Gary L. Gilmore

Ex vivo expansion of human umbilical cord blood and peripheral blood CD34+ hematopoietic stem cells

The proliferation and expansion of human hematopoietic stem cells (HSC) in ex vivo culture was examined with the goal of generating a suitable clinical protocol for expanding HSC for patient transplantation.HSC were derived from umbilical cord blood (UCB) and adult patient peripheral blood stem cell collections. HSC were stimulated to proliferate ex vivo by a combination of two growth factors, flt-3 ligand (FL) and thrombopoietin/c-mpl ligand (TPO/ML), and assessed for expansion by flow cytometry.Ex vivo expansion cultures of UCB were maintained for prolonged periods (up to 16 weeks), and sufficient HSC were generated for adult transplantation. In contrast to UCB, FL + TPO/ML did not significantly increase CD34(+) peripheral blood stem cell (PBSC) numbers.UCB-HSC can be expanded in culture to numbers theoretically adequate for safe, rapid engraftment of adult patients. Additional studies are needed to establish the functional activity of expanded UCB-HSC.

Protective effects of BB-10010 treatment on chemotherapy-induced neutropenia in mice.

Chemotherapy-induced neutropenia is a major dose-limiting factor in the management of cancer patients. Most chemotherapeutic agents are active against proliferating cells, interfering with DNA replication and/or mitosis. A number of chemokines, notably macrophage inflammatory protein-1 alpha [MIP-1alpha], have been reported to induce cell-cycle arrest in immature hematopoietic progenitors, raising the possibility that chemokines, such as MIP-1alpha, could be used to reduce or even eliminate the hematologic toxicity of cycle-active chemotherapy. We tested the effectiveness of BB-10010 [a genetically engineered analog of human MIP-1alpha] in vivo against three different cytotoxic drugs [cyclophosphamide (Cy), 5-fluorouracil (5-FU) and cytosine arabinoside (Ara-C)] commonly used in cancer therapy. BB-10010 treatment reduced the toxicity of all three agents, though the precise mode of protection varied with the cytotoxic drug used. BB-10010 reduced the neutropenic interval in Cy-treated mice without affecting the neutropenic nadir, whereas the absolute neutrophil counts [ANC] of both 5-FU and Ara-C treated mice were significantly higher throughout the neutropenic interval for mice receiving BB-10010 prior to chemotherapy. These findings indicate that the ability to manipulate the cell cycle of hematopoietic progenitors with chemokines, such as BB-10010/MIP-1alpha and other negative regulators, may be exploited to reduce chemotherapy-induced neutropenia; furthermore, the fact that BB-10010 is effective against several different cytotoxic agents is cause for guarded optimism that this approach may be generally applicable, and, once optimized for patient use, may prove to be of significant clinical benefit.

Synthesis of Reactive Polymers for Acrolein Capture Using AGET ATRP.

Acrolein is a toxic metabolite of the anticancer agent cyclophosphamide (CP). Current strategies to mitigate acrolein toxicity are insufficient, and in this brief article, we report the synthesis of well-defined low molecular weight block copolymers using activators generated by electron transfer atom transfer radical polymerization (AGET ATRP) capable of reacting with the cytotoxic small molecule acrolein. Acrolein reactivity was introduced into the block copolymers via incorporation of either (a) aminooxy or (b) sulfhydryl groups. The cytoprotective effect of the polymers was compared to sodium 2-sulfanylethanesulfonate (mesna) the current gold standard for protection from CP urotoxicity, and we found that the polymers bearing sulfhydryl moieties demonstrated superior cytoprotective activity.

Viability and Potency of Hematopoietic Progenitor Cells after Prolonged Cryopreservation at –80°C

Peripheral blood HPC were collected from 28 donors at our institution between April 1994 and June 2002 using the Baxter CS-3000 blood cell separator. Patient characteristics are shown in table 1 . Only 3 donors of the HPC products were alive at the time of analysis of the products (UPN 947, UPN 1,000 and the normal unrelated donor); the recipient of the unrelated-donor HPC product was deceased at the time of analysis. On the day of the third transplant, the UPN 947 sample was taken directly from the HPC product infused at the bedside. The UPN 1,000 sample was obtained from a remaining unit stored in the same cassette as the infused product and analyzed 231 days after the third transplant. Patients provided inPeripheral blood hematopoietic progenitor cells (HPC) collected by apheresis and cryopreserved constitute the major source of stem cells for autologous transplantation, which is widely used to treat hematological malignancy [1, 2] . Controlled-rate freezing, using dimethyl sulfoxide (DMSO), with or without hydroxyethyl starch (HES) as cryoprotectant, and storage of products in liquid nitrogen (LN) is the most widely used method of cryopreservation and storage of HPC [3, 4] . The method requires specialized equipment, a constant supply of LN and carries the risk of cryogenic injury to laboratory personnel. An alternative method of cryopreservation with 5% DMSO, 6% HES and 4% human serum albumin (HSA) was described by Stiff et al. [5] , utilizing non-controlledrate freezing and storage in a mechanical freezer at –80 ° C. HPC cryopreserved by this method provided sustained engraftment in 68 of 72 patients after high-dose chemotherapy [6] . However, the effect of storage on HPC activity for 1 5 years under these conditions has not been examined. We examined the viability and hematopoietic colony-forming activity of HPC products stored in DMSO/HES/HSA at –80 ° C between 7.1 and 14.9 years. The mean number of days of storage of the HPC product at –80 ° C was 4,254 (range 2,582–5,433, standard deviation 563). Received: November 25, 2010 Accepted after revision: March 10, 2011 Published online: May 17, 2011

Differential CD34+ engraftment of nod-scid mice with ex vivo expanded human CD34+ cord blood stem cells correlates with CD38 phenotype

Abstract Ex vivo expansion of cord blood [CB] hematopoietic stem cells [HSC] may increase the number of patients eligible for allogeneic transplants, by making CB a reliable source of HSC for transplantation. We previously showed that CB-HSC can be significantly expanded with flt-3 ligand [FL] and c-mpl ligand [ML]; 10 6 input CD34 + cells increase to 10 8 CD34 + cells by 8-9 weeks of culture. However, it is essential to know the function of expanded CB HSC cells prior to clinical usage. To assess the in vivo function of expanded CB HSC, cells from expansion cultures were injected into irradiated [375 cGy] non-obese diabetic/severe combined immune deficient [NOD- scid ] mice. Uncultured CD34 + UCB served as engraftment controls. Engraftment was analyzed 5-8 weeks post-injection by two-color staining of bone marrow cells with antibodies to human CD45 [FITC] and selected CD antigens [PE]. Cells grown from 2 to 16 weeks engraft NOD- scid mice, as evidenced by human CD45 + cells in the marrow, giving rise to mature human leukocytes. Surprisingly, cells from 2-week cultures generate few CD34 + cells in the marrow of NOD- scid mice, while mice engrafted with cells from cultures ≥3 weeks appear similar to uncultured UCB controls. Kinetic analysis of CD38 expression on ex vivo expanded CB-HSC using 3-color flow cytometry shows that the CD34 + cells from uncultured UCB are predominantly CD38 lo , but there is a shift to CD34 + /CD38 + cells during the first 2 weeks of culture. At later time points, the CD38 + population diminishes, so that virtually all CD34 + cells are CD38 lo . Since CD34 + /CD38 + stem cells are considered more mature than CD34 + /CD38 lo HSC, our results correlated CD38 expression to CD34 + engraftment potential. These findings indicate that short-term cultures (10-14 days) lead to a decline in CB HSC activity and impaired engraftment, but extended culture conditions lead to a return of CD34 + /CD38 lo stem cells, and restoration of normal engraftment potential by these cultured CB HSC.