microenvironment and its relation to treatment

Abstract Multiple myeloma is the most common haematological malignancy yet currently it remains incurable. For decades the mainstay in therapy has been non-targeted approaches including genotoxic agents and immunosuppressants. With myeloma predominantly affecting an elderly population, who are vulnerable to aggressive therapy, these non-specific approaches have resulted in poor survival. However, in recent years an explosion of collaborative research into myeloma has identified molecular interactions between myeloma cells and the bone marrow microenvironment as promoting myeloma development and associated complications such as bone lesions due to osteolysis. At the same time, a better understanding of the adhesion molecules, cytokines and signalling pathways involved in myeloma has led to the development of new targeted therapies, which are improving the quality of life for patients and significantly extending median patient survival. This review explores the current understanding of molecular pathways that promote myeloma progression and lead to bone destruction, with particular reference to the influence of interactions with the bone marrow microenvironment. It describes molecular targets for therapy with reference to the new therapeutics and their improved efficacy. While the outlook for myeloma patients has improved in recent years as a result of these new approaches, drug resistance remains a problem and future therapies will also need to address the molecular mechanisms of resistance in order to improve further the outcome for patients with this disease.

Toxicity testing: the search for an in vitro alternative to animal testing.

Abstract Prior to introduction to the clinic, pharmaceuticals must undergo rigorous toxicity testing to ensure their safety. Traditionally, this has been achieved using in vivo animal models. However, besides ethical reasons, there is a continual drive to reduce the number of animals used for this purpose due to concerns such as the lack of concordance seen between animal models and toxic effects in humans. Adequate testing to ensure any toxic metabolites are detected can be further complicated if the agent is administered in a prodrug form, requiring a source of cytochrome P450 enzymes for metabolism. A number of sources of metabolic enzymes have been utilised in in vitro models, including cell lines, primary human tissue and liver extracts such as S9. This review examines current and new in vitro models for toxicity testing, including a new model developed within the authors’ laboratory utilising HepG2 liver spheroids within a co-culture system to examine the effects of chemotherapeutic agents on other cell types.

Development of Lymphocytotoxic and Platelet Reactive Antibodies: A Prospective Study in Patients with Acute Leukaemia

Abstract. Lymphocytotoxic (LCT) and platelet reactive (PR) antibody (Ab) responses were serially determined in 49 patients with acute leukaemia. LCTAb were found in 20 patients and occurred in 13 patients with acute myeloid leukaemia and 7 patients with acute lymphoblastic leukaemia. Four differing patterns of LCTAb responses could be defined. Thirteen of 22 subjects showed marked reduction or loss of LCTAb. Indirect platelet immunofluorescence, measured by flow cytometry, provided the most convenient means of detecting PRAb which were found in 11 subjects and generally showed moderate or weak reactivity.