Collection of fetal blood for stem cell research and therapy.

Stem cell research has generated novel therapeutic opportunities at the expense of new ethical and legal problems. Its promoters recommended early clamping of the umbilical cord to maximize the amount of acquired fetal blood. Fear has been expressed, therefore, that the donor could be compromised by this approach. Actually, the problem is more complex than generally assumed. In certain clinical situations the neonate may benefit from or become harmed by additional blood volume. Gravity influences the direction of umbilical blood flow and, thus the consequences of early or delayed cord clamping. Therefore, vaginal birth promotes blood flow from the placenta to the fetus, whereas delivery by cesarean section usually has the opposite effect. Largely ignored in the course of the relevant debates, the above facts require consideration. The controversy may be beneficial in the long run by drawing attention to this relatively neglected aspect of perinatal medicine.

Pooled umbilical cord blood as a possible universal donor for marrow reconstitution and use in nuclear accidents.

Human umbilical cord blood has been shown to be an effective source of stem cells for marrow reconstitution in pediatric patients. Unfortunately, the quantity of stem cells obtained from an individual donor can be quite limited in both the total volume and the numbers of stem cells per ml of cord blood. HLA matching further limits the availability, but recent publications indicate close matching may be unnecessary. Therefore, if cord blood from different donors can be combined, larger numbers of stem cells can be available for clinical use provided pooling does not produce a negative effect. Storage of single cord blood specimens at 4 degrees C for 10-21 days in gas permeable bags produced an apparent increase in the percentage of immature cells (CD34, CD117, GPA) and mitotic activity (S+G2/M cells) over day 1. With similar storage of pooled specimens there was a further increase in the number of immature colonies cultured, CD34, CD117, GPA, S+G2/M cells. In addition, nucleated red blood cells increased over the mean values obtained from single cord blood samples. Our previous studies have indicated that large numbers of human mononuclear cells are necessary to reconstitute an irradiated animal model. By combining multiple samples of human cord blood, adequate numbers of stem cells could be pooled for use in adults and would provide cells for megadose therapy, including those patients that had accidentally received lethal irradiation.

Human umbilical cord blood cells ameliorate Huntington's disease in transgenic mice.

Human umbilical cord blood mononuclear cells given in megadose quantity (71-74 x 10(6) and 100-105 x 10(6)) were able to increase the life span of B6CBA-TgN(Hdexon1)62Gpb mice (Huntington disease) from an average of 88 days to 97.8 and 103.4 days respectively. The rate of weight loss, which begins in these mice before the onset of symptoms of chorea, was far less in the animals receiving human cord blood mononuclear cells (p<0.01) than the weight loss in untreated control mice. With the full understanding that additional histological and behavioral studies are indicated, but because of the potential clinical significance, a brief report is being submitted.

Human umbilical cord blood cells ameliorate Alzheimer's disease in transgenic mice.

Having had success in extending the life of mice with a transgene for amyotropic lateral sclerosis (SOD1) mice and Huntington's disease (Hdexon1), we administered megadoses of human umbilical cord blood mononuclear cells to mice with Alzheimer's disease. These mice have an over-expression of human Alzheimer amyloid precursor protein (APP), die early and develop a CNS disorder that includes neophobia. When given 110 x 10(6) human umbilical cord blood mononuclear cells, these mice (HuAPP 695.SWE) had considerable extension of life with a p value of 0.001 when compared to control animals.

The potential for the use of mononuclear cells from human umbilical cord blood in the treatment of amyotrophic lateral sclerosis in SOD1 mice.

The SOD1 mice (transgenic B6SJL-TgN(SOD1-G93A)1GUR) have a mutation of the human transgene (CuZn superoxide dismutase gene SOD1) that has been associated with amyotrophic lateral sclerosis (ALS). In a preliminary study, we demonstrated that a megadose of human umbilical cord blood mononuclear cells given intravenously after 800 cGy of irradiation could substantially increase the life span of SOD1 mice. This report is an attempt to confirm and expand the preliminary findings. By repeating the study and raising the number of human cord blood cells from 33.2-34.0 x 10(6) to 70.2-73.3 x 10(6) there was a further significant increase in the life span of the SOD1 mice. The average life of the controls was 123.5 days while that of mice receiving the larger megadose of cells was 162 days. While all the controls were dead by 130 days, the treated group receiving 70.2-73.3 x 10(6) cells had one animal living up to 187 days and one 210 days. In order to obtain a megadose of cells, pooled blood from different donors was used and did not appear to have a negative effect, but indicated a beneficial effect on survival. The clinical significance of these findings may extend beyond the potential treatment for amyotrophic lateral sclerosis. This study confirms and extends the preliminary study whereby increasing the dose of human umbilical cord blood cells we were able to substantially further increase the survival of SOD1 mice.

Human umbilical cord blood effect on sod mice (amyotrophic lateral sclerosis).

In previous studies we observed that human umbilical cord blood (HUCB) could have a protective effect on the onset of disease and time of death in MRL Lpr/Lpr mice which have an autoimmune disease that may be considered similar to human lupus. We believed a temporary xenograph may have occurred in these animals with the disease process delayed and the life span markedly increased. When HUCB is stored at 4 degrees C in gas permeable bags, there is a decrease of the cell reaction in mixed lymphocyte cultures. The blood, however, maintains a significant number of cells capable of producing replatable colonies. This study attempted to determine the effect of HUCB on SOD1 mice (transgenic B6SJL-TgN(SOD1-G93A)1GUR), which have a mutation of the human transgene, (CuZn superoxide dismutase gene SOD1) that has been associated with amyotrophic lateral sclerosis. We previously developed evidence that the survival of lethally irradiated mice was related to the number of human mononuclear cells administered. In the present study, we decided to investigate the effect of a relatively large dose of human mononuclear cord blood cells on SOD1 mice subjected to a sublethal dose of irradiation preceded by antikiller sera (rabbit anti-asialo). The SOD1 mice show evidence of paralysis at 4 to 5 months. The average expected lifetime of these mice is reported to be 130 days (Jackson Laboratory). In this experiment, there were 23 mice. Two mice died before the onset of paralysis. The remainder were divided into three groups: group I: control group of 4 untreated mice; group II: an experimental group of 6 mice treated with antikiller sera, 800 cGy irradiation plus 5 x 10(6) congenic bone marrow mononuclear cells; group III: another experimental group of 11 mice treated with antikiller sera, 800 cGy irradiation plus 34.2-35.6 x 10(6) HUCB mononuclear cells, previously stored for 17-20 days at 4 degrees C in gas permeable bags. The results were as follows: the average age at death was: (I) 127 days for the untreated control group, (II) 138 days for the group that received 800 cGy of irradiation and congenic bone marrow (BM) and (III) 148 days for the group that received irradiation and HUCB. (P < 0.001 HUCB vs control, p < 0.01 HUCB vs BM). The longest surviving mouse in each group was 131, 153, and 182 days old respectively. In summary, large doses of HUCB mononuclear cells produced considerable delay in the onset of symptoms and death of SOD1 mice. These preliminary results may not only indicate that amyotrophic lateral sclerosis is an autoimmune disease, but may also indicate a possible treatment for a devastating disease and possibly others.

The feasibility of using blood bank-stored (4 degrees C) cord blood, unmatched for HLA for marrow transplantation.

The main purpose for storing large numbers of umbilical cord blood (CB) units by cryopreservation is to obtain a close HLA match for use in bone marrow transplantation. The use of partially matched or unmatched CB has been suggested, and publications about the success of 3 antigen mismatches have given some credence to this suggestion. Graft vs host disease still is considered a major barrier for successful CB transplantation. The cost per frozen CB unit of approximately $15,000 considerably limits its availability in developing countries. Eleven human umbilical cord specimens were stored in gas-permeable bags at 4 degrees C for up to 3 weeks. Clonal growth, replating efficiency in methylcellulose cultures, differential count, and flow cytometric immunophenotyping results were examined at intervals up to 21 days. Mixed lymphocyte cultures were evaluated on 13 similarly stored specimens at intervals up to 14 days. When plated at 1, 10, and 21 days, the combined percentage of the more primitive colonies increased on days 10 and 21. Replating efficiency of blast cell colonies when stem cell factor was added was 81.2% and 67.8% on days 10 and 21, respectively. When mononuclear cells were immunophenotyped, the mean percentage of CD34+ and CD117+ cells, considered primitive stem cell markers, increased significantly from day 1 to day 21. The ability of stored CB cells to respond to phytohemagglutinin or alloantigens decreased progressively from day 1 to day 14. By day 14, the reactivity of CB responder cells, in mixed lymphocyte cultures, to fresh allogeneic CB stimulator cells declined significantly. These findings suggest that CB can be stored in existing blood bank facilities and retain its hematopoietic potential for transplantation. Furthermore, it may be feasible to combine individual CB samples to provide a sufficient number of viable stem cells for transplantation, substantially expanding the number of potential recipients.

Effects of cord blood transfer on the hematopoietic recovery following sublethal irradiation in MRL lpr/lpr mice.

The potential of cord blood (CB) to serve as a rich source of stem cells and stem cell factors is receiving increasing attention. In addition, perhaps because of the early ontogeny of these cells or the lack of surface antigens, cord blood stem cells do not appear to require close identity with the recipient. In the present pilot study, we investigated the presence of a hematopoiesis enhancing effect (HEE) by assaying the ability of human cord blood cells to augment hematopoiesis across a species barrier. For these experiments, autoimmune-prone MRL-Ipr/Ipr mice were exposed to sublethal levels of irradiation and cord blood administration to study the role of factors present in human cord blood in augmenting the rate of lymphopoiesis. This strain was chosen because of the increased presence of peripheral T and B subpopulations, namely the B-1 and CD4/CD8 double negative T-cell subpopulations, which do not arise directly from bone marrow precursors, but rather accumulate with age. MRL-Ipr/Ipr mice were sublethally irradiated and reconstituted with syngeneic bone marrow (BM) cells or with human cord blood cells or peripheral blood mononuclear cells (PBMC), or were left unreconstituted. At 2 weeks post-treatment, lymphoid populations in the spleen and lymph nodes were studied as a measure of hematopoiesis. Factors present in cord blood were able to augment hematopoiesis over that which occurred endogenously. At 2 weeks postirradiation, recipients of BM cells displayed the fastest rate of peripheral lymphoid recovery, nonreconstituted mice showed the slowest lymphoid recovery, and recipients of cord blood recovered their lymphoid populations at an intermediate rate. Similarly, myelopoiesis was increased in irradiated SJL/J recipients of human cord blood. Thus, human cord blood cells appear to produce/induce factors that may act as an adjunct to increase stem-cell activity.

Endogenous hematopoietic reconstitution induced by human umbilical cord blood cells in immunocompromised mice: implications for adoptive therapy.

Human umbilical cord blood (HUCB) cells show promising advantages over bone marrow (BM) cells for a variety of diseases that require transplantation. We observed that lethally irradiated SJL/J mice given a single injection of HUCB cells survive, whereas vehicle-injected mice do not. Because survival is not due to long-term engraftment of HUCB cells, we used this HUCB/mouse model to investigate additional therapeutic benefits of HUCB cells. We investigated the mechanism by which HUCB cells accelerated endogenous hematopoiesis in mice that received either lethal (9.5 Gy) or lower-dose (8.0 Gy) radiation and then were given a single injection of HUCB mononuclear cells. Compared to irradiated control mice, the lethally irradiated, HUCB-injected group showed significant increases in peripheral white blood cell counts, red blood cell indices, and granulocyte-macrophage colony-forming units (CFU-GM) by 3 weeks. In contrast, no significant differences in these parameters were observed between control and HUCB-injected mice that received the lower dose of irradiation. Moreover, regardless of the radiation dose, only HUCB-injected mice exhibited immune responses comparable to those of age-matched normal mice. The clinical relevance of these observations was determined in long-term, culture-initiating cell assays with human BM stem cells and irradiated (gamma-) HUCB cells. CFU-GM colonies were detectable in cultures containing gamma-HUCB cells by day 15, but were undetectable in cultures without gamma-HUCB cells until day 40, suggesting a hematopoietic stimulatory role for HUCB cells. Overall, the results indicate that in addition to their use for transplantation, HUCB cells also may be used as an adjuvant therapy to enhance hematopoietic reconstitution and immunocompetence of the host. This hematopoiesis-enhancing effect represents a heretofore unrecognized function of HUCB cells.

HLA DR and AB surface antigens correlate with cell shape (surface area).

In order to help explain some of the various phenomena associated with both benign and malignant cells, this study was undertaken to determine if changes in the shape of the cell could alter the recognition of the cell. Non-transformed Human cells, HEL 299, were evaluated for their shape and surface antigens. A direct statistical correlation was found between the two surface antigens HLA AB and DR and the cell shape (surface area). The possible significance of this phenomena in non transformed human cells to neoplastic proliferation is suggested.

Potential for clinical use of viable pluripotent progenitor cells in blood bank stored human umbilical cord blood.

There are indications that a close HLA match may not be necessary when human umbilical cord blood (HUCB) is used to effect a hematopoietic transplant. This was first suggested in 1972 and was further supported by the ability of HUCB to produce mouse survival following lethal irradiation. In China multiple units of HLA unmatched HUCB was utilized successfully in children to effect transplants. A recent publication indicated that newborn rodent blood cells can engraft in adult mice across the non-H-2 [corrected] antigens. Furthermore, there recently have been successful transplants with 3 antigens mismatched HUCB. In this study HUCB was stored in polyolefin blood bank bags at 4 degrees C. The storage was similar to that used in routine blood banking. Clonogenic assays were performed at Day 1 and 21 utilizing various growth factors. Replating efficiency was determined on colonies obtained from cord blood that was stored (non-frozen) for 21 days. The functional ability of day 21 old HUCB was determined by its ability to produce survival of lethally irradiated mice. It was found that approximately two-thirds (62.5%) of single primary blast cell colonies in day 21 stored HUCB could generate various types of secondary colonies. In some instances the secondary colonies were counted as high as 42 total mean colonies per single primary colony. These blast cell colonies (CFU-BL) were able to form single and multi lineage colonies composed of virtually every hematopoietic cell types. Animal survival studies were utilized in an effort to determine possible functional ability and were successful in producing fifty-day survival in 54% of lethally irradiated SJL/J mice and 100% ALB/C mice. This study holds the potential of making HUCB available for purposes of marrow transplantation to all who need it. It could further remove most of the moral and ethical issues related to HUCB, reduce to a fraction the cost in providing stem cells for marrow transplantation and potentially allow HUCB to be handled by existing blood banks.

Potential effectiveness of stored cord blood (non-frozen) for emergency use.

Bone marrow has been used for a number of years to assist patients who have accidentally received potentially lethal levels of irradiation. The intent of the transplant is to replace the victim's own bone marrow that has been injured from the irradiation or to act as temporary support to allow the patient's own marrow to recover. Following the Chernobyl disaster, some victims received bone marrow that was HLA matched or partially matched. However, donor marrows were difficult to obtain in adequate numbers; as a substitute for bone marrow, frozen fetal liver cells were used as a source of hematopoietic stem cells. The use of fetal livers, however, was unsuccessful. Human umbilical cord blood, currently considered an excellent source of hematopoietic stem cells, was not used at Chernobyl. For several years, we have been able experimentally to keep SJL/J mice alive with the use of human umbilical cord blood after the animals received lethal levels of irradiation. This finding suggests that under certain conditions human cord blood does not have to be HLA matched to facilitate rescue from irradiation. In addition, there are reports of unmatched HLA cord blood being used successfully for marrow transplantation. If human cord blood does not have to be matched for HLA, there may be emergency cataclysmic circumstances where the availability of umbilical cord blood may be of considerable value. To simulate a clinical situation such as a nuclear accident, in which human cord blood might serve as a source of stem cells for marrow transplantation, we attempted to rescue immunocompetent mice after 900 cGY of irradiation with the use of (nonfrozen) human cord blood stored in a blood bank. The blood was stored under routine conditions (3-6 degrees C) for 5 and 7 days in special bags that allow transmission of oxygen. Following lethal levels of irradiation, the cord blood was administered to the animals and a significant survival rate was obtained.

The effect of human cord blood on SJL/J mice after chemoablation and irradiation and its possible clinical significance.

There is evidence from the existing published literature that human umbilical cord blood, when used for purposes of bone marrow transplantation, does not necessarily have to be HLA matched in order to be efficacious. These reports include experimental observations on the ability of human umbilical cord blood to rescue lethally irradiated mice and clinical observations from China wherein HLA mismatched umbilical cord blood has been engrafted successfully in children with malignant disease. The study reported herein describes an experimental immunocompetent murine model to determine if human umbilical cord blood can be used to improve survival after chemoablation and irradiation. The animals received chemoablation followed by irradiation, and irradiation alone. The presence of human DNA in these mice following injection of human umbilical cord blood cells was determined, and the immunological status of the animals was evaluated. Animals receiving human umbilical cord blood cells after chemoablation and irradiation had a better mean survival at day 50 than animals receiving syngeneic marrow. Human DNA could be found in various organs, particularly the lung, spleen and liver of the mice for the first 30 days. Thereafter, human DNA became more difficult to detect but trace amounts of human DNA could be found up to one year later. The results of mixed lymphocyte reactions and phenotype analyses for murine T cell markers performed after injection of HUCB cells both indicated endogenous repopulation, and relatively intact immune systems in these mice. Since human umbilical cord blood allowed mice to survive the lethal effects of chemoablation plus irradiation, or irradiation alone, with reconstitution of the animals' own, relatively intact, immune systems, it would appear that HLA mismatched human umbilical cord blood could potentially be used as an adjuvant treatment for patients with advanced malignancies or other diseases for which hematopoietic reconstitution is indicated.

Effect of human cord blood transfer on survival and disease activity in MRL-lpr/lpr mice.

The MRL-lpr/lpr mice have a genetic defect and by 6 months of age usually die after developing severe autoimmune disease and massively enlarged lymph nodes (Cohen, P. L., and Eisenberg, R. A., Annu. Rev. Immunol. 9, 243-269, 1991). Similarly as in some genetic diseases in humans, these mice, if given an appropriate marrow transplant from a mouse not having the defect, will have partial correction of the disease process and an extension of life (Cohen, P. L., and Eisenberg, R. A., Annu. Rev. Immunol. 9, 243-269, 1991; Lenarsky, C., Kohn, D. B., Weinberg, K. I., and Parkman, R., Hematol. Oncol. Clin. N. Am. 4, 589-603, 1990). Utilizing human umbilical cord blood as a donor source for marrow transplantation, we have been able to obtain significant correction of the MRL-lpr/lpr genetic defect and double the life span. By 11 months of age, 5 months beyond their usual life span, both the animals receiving congenic marrow (MRL-+/+) and the animals with human cord blood had mild lymphadenopathy, a decrease in double-positive T cells, and an increase in double-negative T cells. Granulomatous vasculitis could be identified in the animals receiving human cells and could not be found in the animals receiving MRL-+/+ marrow.

Magnetic resonance imaging of intraosseous lipomas: a radiologic-pathologic correlation.

Four patients with intraosseous lipomas were studied with magnetic resonance imaging. The imaging features and histology of each tumor were compared. Magnetic resonance imaging was very helpful in establishing a pathologic diagnosis. If a severe degree of involution was present, then the magnetic resonance findings could be ambiguous, making diagnosis more difficult.