Alan K. Smith

Marrow Cell Transplantation for Infantile Hypophosphatasia

An 8‐month‐old girl who seemed certain to die from the infantile form of hypophosphatasia, an inborn error of metabolism characterized by deficient activity of the tissue‐nonspecific isoenzyme of alkaline phosphatase (TNSALP), underwent the first trial of bone marrow cell transplantation for this heritable type of rickets. After cytoreduction, she was given T‐cell‐depleted, haplo‐identical marrow from her healthy sister. Chimerism in peripheral blood and bone marrow became 100% donor. Three months later, she was clinically improved, with considerable healing of rickets and generalized skeletal remineralization. However, 6 months post‐transplantation, worsening skeletal disease recurred, with partial return of host hematopoiesis. At the age of 21 months, without additional chemotherapy or immunosuppressive treatment, she received a boost of donor marrow cells expanded ex vivo to enrich for stromal cells. Significant, prolonged clinical and radiographic improvement followed soon after. Nevertheless, biochemical features of hypophosphatasia have remained unchanged to date. Skeletal biopsy specimens were not performed. Now, at 6 years of age, she is intelligent and ambulatory but remains small. Among several hypotheses for our patients survival and progress, the most plausible seems to be the transient and long‐term engraftment of sufficient numbers of donor marrow mesenchymal cells, forming functional osteoblasts and perhaps chondrocytes, to ameliorate her skeletal disease.

Direct contact between CD34^+ lin^- cells and stroma induces a soluble activity that specifically increases primitive hematopoietic cell production

Perfused human bone marrow (BM) mononuclear cell (MNC) cultures result in a greater long-term culture-initiating cell (LTC-IC) output than parallel CD34+lin- cell cultures, even when CD34+lin- cells are placed on irradiated preformed stroma (IPFS). This difference has been attributed to accessory cell effects that are potentiated by medium perfusion. The present study investigated the relative contributions of direct contact- and soluble-mediated mechanisms of accessory cells in this culture system. CD34+lin- cells within (i.e., in contact with) the MNC accessory cell mixture generated greater LTC-IC output than CD34+lin- cells in contact with IPFS. Incubation of CD34+lin- cells with MNC conditioned medium (CM) resulted in partial restoration of MNC accessory activity, while CM from IPFS had no activity on LTC-IC output. Interestingly, the level of LTC-IC output supported by MNC CM was equivalent to that supported by direct contact with IPFS. CD34+lin- cells were then cultured in Transwell inserts either alone, with IPFS (direct contact), or with IPFS below the insert. Direct contact with IPFS significantly increased the output of cells, CFU-GM, and LTC-IC from CD34+lin- cells. IPFS below the insert also resulted in significantly increased cell and CFU-GM output, but did not significantly affect LTC-IC output. Further experiments using CM from CD34+lin- cells and IPFS cultures showed that LTC-IC supportive activity was present only when direct contact was allowed between CD34+lin- cells and IPFS. ELISA and RT-PCR experiments showed that contact did not induce changes in the levels of several known growth factors, including GM-CSF, IL-1beta, IL-3, IL-6, IL-11, LIF, KL, FL, Tpo, TGF-beta, and MIP-1alpha. These results indicate that direct contact between CD34+lin- cells and IPFS induces soluble activity, which specifically increases LTC-IC output from CD34+lin- cell cultures, providing evidence for a novel direct contact-mediated two-way mechanism of communication between primitive hematopoietic cells and stroma.

Clinical Scale Expansion of Cryopreserved Small Volume Whole Bone Marrow Aspirates Produces Sufficient Cells for Clinical Use

Ex vivo expansion of bone marrow (BM) mononuclear cells (MNC) in a perfused culture system produces stem-progenitor cell type(s) in sufficient number(s) for hematopoietic reconstitution. The limitations in using fresh BM MNC for ex vivo expansion include additional cell processing and inflexibility in patient treatment. Cryopreservation of whole bone marrow (WBM) eliminates processing costs of MNC or CD34+ cell selection and allows for flexibility in patient treatment. We developed a convenient system to cryopreserve and thaw small volume WBM aspirations (n = 13) and then compared the expandability of unprocessed normal cryopreserved/thawed (C/T) WBM to that of fresh BM MNC cultured in the presence of erythropoietin, PIXY 321, and Flt3-ligand. Ex vivo expansion potential was retained in WBM aspirates after C/T. When initiated with 225 million viable nucleated cells, clinical scale expansion cultures (n = 6) yielded 9.7+/-2.8 x 10(8) total cells, which contained 10.4+/-5.8 x 10(6) colony-forming units-granulocyte-macrophage (CFU-GM), 1.3+/-1.4 x 10(4) LTCIC, and 2.2 x 10(6) CD34+Lin- cells, sufficient cell numbers for clinical use. These studies demonstrate that ex vivo perfusion culture expansion of unfractionated C/T WBM (< or =30 ml) provides doses of stem-progenitor cells similar in composition to expanded fresh BM MNC, previously demonstrated to achieve hematopoietic reconstitution.

Ex Vivo Hematopoietic Cell Expansion for Bone Marrow Transplantation

Publisher Summary This chapter discusses hematopoietic stem cell (HSC) transplantation. Major advances have been made in the field of ex vivo manipulation and generation of cells for patient care. The development of a new ex vivo cell therapy requires a series of issues to be successfully addressed to achieve the ultimate goal of patient care. It needs development of an optimized biological process that results in the generation of a potentially therapeutic cell population. Implementation of the biological process in a reproducible manner in a single-use, disposable sterile device at clinical scale is required. In addition, it needs the integration of the device into a system that facilitates good manufacturing practice (GMP) quality control. Finally, it requires clinical trials to establish safety and efficacy of the cell therapy. The new cell therapy will become viable for widespread clinical practice only when each of these issues has been addressed. This chapter describes developments addressing each of these issues, focusing on HSC transplantation as an example and discusses the approach of ex vivo expansion of hematopoietic cells to either augment or replace current HSC transplantation methods.