High-Speed Cell Sorter Isolates Pure Stem Cells For Use in Autologous Transplantation Patients

February 1, 1995

NASHVILLE--An experimental high-speed clinical cell selection device has been shown to be capable of isolating a pure population of hematopoietic stem cells (HSCs), essentially free from cancer cells, and the machine's developer (SyStemix, Inc., Palo Alto, Calif) has received FDA allowance for an active IND (investigational new drug) for clinical testing of HSCs purified by cell selection in cancer patients who are undergoing transplantation.

NASHVILLE--An experimental high-speed clinical cell selectiondevice has been shown to be capable of isolating a pure populationof hematopoietic stem cells (HSCs), essentially free from cancercells, and the machine's developer (SyStemix, Inc., Palo Alto,Calif) has received FDA allowance for an active IND (investigationalnew drug) for clinical testing of HSCs purified by cell selectionin cancer patients who are undergoing transplantation.

Speaking at a scientific session of the American Society of Hematology(ASH) meeting, Chris Reading, PhD, said that the high-speed fluorescence-activatedcell sorting (HS-FACS) process can isolate HSCs at speeds of 15,000to 30,000 cells per second, five to 10 times faster than conventionalsorters. The isolated HSCs retain the ability to self-renew andpropagate, he noted.

The sorter can select HSCs, and thus deplete tumor cells simultaneously,and can process stem cells in large enough numbers to be of therapeuticvalue, Dr. Reading said. Although the mobilization of stem cellsinto the peripheral blood varies widely among patients, SyStemixexpects to be able to sort the required number of cells for transplantation in a few hours.

Dr. Reading, director of cell procesing at Systemix, reported that in mobilized peripheral blood samples from multiple myelomapatients,processing with the high-speed sorter created a 90% purepopulation of HSCs.

In five such samples,flow cytometric analysis showed a tumor burdenin the starting material of 100,000,000,000 cells (range,100,000,000to 4,000,000,000). In ten such samples, after high speed cellsorting , myeloma cells were reduced to below the level of quantitation(1 in 1,000,000 or 0.001%).

The isolated HSCs are defined by the presence of cell-surfacemarkers CD34 and Thy-1 (which are lost as a cell develops intoa mature blood cell) and the absence of other surface markersfound on lineage-committed or mature cells (lineage negative orLin-).

In contrast to CD34+Thy+Lin- populations isolated in the SyStemixprocess, CD34+ populations, targeted in other cell selection devices,can be described as mixed populations that contain progenitorcells, a varying number of stem cells, and possibly cancer cells.It is the CD34+Thy+Lin- cell that is the self-renewing and pluripotentstem cell, he said.

SyStemix has received patents for both the identification methodfor HSCs and the resulting composition of the HSC.

How the Process Works

The process begins with mobilization of stem cells in the peripheralblood with high-dose cyclophosphamide and GM-CSF. "In someof these samples, we've seen stem cell levels as high as 10% and12% ," Dr. Reading said.

Platelets, red blood cells, and mature granulocytes are removedby counterflow elutriation centrifugation, which increases theCD34+Thy+Lin- concentration by about threefold. Monocytes, residualred blood cells, and maturing granulocytes are removed by lysis,which further increases the target population.

The remaining cells are stained with monoclonal antibodies againstCD34, CD14, CD15, and Thy-1, and isolated in the sorter, givingthe ultimate 90% pure HSC population. The purified cells can thenbe cryopreserved for autografting.

Studies utilizing three assays, including testing in SyStemix'proprietary SCID-hu mouse bone marrow model, showed that the purifiedHSCs retain biologic activity, Dr. Reading said.

US Trial Goes Forward

The FDA has allowed a phase I/II clinical trial of hematopoieticstem cells, purified from mobilized peripheral blood via SyStemix'high-speed cell sorter, as transplants in multiple myeloma patients.

The US trial, under the direction of Bart Barlogie, MD, PhD, directorof research, Arkansas Cancer Research Center, Little Rock, willtest the safety and rapid engraftment capacity of these purifiedcells. Similar clinical trials will also be performed in Europe,according to SyStemix.

The company hopes that the purified SyStemix product may substantiallyreduce the risk of graft-induced relapse caused by the presenceof tumor cells in conventional transplants.

SyStemix has built GMP (Good Manufacturing Practices) cell processingcenters in Palo Alto, Calif, and Lyon, France, to perform thecell isolation procedures for these trials.