To our knowledge, this is the first dose finding study using allogeneic BMMSC in ALC. Study has shown that it is feasible to administer stempeucel® at a dose of 2.5 million & 5 million cells/kg body weight through the hepatic artery in ALC. The procedure was reasonably tolerated well in majority of patients though one patient in the 5 million cells/kg dose group developed hepatic artery dissection during catheterization. There were higher incidents of infections in patients who received 5 million cells/kg body weight compared to control group even though not all were attributed to stempeucel® by the investigators.
Hepatic artery catheterization has been in practice since 1960s for administering anticancer chemotherapy (32, 33) and hepatic arterial dissection is a known complication of this procedure. In a report by Habbe et al, six incidents of hepatic artery dissections were observed in 100 attempted hepatic arterial catheter placements for administering chemotherapy (34). In another study of chemotherapy for hepatic malignancy, there was one incidence of hepatic artery dissection out of 28 patients (35). Similarly, intraarterial chemotherapy resulted in one case of hepatic artery dissection out of 30 patients (36). In a phase 1 study of bone marrow mononuclear therapy in cirrhosis in 8 patients, there was one incidence of hepatic artery dissection (37). Thus, the incidence of this complication in our study (one in 20 patients) is comparable to that reported in studies involving hepatic artery catheterization including those intended for stem cell delivery.
The DSMB opined that there is increased incidence of infection in the cell group compared to that of control. Theoretically, MSC can lead to enhanced susceptibility to infection through immunomodulatory function, particularly in patients with liver cirrhosis due to immunosuppression. It is possible that the increased rate of infection in this study was also because of preexisting immunosuppression due to cirrhosis in these patients. Infection and its complications were seen in similar studies involving administration of stem cells in liver cirrhosis. In a study by Sharma et al, one patient died on the 88th day post CD34 + cell transplantation due to development of sepsis and hepatorenal syndrome (38). In a case report by Gasbarrini et al, infusion of CD34 + resulted in fatal outcome due to multiorgan failure secondary to bacterial infection (39). Autologous bone marrow cell infusion in patients with liver cirrhosis resulted in fever in all recipients in a study by Terai S et al (40). Two patients developed self-limiting fever within 2–6 hours after UCMSC administration in acute-on-chronic liver failure patients (28). Hence it appears that infections and its complications are a common place in interventions involving cell therapy in morbid liver conditions.
Infections following stem cell administration have been seen in non-cirrhotic conditions also. In a phase I study using autologous BMMSC for therapy of allograft rejection following renal transplantation, 3 out of 6 patients developed opportunistic viral infection (41). This has been speculated to be due to the immunosuppressive effects of MSC (42). In Graft versus host disease patients, MSC therapy has raised concerns over infections as a complication (43). However, a meta-analysis of MSC studies showed that there was no difference between MSC and control groups in terms of occurrence of infection (44). The same report revealed a significant increase in transient fever in MSC group compared to control probably due to acute inflammatory reactions to particular MSC preparations.
Paradoxically, BMMSCs are thought to be protective against infectious diseases through direct effects on pathogens or indirect effect on the host. While they reduce proinflammatory cytokine and chemokine induction and reduce the migration of proinflammatory cells into sites of injury in the host, they also exert antimicrobial effect on the infectious agents (45). Mechanism of antimicrobial effects include indoleamine 2,3-dioxygenase expression induced by inflammatory cytokines (46) and secretion of cathelicidin LL-37 antimicrobial peptide (47). Antifungal effect has also been demonstrated by IL-17 producing subset of MSC (48). Beneficial role of MSC has also been discussed in tuberculosis, through immunomodulatory functions favorable to the host and down regulation of host susceptibility to infection (49). Sepsis, which is a deranged response of host immune mechanism to microbial invasion, results in organs damage. MSC has been considered a suitable agent to be tested for sepsis because of its antibacterial, immunomodulatory effect, antiapoptosis and regenerative response (50). Extensive preclinical studies have demonstrated efficacy of MSC in animal models of sepsis (51, 52). One clinical trial has also been initiated using MSC in septic shock (53). Arango-Rodriguez has reviewed the mechanisms through which MSCs can facilitate infection in the recipient as well as literature suggesting that MSC may reduce infection (54). Conflicting opinions about role of MSC in infection are probably because of the heterogenicity in MSC with respect to its source, dose, route of administration and the disease condition in which it is administered.
Cell therapy can be administered to liver cirrhosis patients through different routes: peripheral vein, portal vein, spleen and hepatic artery. Intravenous delivery has been commonly used for administration of cells in liver cirrhosis patients. Portal vein catheterization has technical challenges due to ascites in these patients and additional risk of portal vein thrombosis and subsequent variceal bleed. Intrasplenic route has been used by few studies for cell administration in liver cirrhosis (25, 55). Hepatic artery catheterization was chosen in our study, owing to higher proportion of cells possibly lodging in liver. Other than dissection, the potential complication of this route of delivery is worsening of liver function due to embolization. Deterioration of liver function was not seen following cell administration in this study, ruling out liver damage due to cell embolization. There were no other immediate complications directly attributed to stempeucel®. Study by Mohamadnejad et al was prematurely stopped because patients developed complications renal failure and radio-contrast nephropathy, and concluded that injection of CD34 + cells through hepatic artery was probably unsafe (56). However, later studies involving CD34 + cells infusion through hepatic artery did not show such safety issues with administration of CD3 + cells through hepatic (38, 57–59).
This study was designed primarily for assessing the safety and feasibility of administering stempeucel® through the hepatic artery in ALC. As a secondary objective, we also explored possible efficacy and dose-response. Efficacy was seen only in quality of life of patients who received 2.5 million cells/kg dose of stempeucel® compared to control as seen in few mental component scores of SF-36. This may be partially due to open label nature of the study. SF-36 improvement was seen in a study by Salama et al following haematopoietic stem cells therapy in end-stage liver disease patients (60). Quality of life improvement associated with clinical improvement was seen in a study by Kim et al evaluating autologous bone marrow infusion in advanced liver cirrhosis (61). The lack of obvious clinical efficacy seen in this study may be attributed to three reasons: Firstly, the eligibility criteria included patients in the higher severity of liver cirrhosis (Child Pugh class B and C). It is possible that these patients were already in advanced stage of the disease and not amenable for cell therapy. Probably cell therapy has to be attempted at an early stage of disease like alcoholic hepatitis, rather than at a late stage when cirrhosis has already set in. The alcoholic hepatitis stage may help better homing of cells because of the local inflammation. In the advanced stage, it may be difficult for the cells to home to the site of action since there is no active inflammation. Secondly, the starting dose selected for this study (2.5 million cells/kg body weight) might be in the upper end of therapeutic range; higher doses potentially leading to deleterious effects. Lastly, in-spite of strongly conveying the need of alcohol abstinence, some patients might have consumed alcohol during the study, which might have negatively affected the clinical outcome. While most pilot studies involving stem cells in liver cirrhosis had successful outcomes, few studies had negative results. Mohamadnejad, who pioneered the MSC administration in liver cirrhosis with several successful pilot studies, found no benefit of intravenous BMMSC administration compared to placebo in a randomized trial (62). Recently, a double blind study by the same group using BMMNC administered through portal vein in decompensated cirrhosis showed overall no benefit albeit a transient benefit at 3 months (63).
Evidence of efficacy of stem cells requires demonstration of tissue regeneration in addition to proving clinical benefit. Tucker et al recommended a triad of outcome measures for cell therapy trials: demonstration of mechanism of action in terms of cellular response, clinical evidence of improvement and structural benefit (64). This translates to clinical and biochemical improvements in liver cirrhosis, which are easier to demonstrate and structural changes through histopathology of liver tissue, which is a complex procedure. Liver biopsy is challenging especially in cirrhotic patients though several studies have included this procedure. Terai et al has shown that there was improvement in serum albumin, total protein, AFP and proliferating cell nuclear antigen in liver biopsy after 4 weeks of autologous BMMNC infusion therapy (40). Zhang et al showed improvement in ascites, liver function, MELD score in addition to decrease in liver fibrosis markers (27). Kim et al have demonstrated increasing activation of hepatic progenitor cell compartment, hepatic progenitor cell differentiation, and improvement in Child Pugh scores (61). Interestingly, 80% patients showed increase in liver volume as per Magnetic Resonance Imaging. Jang et al have seen histological improvement in 6 out of 11 patients who received BMSC through the hepatic artery for ALC (23). Enhanced angiogenesis was seen in follow-up liver biopsy specimens after boost infusions of mobilized peripheral blood stem cells in decompensated alcoholic cirrhosis in a study by Yannaki et al (65). In the present study, we did not conduct liver biopsy at 6 months follow-up. It is debated that small tissue sample may not be adequate representation of liver pathology and may be subject to sampling error and intra-observer variation (66, 67). Hence, Fibroscan (Transient Elastography) which is a non-invasive technique of assessment of liver stiffness was employed in this study. This method has been validated for diagnosis of liver cirrhosis (68) and was found to be reproducible in patients with chronic liver disease (69). Hence Fibroscan is considered to be an option instead of liver biopsy (70). In this study, there was no change from baseline in Continuous Attenuation Parameter and liver stiffness indicating there is no worsening or improvement in liver fibrosis. To our knowledge, Fibroscan has not been used in published studies involving cell therapy in liver cirrhosis, though the REALISTIC study protocol evaluating CD133 + cells incorporates this technique (71).
Several approaches have been evaluated for improving efficacy of cell therapy in liver cirrhosis. Animal studies have shown that pretreatment of MSC with injured liver cells has improved the ability of MSC for homing and hepatic differentiation (72). Amer et al differentiated the MSC towards hepatocytes by pretreating them with HGF before infusion via intrahepatic or intrasplenic routes in patients with end-stage liver cell failure due to chronic hepatitis C and found improvement in cell treated group (55). However, El-Ansary et al found no difference in efficacy between MSCs differentiated to hepatocytes and undifferentiated MSCs in hepatitis C virus induced liver cirrhosis (24). Salama et al administered granulocyte-colony stimulating factor (G-CSF) daily for 5 days before administering MSC through the peripheral vein (73). Recently, co-administration of MSC with PPAR gamma agonists has been tried with encouraging results (74). Repeat injection has been tried in cell therapy studies. Jang et al administered autologous BMMSC at baseline, and again after 4 weeks and found that histological improvement was seen in 6 out of 11 patients and Child Pugh score improved in ten patients (23). In a study by Zekri et al, liver cirrhosis patients were randomized to receive one session of autologous haematopoietic stem cells followed by MSC, two sessions of similar treatment separated by 4 months or control (75). It was observed that while one session group showed improvement in serum albumin, bilirubin and INR values till 6 months, two session group sustained improvement till 12 months. Zhang et al have tried UCMSC administration thrice, using peripheral vein and showed clinical improvements and MELD scores (27). REALISTIC study aims to evaluate improvement in disease severity using GCSF alone or G-CSF followed by repeated infusion of CD-133 + cells compared to standard protocol of care alone (71).
The transplantation of MSC showed therapeutic potential for liver function improvement according to recent experimental studies and human studies. Although they remain unclear, the major potential mechanisms have been proposed as a twofold; one is the improvement of the microenvironments through paracrine effects, and the other is the replacement of functional hepatocytes (76).
Dose response relationship has to be established in any drug product development. However, it is still unclear whether classical dose-response exists with cell therapy. Review of cell therapy studies in heart disease has shown that the dose response was inconsistent and contradictory in terms of dose of administered cells and clinical response, both in preclinical and clinical setting (77). Most clinical trials apply MSCs according to the body weight of patients (n = 9, 0.5–3 × 10(6)/kg as a single dose), while others apply MSCs according to the total quantity of cells (n = 7, 1–20 × 10(7)) (78). In liver cirrhosis trial using bone marrow cells, Lyra et al noted that there was no correlation of number of cells and clinical improvement (79). However, in a dose ranging study using 5X10(5), 1X10(6) and 2X10(6) cells/kg of CD34 + cells, Nakamura et al have observed improvement in patients who received middle or higher dose (59). It is important to accept that conclusion on dose response cannot be drawn from different trials. Among the recent clinical trials involving applying MSCs to treat liver diseases, the total number of MSCs used was from ~ 10(7) − ~ 10(9), regardless of which method was chosen to deliver MSCs (78).
There are few limitations for this study. First, we have not traced the cells within the body using radioactive technology owing to the inherent complexity of the procedure. Second, we do not have follow-up liver biopsy data. Third, the control group did not receive sham intervention or placebo injection due to ethical reasons. Fourth, the sample size was limited to 20 patients in each dose level, which is insufficient for meaningful detection of efficacy. Lastly, incorporation of a biomarker of liver regeneration like AFP would have provided insights into possible mechanism of action.